Medicine in Drug Discovery 12 (2021) 100110

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Medicine in Drug Discovery

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Research Paper

Engineering nanoparticles to overcome the mucus barrier for drug delivery:

Design, evaluation and state-of-the-art
Chang Liu a,b,1, Xiaohe Jiang a,b,1, Yong Gan a,b,c, Miaorong Yu a,⇑
a
State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China
b
University of Chinese Academy of Sciences, No. 19A Yuquan Road, Beijing 100049, China
c
NMPA Key Laboratory for Quality Research and Evaluation of Pharmaceutical Excipients, National Institutes for Food and Drug Control, Beijing 100050, China

A R T I C L E I N F O A B S T R A C T

Keywords: Mucus is a viscoelastic hydrogel that lubricates and protects mucosal tissues such as the eyes, lung, gastroin-
Mucus barrier testinal tract, and vagina. Nanoparticle‐based drug delivery systems (DDSs) can be easily trapped in the mucus
Mucosal drug delivery network by steric obstruction and/or binding interactions and then are efficiently cleared away with rapid
Nanoparticles mucus turnover, thereby limiting mucosal drug delivery efficacy. Modulating the physicochemical properties
Particle tracking experiments
of nanoparticles (NPs), including size, surface hydrophilicity and charge, shape, and rigidity, is an effective
Mucus‐penetrating nanoparticles
method to overcome the mucus barrier. This review first describes the protective barrier properties of mucus
and how mucus affects the fate of NPs. It then reviews the techniques for evaluating particle movement in
mucus, including macroscopic diffusivity and microcosmic diffusion mechanisms. The state‐of‐the‐art strate-
gies exploited by researchers to overcome the mucus barrier by using NPs are discussed. Finally, the dilemma
and solutions of balancing both mucus penetration and cellular uptake barriers are discussed.

1. Introduction absorption and improved localized treatment [5,6]. Recently, rod‐

shaped NPs [7] and semi‐elastic NPs [8] were reported to rapidly tra-
In recent decades, nanotechnology has been widely applied in phar- verse the intestinal mucus to achieve enhanced oral bioavailability.
maceutical research for the fabrication of efficient drug delivery systems These findings suggest the feasibility to modulate the physicochemical
(DDSs). Nanoparticles (NPs) can enhance drug solubility, increase drug properties of NPs to overcome the mucus barrier.
accumulation at the target sites, and enable co‐delivery of multiple ther- In this review, we first describe the mechanisms by which the
apeutics [1]. However, NPs inevitably face various biological barriers mucus limits the delivery efficacy of NP‐based DDSs. Then, recent
during their transportation in the body, which extremely restrict the advances in methods for the evaluation of particle movement in mucus
delivery efficacy of NP‐based DDSs [2]. Mucus, a viscous and sticky are discussed, as well as the latest developments in the design of NPs to
hydrogel layer that lines on the surface of mucosal tissues such as the overcome the mucus barrier. Subsequently, we highlight the dilemma
eyes, lung airways, and gastrointestinal tract (GIT), is a typical kind of in the development of NPs to overcome both the diffusion barrier of
biological barriers [3]. Mucus is mainly composed of crosslinked and mucus and the absorption barrier of cell, and introduce the corre-
entangled mucin fibers, forming a mesh‐like structure. Thus, mucus sponding strategies to balance the two barriers.
has evolved its function to protect the living organisms against foreign
particles via steric obstruction and/or binding interactions. NPs deliv-
ered to the mucosal tissues are easily trapped and cleared by mucus, 2. Mucus as a critical barrier to NP-based DDSs
thereby limiting the effectiveness of such DDSs [4].
In order to overcome the mucus barrier, mucoadhesive and mucus‐ 2.1. Mucus distribution, composition and structure
penetrating NPs have been proposed successively. For example, Hanes
et al. have pioneered in the development of mucus penetrating NPs Mucus, lining on the surface of mucosal tissues such as the eyes, air-
(MPPs) by decorating NPs with a high density of low‐molecular‐ way, GIT and female reproductive tract (Fig. 1A), is a viscoelastic
weight poly(ethylene glycol) (PEG), which enabled promoted drug hydrogel protecting living organisms against infectious agents [9].

⇑ Corresponding author.
E-mail address: mryu@[Link] (M. Yu).
1
These authors have contributed equally to this work.

