Oral Drug Delivery for Immunoengineering

Abstract

The systemic pharmacotherapeutic efficacy of immunomodulatory drugs is heavily influenced by its route of administration. A few common routes for the systemic delivery of immunotheraputics are intravenous, intraperitoneal, and intramuscular injections. However, the development of novel biomaterials, in adjunct to current progress in immunoengineering, is providing an exciting area of interest for oral drug delivery for systemic targeting. Oral immunotherapeutic delivery is a highly preferred route of administration due to its ease of administration, higher patient compliance, and increased ability to generate specialized immune responses. However, the harsh environment and slow systemic absorption, due to various biological barriers, reduces the immunotherapeutic bioavailability, and in turn prevents widespread use of oral delivery. Nonetheless, cutting edge biomaterials are being synthesized to combat these biological barriers within the gastrointestinal (GI) tract for the enhancement of drug bioavailability and targeting the immune system. For example, advancements in biomaterials and synthesized drug agents have provided distinctive methods to promote localized drug absorption for the modulation of local or systemic immune responses. Additionally, novel breakthroughs in the immunoengineering field show promise in the development of vaccine delivery systems for disease prevention as well as combating autoimmune diseases, inflammatory diseases, and cancer. This review will discuss current progress made within the field of biomaterials and drug delivery systems (DDS) to enhance oral immunotherapeutic availability, and how these new delivery platforms can be utilized to deliver immunotherapeutics for resolution of immune‐related diseases. This article is protected by copyright. All rights reserved.

Full text

REVIEW
Oral drug delivery for immunoengineering
Tien Le
1
| Brian Aguilar
2
| Joslyn L. Mangal
3
| Abhinav P. Acharya
1,2,3,4,5
1
Chemical Engineering, School for the
Engineering of Matter, Transport, and Energy,
Arizona State University, Tempe, Arizona, USA
2
Biomedical Engineering, School of Biological
and Health Systems Engineering, Arizona State
University, Tempe, Arizona, USA
3
Biological Design, School for Biological and
Health Systems Engineering, Arizona State
University, Tempe, Arizona, USA
4
Materials Science and Engineering, School for
the Engineering of Matter, Transport, and
energy, Arizona State University, Tempe,
Arizona, USA
5
Biodesign Center for Immunotherapy,
Vaccines and Virotherapy, Arizona State
University, Tempe, Arizona, USA
Correspondence
Abhinav P. Acharya, Chemical Engineering,
School for the Engineering of Matter,
Transport, and Energy, Arizona State
University, Tempe, AZ 85281, USA.
Email: abhi.acharya@asu.edu
Funding information
National Institute of Health, Grant/Award
Numbers: R01AI155907, R01AR078343
Abstract
The systemic pharmacotherapeutic efficacy of immunomodulatory drugs is heavily
influenced by its route of administration. A few common routes for the systemic
delivery of immunotherapeutics are intravenous, intraperitoneal, and intramuscular
injections. However, the development of novel biomaterials, in adjunct to current
progress in immunoengineering, is providing an exciting area of interest for oral drug
delivery for systemic targeting. Oral immunotherapeutic delivery is a highly preferred
route of administration due to its ease of administration, higher patient compliance,
and increased ability to generate specialized immune responses. However, the harsh
environment and slow systemic absorption, due to various biological barriers, reduces
the immunotherapeutic bioavailability, and in turn prevents widespread use of oral
delivery. Nonetheless, cutting edge biomaterials are being synthesized to combat
these biological barriers within the gastrointestinal (GI) tract for the enhancement of
drug bioavailability and targeting the immune system. For example, advancements in
biomaterials and synthesized drug agents have provided distinctive methods to pro-
mote localized drug absorption for the modulation of local or systemic immune
responses. Additionally, novel breakthroughs in the immunoengineering field show
promise in the development of vaccine delivery systems for disease prevention as
well as combating autoimmune diseases, inflammatory diseases, and cancer. This
review will discuss current progress made within the field of biomaterials and drug
delivery systems to enhance oral immunotherapeutic availability, and how these new
delivery platforms can be utilized to deliver immunotherapeutics for resolution of
immune-related diseases.
KEYWORDS
immunotherapy, oral bioavailability, oral immunotherapeutic delivery, oral vaccination
1|INTRODUCTION
Drug delivery is the process of administering a pharmaceutical com-
pound in order to achieve a therapeutic effect. Oral administration is
a preferred method for delivery because of its convenient and nonin-
vasive delivery of drugs. However, a variety of obstacles limit the effi-
cacy of oral drug administration, namely, the acidic and enzymatic
degradation in the stomach, the range of pH throughout the gastroin-
testinal (GI) tract (pH ranging from 1 to 7), first-pass metabolism, the
steric barrier of the mucosal system and the physical barrier of
the epithelial layers, to name a few. Each of these challenges contrib-
ute to the complex course an immunotherapeutic has to take through
the body's intricate GI tract prior to reaching its target location within
the intestine for absorption and systemic bioavailability. For the
Received: 20 May 2021 Revised: 20 July 2021 Accepted: 25 July 2021
DOI: 10.1002/btm2.10243
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium,
provided the original work is properly cited.
© 2021 The Authors. Bioengineering & Translational Medicine published by Wiley Periodicals LLC on behalf of American Institute of Chemical Engineers.
Bioeng Transl Med. 2022;7:e10243. wileyonlinelibrary.com/journal/btm2 1of18
https://doi.org/10.1002/btm2.10243

