REVIEW
Protein Delivery
Rational Design of Nanocarriers for Intracellular Protein
Delivery
Xiaofei Qin, Changmin Yu, Jing Wei, Lin Li,* Chengwu Zhang, Qiong Wu, Jinhua Liu,
Shao Q. Yao,* and Wei Huang*
Protein/antibody therapeutics have exhibited the advantages of high speci-
ficity and activity even at an extremely low concentration compared to small
molecule drugs. However, they are accompanied by unfavorable physico-
chemical properties such as fragile tertiary structure, large molecular size,
and poor penetration of the membrane, and thus the clinical use of protein
drugs is hindered by inefficient delivery of proteins into the host cells. To
overcome the challenges associated with protein therapeutics and enhance
their biopharmaceutical applications, various protein-loaded nanocarriers
with desired functions, such as lipid nanocapsules, polymeric nanoparticles,
inorganic nanoparticles, and peptides, are developed. In this review, the
different strategies for intracellular delivery of proteins are comprehensively
summarized. Their designed routes, mechanisms of action, and potential
therapeutics in live cells or in vivo are discussed in detail. Furthermore, the
perspective on the new generation of delivery systems toward the emerging
area of protein-based therapeutics is presented as well.
1. Introduction
Proteins display a complex set of functions in the body, such
as catalyzing of biochemical reactions, transporting molecules
within living cell or from one organ to another, formation of
membrane receptors and channels, providing intracellular and
extracellular scaffolding support.[1] Proteins can be used in
many therapeutics by replacing the proteins which are deficient
Dr. X. Qin, Prof. C. Yu, J. Wei, Prof. L. Li, Prof. C. Zhang, Dr. Q. Wu,
Prof. J. Liu, Prof. W. Huang
Key Laboratory of Flexible Electronics (KLOFE) and Institute
of Advanced Materials (IAM)
Nanjing Tech University (NanjingTech)
30 South Puzhu Road, Nanjing 211800, P. R. China
E-mail: iamlli@njtech.edu.cn; iamwhuang@njtech.edu.cn
Prof. S. Q. Yao
Department of Chemistry
National University of Singapore
Singapore 117543, Singapore
E-mail: chmyaosq@nus.edu.sg
Prof. W. Huang
Shaanxi Institute of Flexible Electronics (SIFE)
Northwestern Polytechnical University (NPU)
127 West Youyi Road, Xi’an 710072, P. R. China
The ORCID identification number(s) for the author(s) of this article
can be found under https://doi.org/10.1002/adma.201902791.
DOI: 10.1002/adma.201902791
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or abnormal in the body. For example, the
insulin which were approved by the US
Food and Drug Administration (FDA) in
1982 are the most effective protein thera-
pies for the treatment of diabetes mel-
litus type I and type II. In addition, the
protein therapeutics are also utilized for
augmenting an existing pathway, pro-
viding a novel function or interfering
with organisms. It is well-known that
aberrant mitochondrial function and
protein homeostasis contribute to the
occurrence and development of neurode-
generative diseases, such as Parkinson’s
disease (PD) and Alzheimer’s disease
(AD). Parkin functions as an E3 ubiquitin
ligase and participates in all stages of the
mitochondrial life cycle (from biogenesis
to mitophagy). Previous studies showed
that parkin expression was benefit to
mitochondrial pathology in flight mus-
cles and dopaminergic neurons in PD Drosophila models.[2]
Another protein PINK1, an upstream protein of parkin, was
recently reported to act as an important role in the pathology
of AD. Gene therapy-mediated PINK1 overexpression can pro-
mote the clearance of damaged mitochondria and then alle-
viate amyloid-β peptide accumulation and cognitive loss. Also,
overexpression of parkin was able to rescue all the mutant
phenotypes in pink1 null flies.[3] Recent studies also showed
that protein–disulfide isomerase could alleviate endoplasmic
reticulum (ER) stress and unfolded protein response signaling
pathway, and afford neuroprotection in cellular PD models.[4]
Therefore, the regulation of many proteins is tightly associated
with human diseases including cancer, cardiovascular diseases,
neurological disorders, and viral infection. All these proteins
may function as therapeutic proteins to combat disease in the
future.