[Link]
Received 30 August 2021; Revised 11 October 2021; Accepted 3 November 2021
Available online 10 November 2021
2590-0986/© 2021 The Author(s). Published by Elsevier B.V.
This is an open access article under the CC BY-NC-ND license ([Link]
C. Liu et al. Medicine in Drug Discovery 12 (2021) 100110

Composed of water, electrolytes, lipids and proteins, mucus is charac- namely, large NPs are generally locally restricted via steric obstruction,
terized with thickness ranging from tens to hundreds of microns in dif- and small and inert NPs with diameters below the cutoff pore size of
ferent mucosal tissues (Table 1). For example, the thickness of mucus mesh structure can readily diffuse in mucus via Brownian motion
in human airway is approximately 0.5–50 μm, and it is 16–155 μm in [25]. However, more NPs exploited in research are non‐inert, and they
gastrointestinal mucus. Mucin, a glycoprotein with a diameter of are prone to be trapped in mucus even with small dimensions. This
3–10 nm, is the primary functional component of mucus. The protein finding indicates an additional mechanism to regulate particle diffu-
backbone of mucin is rich in repeats of serine and threonine, where sion. NPs with specific surface properties, such as positive charge
glycosylation often occurs [10], and glycan side chains with terminal and hydrophobicity, can form multiple interactions with mucin fibers;
residues of sulfate and sialic acid contribute to the negative surface for instance, the negative charged surface enables ionic interactions,
charge of mucus [11]. The hydrophobic globular non‐glycosylated hydrophobic globular regions allow hydrophobic interactions, and gly-
regions are rich in cysteine, and can serve as sites for disulfide bond cosylation provides sites for hydrogen bond formation [26]. Through
formation, thereby linking mucin fibers to form a crosslinked, bundled these binding interactions, NPs can be entrapped and immobilized in
and entangled mesh network (Fig. 1B) with pore size ranging from 20 the mesh network regardless of the particle size. Mucus turnover can
to 1800 nm [12]. In addition, mucus in the airway shows remarkably then eliminate entrapped NPs in a dynamic manner via constant secre-
higher viscosity than that in GIT and eyes (Table 1). Thus, the spatial tion and shedding. Therefore, with NP transport suffering from the
difference in mucus properties mentioned above indicates the neces- aforementioned immobilization and clearance, mucus imposes inevita-
sity of precise design for drug delivery system when applied to specific ble barriers for mucosal drug delivery [27]. On account of these parti-
mucosal tissue. cle elimination mechanisms, researchers have developed
corresponding strategies to overcome the mucus barrier, and to pro-
2.2. The barrier properties of mucus mote the diffusion and penetration of NPs for enhanced therapeutic
effects, which is depicted in detail in Section 4 (see Fig. 3).
Apart from common protective functions, mucus also acts as a fil-
ter, regulating the diffusion and exchange of nutrients, macro- 3. Techniques for characterizing particle movement in mucus
molecules and NPs. Broadly speaking, the transportation of NPs in
mucus is controlled by the steric obstruction and binding interactions To date, various techniques have been utilized to evaluate the dif-
owing to the mesh structure as well as the physicochemical properties fusibility of NPs in mucus from macroscopic and microscopic perspec-
of NPs (Fig. 2) [24]. Size exclusion is a universal feature of mucus, tives (Table 2). At a macro level, apart from the observation of
distribution in mucus through fluorescence microscopy in a static
state, dynamic diffusion characterization techniques, including fluo-
rescence recovery after photobleaching (FRAP) and multiple particle
tracking (MPT), have been applied to quantify the diffusivity of NPs
in mucus. The combination of these techniques contributes to compre-
hensively evaluation of the diffusion process. For the microscopic
mechanisms, super‐resolution microscopy (SRM) and microscale ther-
mophoresis (MST) can be used to explore the motion modes of NPs and
the interactions between NPs and mucus, respectively. Unlike conven-
tional tracking techniques that focus on particle diffusivity, SRM
microscopically characterizes the particle transport process, including
morphology change and rotation. Thus, these techniques have become
useful tools to investigate NP‐mucus interactions and evaluate the pen-
etration efficiency of NPs.

3.1. Macroscopic characterization techniques

3.1.1. Static imaging

Fig. 1. Schematic illustration of the mucus distribution and structure. (A) To intuitively display the results of NP diffusion, static imaging is
Mucus is distributed in eyes, nasal cavity, lung, stomach, intestine etc. (B) The often exploited to observe the distribution of NPs in mucus, including
network of mucus is composed of mucin fibers which contain glycosylation
longitudinal penetration depth and lateral diffusion area. However,
regions with negative charge and non-glycosylated hydrophobic regions rich
conventional widefield fluorescence microscopy captures only vague
in cysteine.
two‐dimensional (2D) images of thick specimens [28]. For the acquisi-

Table 1
Properties of mucus in different mucosal tissues.

Mucosal tissue Thickness Pore size Viscosity

Gastrointestinal tract 16–155 μm <200 nm (Mouse) 10–16 mPa-s (Pig)

[13] [14] [15]
Eye ∼3 μm 550 ± 50 nm (Bovine) 2–9 mPa-s
[16] [17] [18]
Airway 0.5–50 μm 500 nm 10 Pa-s (Bovine)
[19] [20] [21]
Vagina Change with menstrual cycles Change with menstrual cycles Change with menstrual cycle
[22] [12] [23]

*Unstated data are measured in human mucus.