purpose of this review, bioavailability is defined as the potential of
orally delivered drugs to reach systemic circulation. These issues are
further escalated and complicate delivery of biologics such as vaccines
and antibodies that need to be delivered systemically for modulating
disease outcomes. Biomaterials and drug delivery systems (DDS) can
play an important role in developing strategies to overcome these
issues and allow for delivery of therapeutics to the immune system
locally in mucosa or systemically. Notably for delivery of fragile immu-
notherapeutics, such as antibodies, mRNA, and DNA, specialized DDS
are required, which can overcome the challenges associated with oral
delivery. While there have been recent reviews that also suggest the
importance of DDS for oral delivery,
1–4
this review will focus on dis-
cussion of immune engineering and immunotherapeutic delivery via
oral route. In this review, we will first discuss the major immunothera-
peutics that can potentially be delivered orally, and challenges associ-
ated with poor oral drug targeting. Next, we will discuss current oral
to systemic delivery strategies, specific delivery mechanisms, and the
promising future of oral drug administration for the systemic modula-
tion of immune responses.
2|IMMUNOTHERAPEUTICS AND THEIR
TARGETS
Human intestines house approximately 10
12
lymphoid cells per meter
and is known to have the highest density of immune cells in the
body.
5
Thus, this tissue provides an attractive target for different ther-
apeutics that can modulate immune-related diseases including cancer,
autoimmune diseases, and infection.
Since the GI tract is inherently tolerance-inducing, it also provides
the opportunity to generate tolerance toward molecules that the body
has not been exposed to previously. For example, intravenously deliv-
ered therapeutics such as checkpoint inhibitors
6,7
or anti-VEGF
8
can
generate neutralizing antibodies, called antidrug antibodies,
9
which
severely reduce their efficacy. Therefore, generating strategies for
presentation of these therapeutics to the oral or mucosal immune sys-
tem provides a unique opportunity to generate tolerance toward
intravenously infused drugs, and thus improve efficacy by diminishing
the generation of antidrug antibodies.
Although, GI tract is naturally tolerance inducing, it is also the site
of entry for most of the pathogens, and developing immune responses
that can eliminate these pathogens in the tolerance-inducing environ-
ment is especially challenging. Therefore, generating DDS that can
orally deliver therapeutics (e.g., vaccines) targeted toward immune
cells such as dendritic cells (DCs) can be highly beneficial. This topic
will be discussed in detail later in this review.
In addition to vaccines, another important class of immunothera-
peutics are interleukins (ILs, e.g., IL-10, IL-4, IL-2), which can have dra-
matic effect on immune responses in the gut. These ILs have their
specific receptors on different immune cells that line the intestine,
and thus provide druggable targets for generating immunotherapy,
which can be either pro- or anti-inflammatory. In addition to ILs,
growth factors can also generate robust immune responses by
proliferating specific type of immune cells. For example, granulocyte
macrophage colony stimulating factor (GM-CSF) can be utilized to
proliferate innate cells (e.g., DCs),
10,11
and is utilized in clinic for treat-
ment of cancer.
12
Targeting GM-CSF specifically to colon tumors can
be achieved by developing DDS that deliver this growth factor at the
site of lesion, thereby making the therapy more effective. Moreover,
small molecules such as rapamycin
13,14
that target the mTOR path-
way
15
if targeted to specific sites of the immune system within the
gut can provide site-specific immune suppression. Lastly, delivery of
specialized probiotics in the GI tract that can modulate the immune
function is another major area where DDS can make a large impact.
Some of the strategies that DDS utilizes to deliver these immunother-
apeutics are discussed in this review.
Despite tremendous promise of DDS there still exists natural GI
tract barriers that have to be overcome if oral delivery is to be consid-
ered for immune engineering. These natural barriers are briefly dis-
cussed below, and for further information on this topic readers are
encouraged to read more specialized reviews.
1,16
3|GI TRACT BARRIERS THAT PREVENT
ORAL IMMUNOTHERAPEUTIC DELIVERY
3.1 |Physiochemical barrier
Orally delivered pharmaceuticals travel through the upper (mouth to
the duodenum of the small intestine) and lower (most of the small
intestine to the large intestine) segments of the GI tract, and the latter
segment contains the most barriers for oral delivery yet houses most
of the drug absorption. As the drug travels through the upper segment
of the GI tract, it encounters the degradative environment of the
stomach (pH from 1 to 3) and is also met by strong proteolytic gastric
enzymes (i.e., lipase, pepsin, amylase).
17
This acidic environment and
increased proteolytic activity within the upper GI tract can lead to the
degradation of drugs before they reach the small intestine for absorp-
tion, therefore, limiting the efficacy of the drug.
18
Furthermore, these
pharmaceuticals must also be able to overcome mechanical stress
(gastric flow) that resist the progression of the drug.
18
Notably, pro-
teins and other large biologics, like immunoglobulins, undergo stability
and absorption challenges due to rapid degradation in the gut.
18
In
one study, bovine milk immunoglobulin exhibited a 96% reduction
in its rotavirus-neutralizing activity in vitro when incubated with pep-
sin at a pH of 2, thus demonstrating the consequential effects of the
GI environment on large biologics.
19
Therefore, it is important that
biologics must be specially modified to endure the natural characteris-
tics of the gut.
Additionally, orally delivered drugs also have reduced systemic
availability as compared to drugs that are delivered intravenously or
intranasally due to the phenomena known as first-pass metabolism.
20
The first-pass effect describes how the concentration of an orally
administered drug is reduced prior to meeting systemic circulation
due to decreased gastric residence time and enzymatic degradation.
20
Kolars et al. demonstrated this effect in their study, where cyclosporin
2of18 LE ET AL.

was delivered to the small bowel of two patients following liver trans-
plantation.
21
Approximately 25% and 51% of total cyclosporin-
derived metabolites were observed in portal blood for the patients
after 60 min of delivery, thus indicating heightened metabolic degra-
dation of cyclosporin.
21
This reduced availability of the drug in the
systemic circulation directly decreases the sustained response that
the oral therapeutics were initially intended to produce. The reduction
of drug available in the systemic circulation is likely attributed to the
drastic physiochemical conditions existent in the GI tract, such as
the steric barriers of the mucosal immune system and the physical
barrier of the intestinal epithelial layer.
18,20
In order to overcome the
first-pass effect, oral drugs are typically administered at a larger con-
centration; however, this then affects the toxicity and efficacy of vari-
ous pharmaceuticals.
20
3.2 |Epithelial barrier as immune defense
Immunotherapeutics must also overcome the challenge of limited traf-
fic time in the GI tract and limited surface available for absorption
(Figure 1, all schematic figures generated using Biorender.com, unless
otherwise stated).
The epithelial layer of the GI tract contains tight junctions that
further regulate the movement of substances within and through this
surface, forming the first line of defense of the immune system.
22
These tight junctions create a barrier that affects both the paracellular
and transcellular transportation of molecules through epithelial tissue,
thus molecules attempting to reach systemic circulation must cater to
the underlying mechanisms of active/passive transport through this
layer.
23
Encountering these challenges therefore reduces gastric resi-
dence time of a pharmaceutical and adds to the overlying challenge of
delivering a sustained effect of orally administered drugs.
Notably, epithelium that act as a defense mechanism can also be
utilized to deliver drugs to the immune system as well. For example,
Pridgen et al. demonstrated that polyclonal IgG Fc conjugated with
poly(lactic acid)-polyethylene glycol nanoparticles could be utilized to
target the Fc receptor (FcRn) presented by the epithelial cells. More-
over, this study showed that these nanoparticles conjugated to IgG Fc
were able to transcytose through the epithelium in vitro.
24
Lastly, this
study also showed that orally administered FcRn-targeted
nanoparticles increased he mean absorption efficiency of the
nanoparticles 10-fold as compared to the control of nanoparticles
that were not targeted to FcRn (Figure 2).
24
This strategy of utilizing
FcRn to target the epithelium can be utilized to generate immunity
against infectious pathogens (e.g., M72 antigen against Tuberculosis),
but also toward generating tolerance in autoimmune diseases
(e.g., collagen for treatment of rheumatoid arthritis).
3.3 |Intestinal microbiota
Microbiota consists of microorganisms that reside in the gut of mam-
mals and are necessary for the maintenance of immune homeostasis
in the gut.
25
Gut microbiota act in a mutually beneficial relationship
with the host to both strengthen the immune system through a series
of microbiota-dependent cascades within the epithelium as well as
allow microbiota to thrive in the mucus.
26
However, such microbiota
can still serve as a threat if the immune system is weakened.
26
Fur-
thermore, it is important to note that while the GI tract serves an
essential function in the digestion of foods through the existence of
specific characteristics within its dynamic environment and complex
regulative mechanisms, it can also impair the efficacy of orally admin-
istered pharmaceuticals.
25,26
Interestingly, microbiota which prevent the drug permeation
through the gut, also can be used as a therapeutic themselves for
immunotherapies. For example, Lin et al. demonstrated that a probi-
otic, Escherichia coli Nissle 1917 (EcN) can be delivered to the Peyer's
patches that can then induce anti-inflammatory responses in the
intestine (Figure 3). Interestingly, this study took advantage of
β-glucan embedded on yeast membrane to target the M cells, by coat-
ing EcN with yeast membrane.
27
Importantly, this study demon-
strated that delivery of yeast membrane coated EcN (EcN@YM) when
delivered orally, could localize to the Peyer's patches (immune organ),
where they can generate an immune response to prevent degradation
of the intestinal barrier.
27
3.4 |Mucosal immune system of the gut
Among the first lines of immunological protection in the GI tract is the
specialized mucosal layer that lines the surface of the epithelium.
The mucosal layer is a gel-like structure composed of glycoproteins
called mucins which are secreted by goblet cells that line the intestine.
28
Unfortunately, the constant production of mucus in the GI tract also
largely reduces the availability of orally administered therapeutics to
their targets. The mucosal system functions as a specialized immune
defense system of the GI tract by detecting luminal foreign entities and
either removing or neutralizing them while protecting the body's natural
microbial flora.
29,30
Interestingly, mucosal surfaces vary in thicknesses
along the GI tract due to the structure of their charged glycoproteins.
This forms a steric barrier that restricts movement throughout its layer
and drugs must permeate the mucosal barrier before entering systemic
circulation.
31
Mucus is constantly being secreted and cleared quickly,
thus trapping and removing foreign structures jointly and decreasing
residence time of delivered drugs.
31
The gut mucosa poses a particularly
significant challenge for immunotherapeutics, such as antibodies and
other large proteins, due to its dynamic nature and steric barriers. Par-
ticularly, it was shown that the diffusion coefficient decreased with
increasing molecular weight of proteins when tested in vitro in porcine
intestinal mucus, thus demonstrating the size-dependent barricade of
the steric mucosal barrier.
32
Large proteins, especially antibodies, are
also found to bind with mucins through electrostatic forces or strong
hydrogen bonds thus essentially being trapped and unable to reach sys-
temic circulation.
33,34
Therefore, for developing oral to systemic immu-
notherapeutics it is necessary to design them so that these are able to
overcome the mucosal barriers.
LE ET AL.3of18