Until now, the protein market grows so rapidly that many
other protein therapeutics, including Fabrazyme (agalsidase beta
for Fabry disease), PEGINTRON (pegInterferon-α2b for hepa-
titis C), and Cotazym (pancrelipase for cystic fibrosis or pan-
creatic insufficiency) have been approved for clinical use.[5] The
market of the therapeutic proteins holds tremendous potential
and it is estimated that by the end of 2019, the global market for
bioengineered protein drugs is valued at $222.7 billion, which
include therapeutic purpose, protein vaccines, and protein-based
diagnostics.[6] Compared with small molecules, proteins possess
unique advantage properties including less toxicity, high substrate
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specificity, low susceptibility to multidrug resistance and less

side effects to interfere with normal biological processes, and Lin Li received his B.Sc. and
last but not the least, the clinical development and FDA approval Ph.D. degrees in chemistry
time of protein therapeutics may be faster than that of small- from Anhui University in
molecule drugs.[1,5] However, there are several unfavorable 2004 and 2009, respectively.
physicochemical properties limit their usage of therapy, such He carried out postdoctoral
as entrapment into lysosomes, fragile tertiary structure which studies with Professor Shao
susceptibility to enzymatic degradation, easy aggregation when Q. Yao at National University
administered into serum, loss of activity, easy cause of an of Singapore (NUS). He
immune response, large molecular size which would be recy- obtained his current pro-
cled to the surface of the cells, poor penetration of the mem- fessor position in Nanjing
brane due to the electrostatic repulsion and poor stability in the Tech University (NanjingTech)
gastric low pH environment.[7–12] Furthermore, many proteins in 2015 after joining
(e.g., insulin, growth hormone, and oxytocin) have short half- Academician Huang’s group. His current research inter-
lives because of the fast renal clearance, and frequent injections ests are synthetic small-molecule indicators for monitoring
cause patient’s uncomfortable and inconvenience, representing biological/chemical species in physiological environments,
an immense challenge to modern proteins therapy.[13,14] especially in the mitochondrion.

2. Strategies for Intracellular Protein Delivery Shao Q. Yao is a Chinese-

born chemist currently
Direct delivery of proteins into the cells encounter many obsta- residing in Singapore. A
cles, such as serum instability and membrane impermeable graduate of Ohio State
due to electrostatic repulsions. To solve these issues, various (B.Sc. Honors, 1993) and
nanoparticles (NPs) are designed and utilized to enhance their Purdue (Ph.D., 1998), he
physical and biological stability, promote cell and tissue penetra- joined National University of
tion and effectively aide intracellular protein delivery.[15] A wide Singapore (NUS) in 2001 and
array of nanocarrier systems including lipid, polymers, protein is currently a professor at the
complex, inorganic materials have been shown to be prom- Department of Chemistry.
ising strategies.[16–19] In these strategies, different methods are His current research interests
applied to load the delivery system with the targeted proteins, broadly fall in the field of
such as genetic modification, physical adsorption, or chemical “catalomics,” in which the aim is to use tools in chemical
conjugation. In addition, these protein delivery systems based biology and proteomics, bioimaging, and materials for
on NPs could be engineered with various ligands for targeting to proteome-wide interrogation of enzymes for potential
the desired subcellular compartments like cytosol, nucleus and therapeutics.
mitochondria. Moreover, recent studies have reported that engi-
neered nanoparticles can be tailed with integration of stimuli
triggers to make protein delivery with both spatiotemporal- and
dosage-controlled manners possible, facilitating release of drug Wei Huang received his
with desirable pharmacokinetics.[20] In the last few years, there Ph.D. degree from Peking
was an increasing interest in delivering various systems of pro- University (1992). He began
teins. In this review, we will thoroughly summarize the research his postdoctoral research
progress of nanoparticle-based delivery system in native pro- in National University of
tein delivery, especially focus on the recent studies. We will Singapore (NUS, 1993);
survey representative examples of various materials and present moved to Fudan University
diverse approaches for protein delivery to complement previous where he founded the
reports. This includes the areas of polymeric nanoparticles (nat- Institute of Advanced
ural polysaccharides and synthetic polymers), lipid-based nano- Materials (IAM, 2002); was
carriers, protein-based complex, cell-penetrating peptide-based appointed as the Deputy
nanocarriers, exosome-based protein delivery system, metal- President of Nanjing
organic framework (MOF) composites and inorganic nanoparti- University of Posts and Telecommunications (NUPT,
cles (silica, gold, magnetic and carbon) (Figure 1). 2006). He assumed his duty as the President of Nanjing
Tech University (NanjingTech, 2012) and was appointed
as Deputy President and Provost of Northwestern
3. Nanoparticle-Mediated Protein Delivery Polytechnical University (NPU, 2017). His research inter-
ests include organic/flexible electronics, nanomaterials,
3.1. Polymeric Nanocarriers for Protein Delivery
and nanotechnology.