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C. Liu et al. Medicine in Drug Discovery 12 (2021) 100110

Fig. 2. Schematic illustration of filtration effects on NPs by mucus. (A) NPs larger than the mucus pore size are hindered from penetration, while smaller
counterparts can readily diffuse through mucus layer. (B) Non-inert NPs can form various interactions (hydrophobic and ionic interaction, hydrogen bond etc.)
with mucin fibers, thus trapped in the mucus network.

the particle diffusion process can be reflected over long time and dis-
tance scales. For example, Murgia et al. [31] deposited carboxylated
NPs on porcine respiratory mucus and used CLSM to image the distri-
bution of NPs in mucus at a depth of 25–40 μm. The 2D distribution of
differently sized NPs at different depths was shown to compare the NP
penetration capacity. Moreover, the cross sections of z‐stacks intu-
itively showed the penetration depth of NPs.

3.1.2. Fluorescence recovery after photobleaching (FRAP)

Although penetration depth and movement area show the perme-
ability of NPs in mucus, their diffusion parameters such as diffusion
rate need to be further measured by dynamic characterization tech-
niques that allow real‐time monitoring. FRAP is a straightforward tool
to investigate the diffusion of proteins as well as NPs in living cells or
biological media such as mucus [32]. In FRAP experiments, particles
labeled with fluorescence in certain regions are photobleached by an
intense laser beam, and subsequently, the unbleached surrounding
particles diffuse into the bleached region, resulting in fluorescent
recovery [33]. The process only takes a few minutes and is monitored
by confocal microscopy at low laser power, acquiring the parameters
including the mobile fraction (Mf), diffusion coefficient (Deff) and half
time of recovery (t1/2) calculated from recovery curves. Lehr et al. [31]
Fig. 3. Schematic illustration of engineering strategies for MPPs to overcome utilized FRAP to investigate the penetration ability of polystyrene NPs
mucus barrier. Shape, rigidity, size and surface property can be modulated to of different size in pig pulmonary mucus. The diffusion coefficient was
facilitate the mucus penetration of NPs. higher for 100 nm NPs than for 200 and 500 nm NPs, while there was
a higher mobile fraction for 200 nm NPs than for 100 nm NPs. The
contradictory results indicated that there might be an interaction
between NPs and mucin fibers, which led to anomalous diffusion.
tion of clear 2D images and the entire three‐dimensional (3D) distribu-
tion images, confocal laser scanning microscopy (CLSM) is extensively
utilized because of its high resolution and optical sectioning perfor- 3.1.3. Multiple particle tracking (MPT)
mance [29]. The fluorescence‐labeled NPs are imaged at a series of Over the past three decades, MPT has matured into an intuitive and
depths, and a stereo image are further reconstructed to show the 3D powerful technique to acquire qualitative and quantitative information
distribution of NPs in mucus [30]. With the aid of time‐lapse imaging, on particle diffusion in complex biological media [34]. Different from

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C. Liu et al. Medicine in Drug Discovery 12 (2021) 100110

Table 2
Techniques exploited to characterize particle diffusion in mucus.

Technique Function Result Parameter References

FRAP Macroscopic diffusivity Dynamic diffusion observation Mf, t1/2 [20,21]

Deff
MPT Particle tracking Deff, MSD [15,22–35]
Static imaging Particle distribution observation Penetration depth, Lateral diffusion area [20,21,24,25,28–32,36,37]
SRM Microscopic diffusion mechanism Single NP monitoring Movement mode, Morphologic variation [28–31]
MST NP-protein interaction quantification KD [38]

*FRAP, fluorescence recovery after photobleaching; MPT, multiple particle tracking; SRM, super-resolution microscopy; MST, microscale thermophoresis; Mf,
Mobile fraction; Deff, effective diffusion coefficient; t1/2, half time of recovery; MSD, mean-squared displacement; KD, dissociation constant.

FRAP, which features ensembled‐averaged diffusion, MPT can track following their incubation with mucus solution [43]. However, these
the movement of both single and multiple particles at high indirect evaluations cannot meet further requirements of microscopic
temporal‐spatial resolution, obtaining the mean‐squared displacement mechanism exploration; thus, quantitative techniques capable of
(MSD). Thus, MPT allows the distinguishment of individual particles determining the interactions between NP and mucus are urgently
with diffusivity varying by several orders of magnitude [35]. Because needed.
of its excellent performance in analyzing the diffusion heterogeneity, MST exploits infrared laser to create a temperature gradient where
MPT was pioneered by Hanes et al. [36] to investigate particle diffu- molecules undergo directed movement and the properties of mole-
sion in complex biological media such as the highly viscoelastic cules, such as size, charge and hydration shell, largely influence their
mucus. For MPT study, fluorescein‐labeled NPs are mixed with mucus, directed diffusion detected in localized temperature fields. Because of
and the diffusion process is captured using a fluorescence microscope the high sensitivity to changes in molecular properties caused by
equipped with a high‐resolution digital CCD camera [37]. After analy- molecular binding, MST is a powerful tool to quantify biomolecular
sis of time‐resolved trajectories, both the Deff and transport modes can and NP‐protein interactions [44]. Oriol et al. [45] performed MST
be obtained [38]. For example, Hanes et al. [6] fabricated DNA NPs experiments to determine the affinity between protein and silica
with different poly(ethylene glycol) (PEG) coating densities (0%, NPs. They compared the binding affinity KD of human serum albumin
40%, 60%) and evaluated their diffusion via MPT in human sputum (HSA), transferrin (Tf) and human fibrinogen (Fib), which were fur-
samples. PEGylated NPs showed higher MSD values than non‐ ther used as parameters in a coarse‐grained model, to better under-
PEGylated NPs, suggesting that PEG coating can facilitate navigation stand protein‐NP corona formation. Although there has not been any
of NPs through mucus. report that MST is utilized to characterize NP‐mucin interactions, it
is a promising technique and may contribute to studying the micro-
3.2. Microscopic characterization technology scopic mechanism of NP diffusion in mucus in the future.