Notably, Howe et al. demonstrated that not only the route of
administration (systemic vs. oral) of therapeutics but also whether the
therapeutic is associated with a nanoparticle is important for
the development of an immune response. Specifically, this study dem-
onstrated that oral delivery of ovalbumin (OVA) protein alone induce
tolerance, whereas OVA conjugated to nanoparticle induced immuno-
genic response (Figure 4).
35
Moreover, subcutaneous boosting with
OVA further increased the production of IgA titer, which is important
for developing immunity against oral pathogens.
35
These data suggest
that the mucosal immune systems barriers can be taken advantage of
FIGURE 2 Nanoparticles conjugated with IgG Fc can be targeted to the intestinal epithelium. (a–c) FcRn is expressed in different parts
of the intestines. (d) Nanoparticle intestinal uptake in mice shown here in red Source: Reprinted/adapted from Pridgen et al. Sci Transl Med.
2013;5(213):213ra167, © The Authors, some rights reserved, exclusive licensee AAAS. Distributed under a CC BY-NC 4.0 license http://
creativecommons.org/licenses/by-nc/4.0/
FIGURE 1 Drug molecules must bypass various barriers in the intestinal tract to reach systemic circulation. Some of these barriers include
microbiota, mucosa, epithelial cells, and the immune system. Microbiota maintain immune homeostasis in the gut. A double-layered mucosa coats
the epithelium, which is bound by tight junctions. The basement membrane forms a dense layer under the epithelial layer. The gut barrier also
houses several key immunological components (Peyer's patches, lymph nodes, dendritic cells, macrophages, T cells), which play an important role
in preventing foreign pathogens/materials from invading systemic circulation
4of18 LE ET AL.

depending on the type of immunity desired (tolerogenic versus immu-
nogenic). Therefore, stronger IgA-based immunity against mucosal
infections such as SARS-COV2 may be generated by not only immu-
nizing in the non-mucosal tissue (e.g., intramuscular injections) but
also orally with appropriate adjuvants.
The commensal bacteria reside primarily on the outer mucosal
layer and actively interact with the mucin. For example, Bacteroides
thetaiotaomicron, a type of commensal bacteria, has been shown to
increase goblet cell differentiation, which are then responsible for
generating mucin.
36–38
Different probiotic agents, which function to
grow and restore intestinal flora, have also been found to stimulate
mucin protein production and thus help to enhance pathogenic resis-
tance.
39,40
Therefore, it is evident that commensal bacteria play a
significant role in maintaining immunity at the mucosal layer. It is
important to note, though, that the success behind this symbiotic rela-
tionship is still being widely investigated and conflicting evidence sug-
gests that the penetration of commensal bacteria through the
mucosal layer is associated with inflammatory diseases, such as
inflammatory bowel disease or Crohn's disease.
41
Although the outer mucosal layer houses various microbiota that
live in a symbiotic relationship with the host, the inner mucosal layer
is nearly devoid of any bacteria.
42
Structurally, the inner mucosal
layer is more compact than the outer layer, and thus serves more as a
physical line of defense against pathogenic agents or foreign enti-
ties.
42
Drug delivery vehicles, therefore, must also seek new pathways
to combat against the constantly recycled inner and outer layer of
FIGURE 3 Yeast membrane coated Escherichia coli Nissle 1917 (EcN@YM), prevents intestinal barrier impairment from Salmonella infection.
Using the experimental design (a) this study shows that EcN@YM prevented Salmonella mediated submucosal edema (b), depletion of goblet cells
(c), pathological score (d), and an increase in intestinal permeability (e and f) Source: Reprinted/adapted from Lin et al. Sci Adv. 2021; 7(20):
eabf0677. © The Authors, some rights reserved, exclusive licensee AAAS. Distributed under a CC BY-NC 4.0 license http://creativecommons.
org/licenses/by-nc/4.0/
LE ET AL.5of18

mucus.
43
This may involve coating the drug carrier with a specialized
polymer that allows for increased mucosal penetration or
adhesion.
31,44,45
Because the intestinal mucus provides a niche for various types
of microbiota, it is important that the intestinal immune system be
able to differentiate between commensal bacteria and pathogenic
bacteria. The exact method in which a homeostatic environment is
maintained is still unknown, though various studies have shown that
the presence of antigen-presenting cells, specifically DCs, along with
assorted populations of B and T cells within the mucosa are likely
related to the discrimination between commensal and pathogenic bac-
teria.
46
The subset of DCs found in the mucosa have been compared
to similar DCs found in the respiratory tract where they play a notable
role in tolerance, and thus building a specific tolerance toward com-
mensal bacteria.
47
Aside from commensal bacteria, the specialized
immune system in the mucosa can also recognize certain food anti-
gens to help prevent immune responses against the food antigens.
46
Since the mucosa and the intestinal immune system are so tightly
related, malfunctions at the mucosal layer, where immune responses
can be triggered against non-pathogenic bacteria, are the general
basis for intestinal inflammatory diseases, such as Coeliac disease or
Crohn's disease.
46
However, this relationship between the mucosa
and the intestinal immune system demonstrates the opportunity to
target the mucosal layer for oral delivery of drugs. Moreover,
targeting DCs or mimicking the commensal bacteria can be an alterna-
tive approach to deliver immunotherapeutics orally to the mucosal
immune system and potentially treat these inflammatory diseases.
An understanding of each of the above discussed challenges is
necessary to advance the construction of novel biomaterials to tackle
specific barriers in oral administration. Oral DDS takes into account
these barriers in order to improve drug availability for oral to systemic
delivery. Few of the strategies to achieve this include modulating the
epithelial barrier for drug absorption, formulating therapeutics to bet-
ter adhere to the mucosal layer, or targeting specific immune cells in
the gut (Figure 1). These DDS strategies are further discussed in detail
in the following sections with examples expanding the potential appli-
cation of these DDS strategies on immune engineering and immuno-
therapeutic delivery via oral route.
4|MUCOADHESION TO IMPROVE DRUG
DELIVERY
To optimize the amount of drug that is absorbed in the body, it is impera-
tive that the carrier either releases a large amount of its contents in a
short amount of time or releases a known amount throughout a set time.
Currently, there is larger focus in the former, but recent developments of
novel biomaterials have utilized mucoadhesion to increase the residence
time of the drug in the GI tract. This method can enhance the therapeutic
effect by increasing the absorption at the target site and can often be
combined with enteric polymers (e.g., Eudragit) for gastric resistance and
GI targeting.
48
Thiseffectwasdemonstratedinaninvivostudywithdia-
betic rats using orally administered insulin enterically coated with Eudragit
as well as a polymeric mucoadhesive layer consisting of polycarbophil–
cysteine that showed a sustained decrease in blood glucose over a time
period of 80 h before steadily reaching its initial value again. Moreover,
no significant effect was observed when insulin was orally administered
without the polymeric coating.
49
Oral delivery techniques such as these,
show promise in developing oral DDS that may allow for an increased
gastric residence time with a controlled and sustained release of the orally
delivered pharmaceutical (Figure 5). Mucoadhesion utilizes the formation
of chemical bonds, most commonly hydrogenorionicbonds,oreven
stronger covalent bonds between the mucosa and the mucoadhesive
materials to prolong the residence time of absorption in the GI tract.
48
In
addition to expanding the absorption window of an orally delivered drug,
formation of these chemical/ionic bonds in combination with specialized
polymers can enhance permeation and prevent degradation of delivered
agents. Some of the polymers that can achieve this include chitosan; how-
ever, this bond is not strong enough on its own to sustain the
mucoadhesion and thus only slightly increases the residence time.
50
FIGURE 4 Induction of intestinal IgA and serum IgG2c antibody response depends on the immunization route that was used for priming
Source: Reprinted/adapted from Howe et al. PLoS One. 2015;10(2):e0118067, with permission from Creative Commons Attribution (CC BY)
license, PLOS One
6of18 LE ET AL.

However, when mucoadhesive technologies are paired with thiolated
polymers, mucoadhesive properties can dramatically increase, as shown in
an in vitro study demonstrating that thiolated polymers showed signifi-
cant stability as opposed to non-thiolated polymers and did not exhibit
any disintegration behavior over the observation period of 48 h.
51
Thiolated polymers interact with the mucosal layer to form strong cova-
lent disulfide bonds (Figure 2) that promote a more structurally stable car-
rier, in turn increasing the residence time in the GI tract at the target
location.
32
In another study, thiolated chitosan micelles demonstrated up
to a 56-fold higher degree of attachment to intestinal mucosa compared
to unmodified chitosan micelles, thus demonstrating the potential of thio-
lation for improved mucoadhesion and drug delivery.
52
The strategies of mucoadhesion can be utilized to increase residence
time of immunotherapeutics such as cytokines and growth factors. Inter-
estingly,diseasessuchasUlcerative colitis and Inflammatory Bowel Dis-
eases may benefit from local delivery of anti-inflammatory cytokines such
as IL-10
53
and IL-27,
54
and antibodies such as anti-IL-22.
55
The
extended residence of these therapeutics will ensure reduction of inflam-
matory cascade induced by pro-inflammatory cells (e.g., T helper type
1 and T helper type 17) in the intestine, and provide immune
homeostasis.
Interestingly, Chung et al. reported that orally delivered IL-10
releasing poly(lactic acid) microparticles ameliorated local GI poly-
posis in mice. This study was able to demonstrate that these
particles were taken up in the Peyer's patches, which could then
polyp numbers and anemia in mice significantly as compared to the
control.
56
Importantly, this study demonstrated that these parti-
cles could increase the survival of mice significantly as compared
to controls. Therefore, delivery of anti-inflammatory cytokines can
be a powerful tool to locally modulate the immune response to
address chronic diseases.
Another strategy for overcoming the mucosal barrier includes
nanoparticle systems that use specialized mucolytic agents. These are
conjugated on the surface of the particles and have the ability to
cleave mucus substructures, which then allows for the drug carrier
to bypass the mucosal layer.
52
De Sousa et al. examined papain (PAP)
and bromelain (BRO) as mucolytic agents, both of which were shown
to permeate through nine 2 mm intestinal mucus gel segments while
the unmodified nanoparticles were found to only permeate through
the first few segments.
57
The diffusion coefficient of intestinal mucin
also exhibited a 2-fold increase in the presence of PAP and BRO as
opposed to unmodified nanoparticles.
57
These results demonstrate
the ability of mucolytic agents to permeate the mucosal layer in the
GI tract. Since both the PAP and BRO enzymes are digested in
the gastric environment, utilizing enteric coatings would be one idea
to consider for the purpose of ensuring the enzymes are not degraded
by the harsh gastric acidity for the successful delivery of
immunotherapeutics.
FIGURE 5 Mucoadhesive polymer coating on drugs can provide higher residence time in the gut. These mechanisms include disulfide bonds
and ionic interactions with the mucosa
LE ET AL.7of18