3.2.1. Super-resolution microscopy (SRM)

4. Recent advances in NP-based strategies for mucosal drug
Macroscopic techniques describe the diffusivity of NPs, while real‐
delivery
time observation microcosmically can reveal the detailed particle dif-
fusion mechanisms. However, conventional optical microscopy fea-
The transport of NPs is hindered by low residence time in mucosal
tures restricted resolution due to the diffraction limit of 200 nm.
tissues due to fluid wash and/or mucus entrapment based on steric fil-
Thus, they are not able to image the structure of NPs with small dimen-
tration or interaction effects. To overcome the challenge of rapid wash
sions. To date, SRM, including stimulated emission depletion (STED)
off, mucoadhesive strategy is exploited to enhance particle‐mucin
microscopy, structured illumination microscopy (SIM), and micro-
interaction and thus the drug release is prolonged at the mucosa, espe-
scopy equipped with Airyscan, has been developed with nanometric
cially for local delivery. In contrast, for drugs that require system
resolution to image cellular structure [39]. Furthermore, it is also
absorption, MPPs can efficiently cross the mucus and reach the muco-
worth noting that SRM has been exploited to characterize the shape
sal epithelial cell for subsequent cellular uptake [58].
and morphology of synthetic materials [40], such as nanofibers, poly-
meric, lipid and metal NPs, due to its non‐invasiveness, nanoscale
accuracy and the ability of multicolor imaging. Recently, SRM has 4.1. Mucoadhesive NPs
emerged as a powerful tool for investigating the microscopic move-
ment of NPs in mucus [7,8,41,42]. With STED microscopy, Yu et al. NPs experience wash‐off during the traverse through mucus, which
[7] found that nanorods could rotate around the mucin fibers, which largely reduces drug accumulation at the mucosa. Due to the physico-
further contributed to their facilitated diffusion in mucus. Besides, chemical properties of mucin fibers, mucus provides multiple interac-
they also investigated the movement of NPs with different elasticities tions for binding with NPs, such as disulfide bond, macromolecule
in mucus via Airyscan [8]. The trajectories of fluorescence‐labeled NPs entanglement, hydrogen bonding, hydrophobic interaction, electro-
were recorded at high temporal and spatial resolution. Interestingly, static interaction and van der Waals interaction [59]. Based on these
soft NPs deformed irregularly, hard NPs remained spherical in shape, NP‐mucin interactions, mucoadhesive polymers have been developed
and the semi‐elastic NPs deformed into ellipsoids and rotated in the and utilized to prolong the residence time of NPs in mucosal tissues
mucus. [60].
Typical mucoadhesive materials include chitosan, thiomers and
3.2.2. Microscale thermophoresis (MST) poly(acrylic acid) (PAA) [61]. Chitosan, a cationic polymer, can stick
The interactions between mucin and NPs largely impede particle to negatively charged mucin via electrostatic interactions. Yu et al.
penetration efficacy. Therefore, besides observation of movement pro- [62] synthesized dexamethasone‐glycol chitosan (Dex‐GCS) conju-
cess, the exploration of mucin‐NP interaction is also an important char- gates, which further assembled into NPs and significantly prolonged
acterization element which can help predict diffusivity and explain the the retention time of Dex during ophthalmic drug delivery. In addition,
underlying mechanism. Usually, the binding interactions are evaluated Li et al. [63] used chitosan nanoparticles to delivery betaxolol across
by monitoring the aggregation and dynamic size variation of particles the ocular mucus for treatment of glaucoma. It was shown that betax-