The combinatorial delivery of multiple targeting mechanisms
allows for the strength of one agent to compliment the strength of
the other for an effective therapeutic delivery. For example, self-
nanoemulsifying drug delivery systems (SNEDDS) as drug carriers
were designed to increase drug dissolution and solubility due to their
lipophilic nature.
58
Interestingly, SNEDDS cannot adhere to the muco-
sal layer within the GI tract due to the net-negative charge of the
mucus. An overall positive charge can be generated if SNEDDS are
paired with mucoadhesives (e.g., chitosan derivatives) for binding to
the negative mucosal layers, and thus increasing availability of drugs
with poor solubility in these environments. The slightly negative
charge of the mucosal layer explains the partial ionic binding of the
positively charged acyl chitosan to the mucosal layer. This novel sys-
tem has found success with drugs like saquinavir
59
in mice experi-
ments as well as tipranavir
60
and cyclosporin A,
61
which were found
to have enhanced effects with orally delivered drugs as compared to
the control in clinical trials.
62,63
An interesting application of this com-
bination strategy of SNEDDS can be delivery of anti-inflammatory
agents for chronic autoimmune diseases such as type 1 diabetes, mul-
tiple sclerosis, and rheumatoid arthritis. For example, rheumatoid
arthritis patients are required to inject themselves with anti-TNFα
every few weeks to limit the damage by immune cells to the tissues.
Therefore, a strategy that allows for antibodies to be taken orally will
allow for delivery of anti-inflammatory agents from oral to systemic
route, thereby potentially increasing compliance as well. However,
after achieving mucosal penetration, the drug then must overcome
transportation through the epithelial barrier before these immuno-
therapeutics can be delivered systemically, and different methods to
overcome this barrier are discussed below.
5|OVERCOMING THE
PHYSICOCHEMICAL CHALLENGES OF
THE EPITHELIAL BARRIER
Development for the enhancement of drug delivery through the epithe-
lia is largely dependent on the mechanism of permeation across the epi-
thelial barrier. These primarily consist of simple passive diffusion,
carrier-mediated diffusion, active transport, and transcytosis initiated by
epithelial cell-mediated endocytosis.
64,65
Several studies provide evi-
dence for simple passive diffusion as the primary mechanism of drug
permeation.
66–70
Simple passive diffusion involves the law of diffusion
in which a molecule moves from an area of higher concentration to that
of lower concentration and while this may be the most efficient mecha-
nism since it does not require an energy input, many complications can
arise due to varying properties of drug molecules, such as size and
charge.
65
Carrier-mediated diffusion is also another common mecha-
nism of drug absorption and utilizes a transmembrane carrier to trans-
port a drug molecule across the epithelia.
65
Active transport and
transcytosis are not as common as passive diffusion and are significantly
more energy-expending processes. While active transport is not com-
mon for most therapeutics, some examples include levodopa
69
for
Parkinson's disease or fluorouracil
70,71
for cancer.
Transport of these therapeutics is further supported by the
microstructure of the epithelial barrier, namely villi and microvilli.
Since the villi and microvilli are finger-like projections that extend off
the epithelial barrier along the length of the intestine on the apical
side facing the intestinal lumen, it helps to increase the amount of sur-
face area that is available for absorption (Figure 5). Moreover, polar-
ized regions on the apical surface form a specialized network that
allows for sorting and packaging of materials in and out of the cell,
which is particularly important for transcytosis and delivery of drugs
to systemic circulation.
72
Indeed, this process is the primary method
for absorption of many immunoglobulins
73
and proteins.
74
In addition to villi and microvilli, other microstructures of epithe-
lial cells are to be taken into consideration when designing DDS for
therapeutic delivery. For example, tight junctions within the epithelial
barrier make it difficult for large drug molecules (≥6 nanometers) to
pass through the epithelial layer.
75
Two principal routes of absorption
through the epithelial layer are the transcellular route and the para-
cellular route (Figures 1 and 5). The transcellular method involves
transportation across the apical side of the epithelial cell membrane,
transportation within the cell, and subsequent removal at the bas-
olateral side of the epithelial cell.
76
On the other hand, paracellular
route involves permeation of the drugs between the cells.
77
Two main
characteristics of the drugs that determine permeation through para-
cellular route include the charge of the drugs and their size.
75,78
In order to utilize the mechanisms of paracellular drug delivery,
strategies have been designed to enhance absorption and permeation
between the cells. This process entails temporarily breaking down the
epithelial cell membrane barrier or opening up intercellular tight junc-
tions.
79
Surfactants have been extensively researched for their ability
to open epithelial tight junctions for drug permeation. Because surfac-
tants are amphiphilic, the hydrophilic and hydrophobic components
can align themselves at the epithelial interface in order to decrease
surface tension and thus facilitate the transportation of molecules
across the epithelial layer. Although surfactants, such as sodium dode-
cyl sulfate or polysorbate-80 were found to increase drug absorption,
they were also found to produce irreversible membrane damage.
78,80
Various animal studies have shown that transient permeabilizing
agents are less cytotoxic when compared to agents that generate irre-
versible membrane permeation. Examples of such agents include
ethylenediaminetetraacetic acid (EDTA), which function as calcium
chelators, or vehicles comprised of fatty acid chains such as caprate or
laureate, which operate through modulation of filament interactions
in the membrane.
81
Recently, negatively charged nanoparticles have
also been found to enhance membrane permeability apparently with
very little toxicity or permanence.
82
As opposed to surfactants, small and negatively charged
nanoparticles work by enhancing transcellular permeation. Regardless,
permeabilizing agents that disrupt the integrity of the cell membrane
also allow for the opportunity of solutes, other than the targeted drug,
to pass through the membrane, thus compromising clinical implemen-
tation of such agents. Nevertheless, clinical trials have utilized these
agents and found success through the combination of both para-
cellular and transcellular enhancements of permeation. For example,
8of18 LE ET AL.

GI permeation enhancement technology (GIPET) is a formulation that
is being developed by Merrion Pharmaceuticals that has found exten-
sive success in clinical trials and has been pre-approved per United
States Food and Drug Administration (U.S. FDA) standards.
83
The
GIPET formulation utilizes fatty acid compounds to enhance mem-
brane absorption and, in clinical trials, has been shown to increase the
oral bioavailability by 12-fold.
84
Other permeabilizing agents that have
found clinical success include Chiasma's transient permeability
enhancer (TPE) technology, which increases the GI absorption of large
macromolecules, shown through clinical trials with Octreolin for
acromegaly,
85
or Oramed Pharmaceutical's formulations of combining
the delivery of enteric coatings and permeation enhancers
(e.g., EDTA) to amplify absorption of orally delivered insulin.
83
The
success of these technologies is likely attributed to the utilization of
FIGURE 6 Delivery of
Staphylococcal α-toxin via
“motor”-based microparticles
vaccines, enhanced distribution
of the payload throughout the
intestine (a–c), and lead to
increase in IgA titers in mice
(d and e) Source: Reprinted/
Adapted from Wei et al. Nano
Lett. 2019;19;1914–1921, with
permission from Copyright 2019
American Chemical Society
LE ET AL.9of18

transient permeabilizing agents that both work to increase drug
absorption while also working rapidly to reverse any structural effects
and minimize any extensive damage to membrane function.
In addition to synthetic surfactants, Zonula occludens toxin (Zot)
coatings have also been studied for manipulating tight junction open-
ings in the intestine for paracellular delivery. In vitro studies in rabbit
ileum with Zot shows that the effects of pharmaceuticals delivered
orally peaked at around 80 min and with reversible effects in a time-
dependent manner, which demonstrates the reversibility potential for
Zot coatings for oral drug delivery.
86
The mechanism behind tight
junction manipulation by Zot involves a rearrangement of the epithe-
lial cytoskeleton induced by a series of complex protein kinase
C-dependent signaling cascades.
87,88
Notably, Zot works similarly to
zonulin, a protein abundant in the digestive tract that plays a signifi-
cant role in tight junction regulation, both of which bind to the same
receptor on intestinal epithelial cells, suggesting that this may be the
reason for the effectiveness of Zot in epithelial permeation.
88
Impor-
tantly, these studies utilizing chitosan and Zot coatings reported no
apparent toxicity.
50,86
However, further research needs to be done to
further elucidate this mechanism and analyze long-term organ-level
and organism-level toxicity in larger animal models.
This enhancement of active transport of drugs systemically from
oral route can be highly beneficial for treating acute immune system-
related diseases, such as infections. In fact, targeting the mucosal
immune system can be highly beneficial in generating immune
responses against pathogens. For example, Wei et al. demonstrated
that molecular-motors consisting of a magnesium-based core, can be
utilized to deliver vaccines in the gut, by actively transporting the vac-
cine components into the intestinal tissue (Figure 6). In fact, they
demonstrated that the active transport due to the “motor”effect of
the particles allows the payload to be distributed uniformly through-
out the intestinal length.
89
These particles were also able to signifi-
cantly increase IgA antibody titers in the feces as compared to the
controls, which indicates that active transport of vaccines via motor-
based particles led to higher immune response.
89
Therefore, this
strategy of active transport might be beneficial to study mucosa-
related infections, such as Listeria and Helicobacter Pylori, where local
short-term generation of immune responses can have a profound
effect on disease outcomes.
In addition to temporarily opening tight junctions, it is also possi-
ble to increase passive transport using prodrugs strategies. Specifi-
cally, drugs are chemically modified to attribute them the properties
of enhanced passive transport. Prodrugs are administered as inactive
substances and converted to its pharmaceutically active form when
metabolized with favorable physicochemical conditions in the body.
76
Prodrug design entails attaching of hydrophilic groups to enhance
drug solubility and using lipophilic molecules to increase passive diffu-
sion through the epithelial cell membrane.
90
Design of prodrugs must
also be specific to particular properties of the pharmaceutically active
drug, as some prodrugs, if prematurely activated before or during dif-
fusion through the epithelial membrane, can cause trapping of the
active drug within the cells of the membrane, and thus not be able to
produce the full effect at the targeted site. For example, the L-valyl
prodrug of zanamivir (antiviral drug) was shown to improve epithelial
cell permeability with around a 3-fold increase in absorption as
opposed to the acyloxy ester prodrugs that inhibited uptake.
91
Other
mechanisms of prodrug design include targeting transporters located
on both the apical and basolateral side of intestinal epithelial cells to
allow for enhanced drug absorption. The human peptide transporter
1 (PEPT1) based prodrugs are an example of targeting specific uptake
transporters that show promise for oral drug delivery.
90
In another study, it was found that oral delivery of insulin with
the cell penetrating peptide (CPP) penetratin showed up to a
78.6-fold increase in its hypoglycemic effects, lasting up to 18 h, as
compared to the insulin control, giving a pharmacological availability
of 18.2%.
92
Additionally, in situ experimentation found a dose-
dependent increase in insulin absorption in the ileum when adminis-
tered with oligoarginine, another CPP.
93
While the mechanism behind
CPPs are not fully understood, a form of endocytosis has been
proposed as the method of cell penetration and targeted antigen
delivery.
94,95
Since insulin is a peptide, other immunomodulatory pep-
tides and proteins can be potentially delivered using CPP for oral to
systemic delivery; however, this area needs to be researched further,
and is discussed in the following section.
6|CURRENT ORAL TO SYSTEMIC
IMMUNOTHERAPEUTIC DELIVERY
STRATEGIES
Copious research has been done that demonstrates the potential for
various therapeutics that can be used to modulate the immune system
against a host of diseases such as autoimmune diseases and cancer.
96,97
Direct delivery to systemic circulation is usually obtained through
intravenous administration; however, noninvasive routes of adminis-
tration, particularly oral formulations, are especially advantageous in
being cost-effective and efficient methods of drug delivery for higher
patient compliance. Immunotherapeutics can take many different
forms; however, many promising agents, such as monoclonal anti-
bodies and other relevant proteins, as well as genetically modifying
agents, such as siRNA and mRNA, are easily degraded in the presence
of gastric enzymes or impermeable to the complex layered GI immune
system, and thus face decreased absorption and reduced drug effect
with oral administration. In order to overcome the challenges attrib-
uted to oral administration, complex delivery systems and biomaterials
have been, and continue to be, developed to further increase bioavail-
ability and evolve this route into a more viable option. The focus of
these delivery systems and biomaterial strategies specifically targets
the physical and biological barriers en route for orally delivered drugs
to reach systemic circulation.
Natural polymers as well as synthetically formulated biomaterials
have been developed to increase the time spent during absorption in
the GI tract. As previously discussed, chitosan is an example of a natu-
ral polymer that increases the time of absorption for orally delivered
pharmaceuticals. For example, pre-clinical trials using chitosan-based
nanoparticles loaded with 10-Hydroxycamptothecin (HCPT) have
10 of 18 LE ET AL.

found success in increased cell uptake and drug absorption for immu-
notherapy of melanoma
98
and has also been reported to enhance
more efficient drug delivery of peptides, such as with eugenol-loaded
chitosan nanoparticles, which produced anti-inflammatory effects in
an aggressive model of rheumatoid arthritis.
99
The MucoJet is another
example of a novel technology that has found success with in vivo
animal studies to penetrate the mucosal layer via oral administration
and elicit antibody production.
100,101
The MucoJet is a small immuno-
therapeutic delivery system consisting of a small plastic device that
can be activated by the user by providing a pressure termed “click”in
their work (Figure 7).
101
The polymeric membrane dissolves due to
the “click,”causing the water reservoir to contact the chemical pro-
pellant (citric acid and sodium bicarbonate), generating carbon dioxide
gas (Figure 7).
101
This gas production increases the pressure to around
30 kPa, providing the device with sufficient force to penetrate the
mucosal layer and deliver a vaccine solution.
101
Aran et al. utilized this
technology to deliver ovalbumin to rabbits, which produced high titers
of antigen-specific immunoglobulins G and A.
Each of these mentioned strategies provide advanced progress in
increasing the bioavailability of orally delivered drugs, thus, making it
a more attractive means of drug delivery. Although each of these
strategies have made considerable progress, the safety, cost, and clini-
cal translation of these technologies still remain unclear. Therefore, it
is particularly important to address specific challenges of oral adminis-
tration with novel biomaterials to make this route a viable candidate
for immunotherapeutic delivery.
FIGURE 7 (a) The two components of the MucoJet are clicked together by the user prior to administration. The polymeric membrane is then
dissolved. Water contact with the chemical propellant causes a chemical reaction that produces carbon dioxide gas, inducing a sufficient jet
velocity and pressure for the vaccine solution to penetrate the buccal mucosa layer. (b) The MucoJet is delivered by mouth to buccal tissue (left).
Upon penetration through the mucosal layer, the vaccine solution is delivered to antigen-presenting cells (APCs) in the mucosa-associated
lymphoid tissue (MALT) to generate an immune response (right) Source: Reprinted/adapted from Aran et al. Sci Transl Med. 2017;9(380):eaaf6413.
© The Authors, some rights reserved, exclusive licensee AAAS. Distributed under a CC BY-NC 4.0 license http://creativecommons.org/licenses/
by-nc/4.0/
LE ET AL.11 of 18