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C. Liu et al. Medicine in Drug Discovery 12 (2021) 100110

olol could be detected at the surface for up to 12 min due to the trary to the present main function of promoting mucus penetration.
improved mucoadhesion endowed by chitosan. Covalent interactions However, researchers further discovered that the low molecular
are also utilized by bioadhesive materials to adhere to the mucus. weight (MW) of PEG could reduce chain entanglement and that the
For example, NPs with thiol groups on the surface can adsorb to mucus high grafting density could shield particle hydrophobic cores [73].
through disulfide bonds with cysteine residues. Irmukhametova et al. Hanes et al. [50] found that the decoration of high MW PEG
[64] synthesized thiolated organosilica NPs and compared their (10 kDa) could result in strong adhesive interactions with mucus
mucoadhesive properties with PEGylated NPs by evaluating their res- and restrict drug delivery through sputum. While a high density mod-
idence time on bovine ocular surfaces. The results showed that thio- ification of PEG of 2 and 5 kDa facilitated their mucus penetration.
lated NPs exhibited significantly greater adhesion on the cornea They prepared poly(lactic‐coglycolic acid) (PLGA) NPs coated with
even after several washes. Furthermore, the physical chain entangle- PEG5k and evaluated the impact of grafted density on NP penetration
ment of polymers such as PAA with mucin fibers can promote the in mucus [49]. Interactions between PLGA‐PEG NPs and mucin
adherence of NPs to mucus. Qian et al. [65] fabricated nanogels con- revealed that mucin adsorption was reduced as the PEG content
taining PAA as the backbone to deliver paclitaxel for cervical cancer increased from 0 to 5%. MPT was utilized to characterize particle dif-
treatment. The nanogels showed excellent adhesive properties and fusion dynamics in cervicovaginal mucus (CVM), where the naked
achieved favorable residence in the vagina, enhancing the therapeutic PLGA NPs were nearly immobilized, PLGA‐PEG3% was mainly con-
effect of paclitaxel in an orthotopic cervical cancer model. strained, and PLGA‐PEG5% diffused freely. Furthermore, the in vivo dis-
tribution results showed that PLGA‐PEG NPs with higher grafting
4.2. Mucus penetrating nanoparticles (MPPs) density could cover a larger epithelial surface and achieve a uniform
distribution throughout the mucus. In addition, the PEG configuration
Contrary to mucoadhesive NPs, MPPs are aimed at moving freely may translate from mushroom to brush as grafting density increased,
through mucus. Thus, MPPs are expected to avoid mucus turnover suggesting that the brush PEG conformation endowed by high surface
and clearance, and finally reach the underlying epithelium. Mucus‐ density can facilitate particle penetration. As for vaginal delivery,
penetrating strategies focus on designing muco‐inert NPs on the basis Laura et al. [74] covalently attached the low MW PEG to fluorescent
of reducing steric obstruction and the binding interactions with mucus. polystyrene nanoparticles (PS‐COOH) to form MPPs. It was found that
In recent years, in addition to conventional strategies of size these MPPs covered 88% of the vaginal surface, which was 2.9‐fold
[31,66,67], surface hydrophilicity [21,49,51,53–57] and charge mod- higher than the control. Likewise, Katrein et al. [75] functionalized
ifications [43,46–48,68], attention has also been paid to the effects of the carboxylate‐modified polystyrene nanospheres with 2–5 kDa PEG
particle shape [7,41,69,70] and rigidity [8,42,52] on mucus and tested the mobility of NPs in cystic fibrosis airway mucus. The
penetration. results showed that PEG modification significantly improved the diffu-
sion of NPs. In addition to PEG, other hydrophilic polymers, such as
4.2.1. Size Pluronic F127 and poly‐N‐(2‐hydroxypropyl) methacrylamide
NPs can be trapped in mucus due to size exclusion when the diam- (pHPMA), have also been utilized to fabricate MPPs. Li et al. [56] mod-
eter of NPs exceeds the pore size of the mucus mesh structure. Studies ified chitosan NPs with Pluronic F127 and lipids to form core–shell
have been carried out to screen the appropriate particle size for muco- NPs. In the E12 cell model, the evaluation of mucus penetration
sal delivery. Murgia et al. [31] prepared NPs of different sizes (100, showed that F127‐modified NPs exhibited a more uniform distribution
200 and 500 nm) and evaluated their diffusion capacity in pig pul- than naked chitosan NPs. Corresponding to superior mucus‐
monary mucus through mechanical dispersion or aerosol deposition. penetrating ability, the modified NPs also showed excellent oral insu-
Interestingly, NPs with sizes of 100 nm and 200 nm could readily dif- lin delivery efficiency, featuring a relative bioavailability of 7.8% and
fuse after mechanical dispersion, while in aerosol deposition, only a long‐lasting hypoglycemic effect for 10 h. Date et al. [57] utilized flu-
100 nm NPs could successfully enter the interior of mucus. This result orescent polystyrene (PS) as the model NPs to investigate the effects of
suggested that the pore size of the mucus surface is smaller than that of F127 modification. They prepared budesonide‐loaded nanosuspension
the interior. He et al. [67] compared the diffusivity of nanocrystals coated with F127 for effective treatment of inflammatory bowel dis-
(NCs) of different sizes in simulated mucus. The MPT results showed ease (IBD). In vivo experiments showed that the F127‐modified PS
that small NCs (∼250 nm) exhibited higher MSDs than their large NPs were distributed throughout the colon, and could penetrate into
counterparts (535 nm and 1100 nm). Polystyrene NPs were exploited the ulcerated areas. In the colitis model, budesonide nanosuspension
by Bandi et al. [66] to explore the size effect on penetration in intesti- remarkably alleviated weight loss and reduced the infiltration of
nal mucus, and it was shown that smaller NPs (50 nm, 200 nm) pene- immune cells, indicating enhanced drug delivery efficiency due to
trated more effectively than the larger counterparts (500 nm, 750 nm). the F127 coating. Shan et al. [51] designed a nanocomplex containing
Overall, NPs with diameters smaller than the average pore size of insulin and cell penetrating peptide and coated the NPs with pHPMA.
the mucus mesh structure may diffuse faster than the large ones in The formed NPs displayed a larger MSD than unmodified NPs in
mucus. mucus, indicating less hindrance brought by the pHPMA modification.
Moreover, pHPMA could dissociate from the NP surface during the
4.2.2. Surface properties mucus penetration process, which exposed the cationic core and con-
Given the typical hydrophobic properties and negative charge of tributed to subsequent cellular uptake. As a result, the modified NPs
mucin fibers, hydrophilic and zwitterionic materials have been widely exhibited high insulin absorption in diabetic rats, which accounted
used to endow NPs with muco‐inert surface. for the ultimate in vivo hypoglycemic effect.