In addition to oral to systemic delivery of immunotherapeutics,
delivery of immunotherapeutics to the immune system of the mucosa
has gained a lot of interest in recent years. Interestingly, mucosa is the
site where most of the pathogens enter the body (>90%),
102
and
hence it can be advantageous to utilize immunoengineering technolo-
gies to deliver immunotherapeutics to the mucosal immune system.
7|TARGETING M-CELLS FOR
IMMUNOTHERAPEUTIC DELIVERY TO THE
MUCOSAL IMMUNE SYSTEM
Microfold (M) cells are specialized intestinal epithelial cells that are
commonly found in mucosa-associated lymphoid tissue (MALT) and in
gut-associated lymphoid tissue (GALT). In the case of GALT, these
cells specialize in transporting antigens from the lumen of the small
intestine toward lymphoid tissue.
103
More specifically, at the induc-
tive site, M cells transcytose antigens to be processed in the Peyer's
patches, which is compacted with various immune cells, such as DCs,
B cells, and T cells (Figure 8).
103,104
Upon activation, immune cells pro-
duce cytokines and altogether are involved in the release of anti-
bodies for an antigen-specific immune response (Figure 8).
103,105
Since M cells interact with these specialized cells responsible for gen-
erating a vaccine response, they are an ideal target for generating a
vaccine response.
105
Current potent examples of oral vaccines include
rotaviruses, polioviruses, and cholera vaccines.
106
Effective targeting of M cells for oral vaccine delivery can be chal-
lenging due to the structure of the cell and the environment in which
they reside. M cells have the ability to uptake foreign entities and
bypass the apical epithelial layer to directly deliver these foreign parti-
cles to the basolateral layer.
107
Although M cells may be an ideal tar-
get for immunotherapeutic delivery, only 1 in 10 million epithelial cells
in the GI tract are M-cells.
108
While it is possible to amplify the fre-
quency of M cells, it might be advantageous to target existing M cells
for efficient drug delivery.
Various animal studies have taken advantage of this efficacy for
M cell targeting in inducing immunological responses for oral vaccina-
tion at the mucosal surface. For example, M cell targeting using a
ligand reovirus protein sigma1 has been shown to facilitate oral toler-
ance in mice and thus exhibit the key role of antigen uptake and M
cell targeting in mucosal immunity.
109
Studies have also looked at the
specific relationship between M cells and secretory immunoglobulin A
(sIgA), which is one of the main defense mechanisms of the MALT.
107
sIgA plays a significant role in regulating immunity in the mucosal epi-
thelial by binding and removing foreign antigens and pathogens that
are found within the mucosal surfaces. Notably, sIgA is also associated
with helping immune tolerance by binding to dietary antigens and
organisms in the microbiota.
110
Importantly, sIgA that is complexed
with antigens, can also be endocytosed by M-cells via reverse trans-
cytosis.
111,112
This reverse transcytosis then provides the antigens
directly to immune cells, such as DCs, which are present in the
MALT.
111
In addition to sIgA, studies have also exhibited success with
M cell targeting by utilizing Claudin 4 targeting peptide (CPE) which
was found to enhance mucosal IgA responses.
113
Additionally, studies
have also utilized p24 gag antigens linked to sIgA to elicit HIV-specific
immune responses.
114
Plant lectins are another notable method used to target
M-cells. Ulex europaeus agglutinin 1 (UEA-1) lectin binds specifi-
cally to the alpha1,2 fucose residue that is expressed on the apical
surface of the mouse M-cells.
115
This selective binding promotes
rapid uptake of antigens by M cells. Notably, the apical surface of
the M cell faces the lumen, thus, making M cells an optimal target
for oral vaccinations since the lumen is where oral vaccinations
would be found. It is important to note, however, that a major
drawback to utilizing plant lectins are their potential to produce
antinutritional/toxic effects, such as those observed in studies
showing significant weight loss in pigs after being fed Phaseolus
vulgaris agglutinin (PHA), a kidney bean lectin.
116
Plant lectins can also have an affinity for specific glycoproteins
and, therefore, these can be utilized to target glycosylated proteins on
M cells. Glycoproteins are located on cell surfaces to aid in immune
defense and, due to their unique patterns and structures, can also
serve as an identity marker. However, little is actually known about
the structure and function of glycoproteins on M-cells. Interestingly,
M-cells have a distinct glycosylation protein profile as compared to
other localized cells which, in turn, provides a mechanism to differen-
tiate M cells from its surrounding cells.
103
Specifically, the glycocalyx,
which is a form of glycolipid/glycoprotein coating that serves as a bar-
rier between a cell and its surroundings, is thinner on M cells than the
glycocalyx of its neighboring cells.
103
The reduced glycocalyx on M
cells adds to its overall unique structure and allows easier access to
the intestinal lumen for more efficient uptake of antigens, thus making
it a targeted region of interest for the improvement of immunothera-
peutic delivery. Additionally, glycosylation proteins vary in different
locations of the intestine and also differ between species. This can
potentially be used to target specific locations in the small intestine,
for a targeted delivery and localized drug activation. To date, only lit-
tle is known about the types of receptors that exist on the surface of
M-cells for recognition and subsequent endocytosis, therefore, it is
important to further explore how M-cells can be targeted to uptake
specific antigens for immune targeting while avoiding the absorption
of toxic and invasive pathogens.
8|ORAL VACCINATION—STATE OF THE
ART AND ROLE OF IMMUNOENGINEERING
Although there are more than 20 actively administered vaccines in the
United States, only rotavirus, adenovirus, cholera vaccine, and oral
typhoid vaccines are administered orally.
117
Currently, most vaccines
are delivered by intradermal or intramuscular injections, which are
associated with problems such as safety and high cost of mass immu-
nization.
118,119
Unfortunately, vaccines administered either intrader-
mally or intramuscularly, provide only partial, or in some cases, no
protection at the mucosal site, where most (>90%) of the pathogens
access the body.
102
Therefore, targeting and generating mucosal
12 of 18 LE ET AL.

immune responses against pathogenic proteins or self-proteins for tol-
erance can be highly beneficial. Notably, the mucosal immune system
tends to be immunosuppressive and, therefore, provides an attractive
target for generating tolerance inducing vaccines as well. However,
there are very few oral or intranasal vaccines available, and this can
be directly linked to the lack of delivery systems capable of delivering
proteins (antigen) and adjuvants (provides context for vaccines—
immunogenic/tolerogenic) to the mucosal immune system.
Biomaterials, such as microparticle-based systems, can target the
GALT typically by introducing antigens to the inductive sites on
the surfaces of tissues to streamline an immune response to the effec-
tor sites (Figure 6). As briefly discussed in the previous section, anti-
gens that are transcytosed by specialized M-cells are presented to
antigen-presenting cells (e.g., DCs, B lymphocytes, and macrophages)
for the induction of immune responses.
120
Producing a sustained
immune response with mucosal vaccination by targeting DCs can be
challenging, but have found success in mice studies through the mani-
festation of immunologic memory via directly inducing cytotoxic T cell
activation.
121
Another area where mucosa-targeted vaccines can have a major
impact is with autoimmune diseases, where tolerance against antigens
of interest is desired. For example, in autoimmune diseases, such as
rheumatoid arthritis (RA), the body mistakes self-antigens
(i.e., collagen in the case of RA) as foreign, which leads to immune
responses being mounted against the self-antigen. Specifically, deliv-
ery of antigens orally has been tested in clinical settings with mixed
results, and no treatment has yet been approved.
122–124
One potential
avenue to generate a robust tolerance-inducing response is by directly
delivering antigens of interest to the cells of the mucosal immune sys-
tem. Moreover, a formulation that can deliver tolerance-inducing mol-
ecules, to provide context, along with an antigen can also greatly
improve immune responses.
Considerable progress has been made for the development of oral
vaccine delivery systems and has also been tested in pre-clinical
models. For example, chitosan and alginate microparticles can be
taken up by M cells in the Peyer's patches which can directly
be absorbed by the MALT to induce subsequent immune
responses.
125
Polymeric nanoparticles such as poly(lactide-co-glyco-
lide) (PLGA)
126,127
have also found success in inducing immunoglobu-
lin G (IgG) immune response to promote the linked systemic and
mucosal responses necessary for sustained immunity.
128
Other poten-
tial candidates include the encapsulation of antigens or immunomodu-
latory agents using liposomes,
129
bilosomes,
130
bacterial outer
membrane vesicles (OMVs),
131
virus-like particles (VLPs),
132
and
chemically processed pollen grains,
133
which have found pre-clinical
success against viral respiratory diseases or bilosome-entrapped anti-
biotics with success against the bacterium Burkholderia pseu-
domallei.
130
The pre-clinical success of these biomaterials
FIGURE 8 Inductive sites (right) made up of T and B cells within the Peyer's patches and effector sites (left) within the lamina propria
comprise of intestinal immunity within gut-associated lymphoid tissue. M-cells along the epithelia allows for antigen uptake
LE ET AL.13 of 18

demonstrates how important it is to further our understanding for the
enhancement of oral vaccine delivery systems (Table 1).
9|CONCLUSION
Oral routes of administration play a significant role in drug delivery.
They prove to be an effective alternative to injected routes of adminis-
tration due to their high patient compliance and convenience for
achieving a specialized immune response. However, even with their
copious advantages, numerous orally delivered drugs are associated
with low bioavailability. This is generally attributed to degradative con-
ditions and biological barriers, such as the mucosa or epithelia.
134
Nev-
ertheless, particulate systems that utilize various mucoadhesive and
permeabilizing technologies have found success in the clinical transla-
tion of these formulated carriers. However, further research is needed
for these methods to be utilized for immune engineering. These novel
technologies must also be careful to not disrupt or destroy the natural
immune function of the GI tract when drugs are delivered orally and
therefore should be both transient and effective in its design. Sublin-
gual and buccal routes of administration are also effective methods of
immunotherapeutic delivery that differ from the traditional method of
oral drug administration. These routes bypass the first-pass metabolic
effect and allow for rapid onset of effects. However, only few develop-
ments using these delivery methods have been U.S. FDA approved, due
to the uniqueness of its formulations and need for proof of safety and
efficacy. Nevertheless, it is important to consider them as viable
options for immunotherapeutic delivery. Convenience of sustained
administration and high patient compliance make oral routes of admin-
istration more attractive methods of immunotherapeutic delivery, as
opposed to injectable deliveries. It is expected that the future research
in these systems will revolve around immunoengineering concepts for
constructing biomaterials that target various cells and organs of the
immune system while upholding the integrity of the GI tract as a whole.
ACKNOWLEDGMENT
The authors would like to acknowledge funding sources to Abhinav
P. Acharya that supported this work—NIH R01AR078343 and NIH
R01AI155907.
CONFLICT OF INTEREST
There is no conflict of interest.
AUTHOR CONTRIBUTIONS
Tien Le: Conceptualization; writing - original draft; writing-review &
editing. Brian Aguilar: Writing - original draft; writing-review & editing.
Joslyn Mangal: Conceptualization; writing - original draft; writing-
review & editing.
PEER REVIEW
The peer review history for this article is available at https://publons.
com/publon/10.1002/btm2.10243.
TABLE 1 A representative list of biomaterials as oral drug delivery systems
Agent type Examples Functionality
Mucosa targeting Polymers •Thiolated polymers (polycarbophil–cysteine)
51
•Chitosan-stearic acid-thioglycolic acid
52
•Chitosan
98,99
•Formation of non-covalent bonds or stronger
covalent bonds to increase residence time
•Enhance permeability
Mucolytic enzymes •Papain
57
•Bromelain
57
•Conjugated on particle surface to cleave mucus
substructures
•Degraded in gastric environment
Self-
nanoemulsifying
drug delivery
systems
(SNEDDS)
•Captex 300-Kollipor EL-propylenglycol
58
•Homogenous mixtures of oil, surfactant, and co-
solvent to self-emulsify in aqueous medium
•Ideal for poorly water-soluble drugs
M-cell targeting Plant lectins •Ulex europaeus agglutinin 1 (UEA-1) lectin
115
•Possible antinutritional and toxic effects
•High affinity to M-cells as well as glycoproteins
Proteins •Protein sigma1
109
•Claudin 4 targeting peptide (CPE)
113
•Facilitates oral tolerance in pre-clinical studies
Epithelia targeting Transient
permeabilizing
agents
•Ethylenediaminetetraacetic acid (EDTA)
81
•Gastrointestinal permeation enhancement
technology (GIPET)
83
•Chiasma's transient permeability enhancer (TPE)
85
•Enhances transcellular permeation
•Less toxicity associated with reversibility
Surfactants •Sodium dodecyl sulfate (SDS)
78
•Polysorbate 80 (PS-80)
80
•Amphiphilic structure decreases surface tension
•Facilitates epithelial tight junction opening
•Irreversible membrane damage
Bacterial surface
protein
•Zonula occludens toxin (Zot)
50,86
•Rearrangement of epithelial cytoskeleton for tight
junction opening
•Reversible with no significant toxicity in pre-clinical
studies
14 of 18 LE ET AL.

DATA AVAILABILITY STATEMENT
Any information pertaining to this manuscript will be provided by the
authors upon request.
ORCID
Tien Le https://orcid.org/0000-0002-6824-6838
Abhinav P. Acharya https://orcid.org/0000-0002-9361-2764
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How to cite this article: Le T, Aguilar B, Mangal JL,
Acharya AP. Oral drug delivery for immunoengineering. Bioeng
Transl Med. 2022;7(1):e10243. https://doi.org/10.1002/btm2.
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