[Link]. Surface hydrophility. The hydrophobic interactions between [Link]. Surface charge. The negative charge endowed by carboxyl and
the NPs and mucin fibers seriously restrict their diffusion in mucus. sulfate groups in mucin fibers leads to the immobilization of cationic
To reduce hydrophobic interactions and enhance mucus‐penetrating NPs and hinders the drug delivery efficiency [73]. Inspired by the fact
ability, hydrophilic materials have been used for particle coating, that the surface charge of most viruses in nature is neutralized with
which shields hydrophobic surfaces. Polyethylene glycol (PEG), gener- identical amounts of positive and negative charges, researchers have
ally applied to prolong systemic circulation of drugs and NPs, is the introduced electrically neutral surfaces into designed NPs. Betaine
typical polymer to prepare muco‐inert NPs owing to its hydrophilic derivatives, zwitterions containing equal amounts of cation and anion
properties [71]. Initially, PEG was exploited as a mucoadhesive agent groups such as sulfobetaine (SB), phosphorylcholine (PC), and polycar-
because of chain entanglement with mucin fibers [72], which is con- boxybetaine (PCB), have been widely explored to prepare MPPs.

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Han et al. [47] fabricated zwitterionic micelles composed of 1,2‐dis ate the mucus‐penetrating ability of NPs and ultimately elucidated
tearoyl‐sn‐glycero‐3‐phosphoethanolamine (DSPE)‐conjugated PCBs. the superior diffusion behavior from the perspective of microscopic
DSPE‐PCB micelles showed superior mucus‐penetrating ability, with motion.
MSDs 5 and 100 times that of PEG NPs and cationic NPs, respectively, Given the excellent mucus‐penetrating performance of nanorods,
which was attributed to the overall neutral charge surface. Further- the nonspherical shape and even specific aspect ratio of nanorods have
more, PCB, the substrate of the transporter PAT1, mediates efficient been taken into consideration in carrier design for mucosal delivery.
epithelial absorption, consequently improving insulin oral bioavail- For example, C109, a drug against Burkholderia cenocepacia infection,
ability to over 40%. Gao et al. [68] functionalized mesoporous silica was fabricated into NCs with rod‐like shapes to diffuse through cystic
nanoparticles (MSNs) with sulfobetaine 12 (SB12) and deoxycholic fibrosis mucus for inhalation administration [69]. Guo et al. [77] pre-
acid (DC). They investigated the interactions between NPs and mucus pared lovastatin NCs of different shapes and found that rod‐shaped
by determining the variation in zeta potential and size before and after and flaky NCs featured a higher Papp value, which was 3.0 and 2.5
incubating MSNs with mucin solution. For MSNs‐NH2, the zeta poten- times that of their spherical counterparts, respectively. Banerjee
tial decreased and size increased, while there was nearly no change for et al. [78] compared the oral delivery efficiency of rods, spheres and
MSNs@SB12, indicating a weak interaction due to SB12 modification. discs. It was found that the discs exhibited more cellular uptake in
The mucus‐penetrating ability was also evaluated, and the apparent mucus‐secreting cells than the spheres, implying the superior mucus‐
permeability coefficient (Papp) values of MSNs‐S@SB12 were superior penetrating capability of discs.
to those of MSNs‐NH2.
In addition to betaine derivatives, other zwitterionic materials have
4.2.4. Rigidity
also been utilized. For instance, Shan et al. [46] conjugated PC with
Rigidity is another parameter that can be tuned to modulate parti-
DC to form the amphiphilic material DLPC, which could further assem-
cle diffusivity [79]. Inspired by the findings that the elasticity of can-
ble on particle surface. They characterized the diffusion of DLPC NPs
cer cells and red blood cells (RBCs) plays a vital role in their
by FRAP experiments, showing that DLPC NPs displayed a slower flu-
translocation across narrow pores, Yu et al. [8] hypothesized that mod-
orescence intensity decrease because of their higher diffusivity com-
ulating the rigidity of NPs may facilitate their penetration into mucus
pared to poly(vinyl alcohol) (PVA) NPs. After gavage with NPs, the
to improve drug delivery. They prepared poly(lactic‐coglycolic acid)
intestine was excised and visualized under a fluorescence microscopy
(PLGA)‐lipid NPs with different rigidities (soft, semi‐elastic and hard)
to evaluate the overall NP distribution. DLPC NPs covered more of the
and evaluated their mucus penetrating capability. It was shown that
epithelial surface than PVA NPs, suggesting greater mucus‐penetrating
NPs with moderate rigidity (∼50 MPa) exhibited enhanced diffusion
ability and thus gained more access to epithelial cells. Wu et al. [43]
in intestinal mucus. Orally administered semi‐elastic NPs further
developed virus‐inspired NPs (P‐R8‐Pho) containing the PLGA core
gained a higher bioavailability of doxorubicin (Dox) compared to their
and an equally dense coating of cationic octa‐arginine (R8) and anio-
soft and hard counterparts. To investigate the potential mechanisms,
nic phosphoserine (Pho). The interactions between NPs and mucin
MD simulations and SRM were utilized, and the results revealed that
fibers were investigated. The cationic P‐R8 NPs showed more aggrega-
semi‐elastic NPs deformed into ellipsoids exhibiting a rotation‐
tion than P‐R8‐Pho NPs because of electrostatic interactions. And the
facilitated fast penetrating ability. The rigid NPs were not able to
relative Papp were notably higher for P‐R8‐Pho NPs than for P‐R8
deform, and the soft NPs were hindered by interactions with mucins
NPs. Zhou et al. [48] prepared exendin‐4 loaded metal–organic frame-
fibers. In addition, Li et al. [42] synthesized short nanotubes (SNTs)
work (MOF) NPs coated by a hydrogel containing zwitterionic
and cross‐linked short nanotubes (CSNTs) on the basis of self‐
MPDMSA. The neutrally charged Ex@MIL101@Gel ± showed more
assembly of α‐lactalbumin peptides. MPT experiments were performed
robust diffusion in mucin solution than its positively charged counter-
to investigate particle movement in freshly obtained mucus, and the
parts. Moreover, in the E12 cell monolayer model, the 3D distribution
soft SNTs showed rapid penetration compared to the stiff CSNTs, with
of NPs was imaged by CLSM, and the Ex@MIL101@Gel ± group
the former moving frequently in a larger area. The coarse‐grained MD
showed more signal across the mucus layer secreted by E12 cells.
simulations revealed that the rotation dynamics were augmented by
the shear flow and the mesh‐like structure of mucus. Furthermore,
4.2.3. Shape
the relationship between the temperature and rigidity of liposomes
There are a large number of studies reporting the shape effects on
should be taken into consideration. Yu et al. [52] prepared a series
the in vivo fate of NPs, including cellular uptake, blood circulation and
of liposomes with different phase transition temperatures (Tm) and
biodistribution [70]. However, little attention has been given to the
studied their diffusivities in mucus at ambient temperature. It was
behavior of nonspherical NPs in mucus penetration. Mucosal tissues
observed that the liposomes obtained a moderate rigidity when the
harbor a plenty of bacteria with various nonspherical shapes, such as
the ambient temperature was around the Tm of liposomes, featuring
rod‐like bacilli and spiral spirillum. It has been demonstrated that
an optimal permeability. The underlying mechanism lies in the defor-
shape is associated with bacterial motility, and the helical shape has
mation of liposomes; with Tm close to ambient temperature (e.g., body
long been considered an important factor for Helicobacter pylori to
temperature), the liposomes could deform into ellipsoids smoothly and
effectively penetrate through gastric mucus [76].
gain enhanced mobility, thus facilitating fast diffusion. Zheng et al.
Inspired by the shape effect on bacterial motility, shape modulation
also reported that the MSD of NPs in mucus decreased as their elastic-
is emerging as a new approach to overcome the mucus barrier. Yu
ity increased [80].
et al. [7] designed different shaped MSNs with identical surface
charges and compared their mobility in intestinal mucus. MPT exper-
iments showed that nanorods exhibited a significantly higher MSD 5. Dilemmas between mucus penetration and cellular uptake
than nanospheres. The camptothecin‐loaded nanorods further exhib-
ited higher drug permeability across the intestine than their spherical In addition to the mucus barrier, the epithelial cellular absorption
counterparts. To explain the potential mechanism behind the superior barrier also hinders the mucosal delivery of NPs to a certain degree.
diffusivity of rod‐like shapes, they utilized coarse‐grained molecular However, the requirements of the two barriers for particle design are
dynamics (MD) simulations to explore the potential mechanism of this often contradictory and conflicting: (1) decreasing the size of NPs
kind of shape effect. Similar results were obtained, and the analysis could boost mucus penetration; however, smaller NPs may suffer from
revealed that rotation of nanorods could contribute to translational poor cellular uptake; (2) surface modification with hydrophilic materi-
diffusion. The rotation process was further observed and verified by als such as PEG contributes to improved mucus penetration but is ill
STED microscopy. This work combined multiple techniques to evalu- suited for cellular uptake [81,82]; and (3) neutrally charged NPs are

6
C. Liu et al. Medicine in Drug Discovery 12 (2021) 100110

favorable for mucus penetration but are inefficient in cellular internal- tions. Rational design of NPs with proper physicochemical properties,
ization [43]. Therefore, simultaneously overcoming both the mucus including size, surface property, shape, and rigidity, holds promise to
and the cellular uptake barriers is a major challenge in the design of address this critical barrier. We highlight that the properties of NPs for
NP‐based DDSs targeting mucosal tissues. mucus penetration are usually contrary to those for cellular uptake,
To solve these dilemmas, researchers have utilized dissociable leading to a dilemma in mucosal drug delivery. Screening suitable
materials as coatings that can shed from the NP surface after mucus shape and rigidity has been reported to achieve satisfactory balance
penetration to ensure adequate cellular uptake. For example, Wu between the mucus penetration and cellular uptake barriers. Thus,
et al. [43] designed charge reversible NPs composed of cationic modulation of the important physical properties, namely, shape and
octa‐arginine peptide and anionic phosphoserine. These NPs effec- rigidity, is promising to solve the dilemma in a more straightforward
tively penetrated the mucus because of their overall neutral charge, way. Moreover, the combination of several appropriate physicochem-
after which phosphoserine was hydrolyzed and cationic peptide was ical properties should also be taken into consideration to construct
exposed to promote cellular uptake. Similarly, Shan et al. [51] pre- more effective NP‐based mucosal drug delivery. The characterization
pared NPs coated with pHPMA, which dissociated from the particle techniques play an important role in understanding the interaction
surface during mucus penetration, thus facilitating subsequent entry between NPs and mucus and evaluating the transport efficiency of
into cells. Bovine serum albumin (BSA) was utilized as a hydrophilic NPs. Notably, advanced imaging techniques such as SRM can provide
and neutrally charged material to coat cationic liposomes [83]. The insight into the microscopic movement of NPs, such as rotation and
protein corona decorated liposomes exhibited a neutral surface charge shape transformation, which sheds lights in the underlying mecha-
and gained facilitated diffusivity in intestinal mucus. The corona was nisms of particle fast diffusion. Therefore, advanced techniques can
then shed during penetration, which exposed the cationic core and microscopically characterize particle motion modes, and thus inspire
facilitated cellular uptake. The insulin‐loaded corona liposomes fur- the design of efficient NP‐based mucosal DDSs from new perspectives.
ther achieved a high oral bioavailability of 11.9% in diabetic rats.
Manipulating surface polymer conformation of NPs can also confer
CRediT authorship contribution statement
the particles with multi‐functions to overcome multiple barriers in
mucosal tissues. Yang et al. [41] designed anisotropic PEG‐modified
Chang Liu: Writing – original draft, Writing – review & editing.
rod‐like MSNs with brush‐like conformations at the tips and
Xiaohe Jiang: Writing – original draft. Yong Gan: Writing – review
mushroom‐like conformations at the bodies, which resembled the flag-
& editing. Miaorong Yu: Writing – original draft, Writing – review
ellum and fimbriae of germs. The PEG conformations were confirmed
& editing.
via atomic force microscopy (AFM) by detecting the corona on the
nanorods. Anisotropic conformation was obvious when the PEG con-
tent reached 4%, while there was a transition into an isotropic brush Declaration of Competing Interest
conformation as the PEG content increased to 6%. Interestingly, the
high modification density at the tips facilitated penetration of nanor- The authors declare that they have no known competing financial
ods through the mucus, while the low density of PEG at the bodies just interests or personal relationships that could have appeared to influ-
slightly reduced the cellular uptake, ensuring the ultimate efficient ence the work reported in this paper.
mucosal absorption. Furthermore, the microscopic motion pattern of
PEGylated nanorods was investigated by STED microscopy to be “ad- Acknowledgements
hesion hopping”, and the adhesion force between the nanorod body
and cell membrane was detected by AFM, which was verified through The authors would sincerely appreciate the financial support from
MD simulation. The in vivo drug delivery efficiency was further evalu- the National Natural Science Foundation of China (81803445,
ated, and the oral bioavailability of saquinavir was 21.9‐fold higher 81773651, and 82073773), and NN‐CAS foundation.
than that of crystalline saquinavir.
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