Due to the good biocompatibility and biodegradability,
many polymers can be referred as biomaterials to produce

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Figure 1.  Schematic illustration of various material-based nanocarriers for intracellular protein delivery.
nanoformulations for proteins delivery. Some of them (e.g.,
chitosan (CS) and alginate) have unique feature of adhering to
the mucosal surface and transiently opening the tight junctions
(TJs) between epithelial cells. Moreover, polymers can be easily
modified with functional ligands, which could enhance their
physiochemical properties or improve their cellular uptake for
targeting to the cells.[21] To prepare polymeric nanocarriers with
targeted proteins, various techniques are applied for designing
polymer-based nanoformulations, including emulsion, sur-
factant free emulsion, interfacial polymerization.[22,23] It is well
known that polymers can be classified as natural polymers and
synthetic ones. Polysaccharides including chitosan, cytodex-
trins, alginate, dextran, and their derivatives are one kind of
most important natural polymers, which have been widely used
for intracellular protein delivery.
3.1.1. Polysaccharide-Based Nanoparticles
Chitosan and Their Derivative-Based Nanoparticles: Chitosan is
referred as a family of linear, cationic amino saccharides and
produced by the partial deacetylation of chitin. They are natu-
rally abundantly available polymers composed of randomly
distributed β (1 → 4)-linked N-acetyl-d-glucosamine and d-glu-
cosamine units. Generally, they are isolated at commercial
scale from waste of crustacean shells and fungi. Because of
its promising properties, such as biodegradability, low toxicity,
antimicrobial, nonimmunogenicity, muco-adhesive feature
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and ability to open up epithelial TJs, making it an excellent
choice for many biomedical applications such as wound
dressing, biosensor, tissue engineering application and drug/
protein delivery.[24–28] Various approaches have been used to
prepare chitosan-based nanocarriers, including emulsified sol-
vent diffusion, ionic gelation, microemulsion, reverse micelle
formation, complex coacervation, and polyelectrolyte com-
plexation (PEC).[29] For example, by using the precipitation–
coacervation technique, Zaharoff and co-workers reported that
the chitosan-based nanoparticles could encapsulate various
fluorescence-labeled proteins including FITC-labeled bovine
serum albumin (BSA), ovalbumin (OVA), concanavalin A
(Con A), and insulin.[30] In addition, the size and polydisper-
sity index (PDI) of nanocarriers, protein encapsulation effi-
ciency and protein release rates have been demonstrated to
be influenced by the molecular weight and concentration of
CS, precipitant salt concentration, and proteins size. The com-
position of precipitant salt containing weak anions resulted
in high PDI particles. With increasing the concentration
and intrinsic viscosity of CS, the particle size was increased,
subsequently leading to the increase of the protein loading
efficacy and release rates. To improve the cellular up and per-
meation of protein both in vitro and in vivo, Zhang and co-
workers introduced arginine-rich cell-penetrating lipopeptides
(SAR6EW) and synthesized SAR6EW/CS/insulin loaded NPs.
Their results demonstrated that the SAR6EW functionalized
NPs improved the internalization of insulin into Caco-2 cells
via adding clathrin and caveolae mediated endocytosis and
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showed much better hypoglycemic effect as compared with
unmodified NPs in diabetic rats.[31]
As a useful tool for functional protein delivery, chitosan-
based PEC is formed by charge interactions between positive
charged chitosan and negative charged polyelectrolytes (e.g.,
tripolyphosphate, alginate, hyaluronic, carrageenan, pectin,
gellan gum, and poly-ϒ-glutamic acid). It has proven to be
a simple and mild method to fabricate protein delivered NPs
as it does not require organic solvents or sonication, thus can
provide protection to the encapsulated proteins.[32,33] Hu et al.
developed a chitosan-based PEC delivery system consisting of
carboxymethyl pachyman (CMP) and CS.[34] CMP is negatively
charged polysaccharides which could interact with CS to form
CMP/CS NPs through PEC method. The diameter of CMP/CS
NPs was in the range of 100–200 nm as the BSA being loaded
into this nanocarrier with encapsulation efficiency of 52.9%.
However, no further in vitro or in vivo assay was performed
to confirm the enzymatic activity of protein. Generally, pro-
teins were encapsulated in CS-tripolyphosphate (TPP) NPs by
interacting with both the positively charged CS and negatively
charged TPP. Furthermore, the physicochemical characteristics
of NPs were highly influenced by the deacetylation degree of
chitosan, the ratio of TPP and chitosan, ionic strength and pH.
Recently, Stie et al. prepared CS-TPP NPs. Two proteins BSA
and p53 with different isoelectrical points could be success-
fully encapsulated in the NPs and effectively internalized by
the SK-Mel 28 cells.[35] Moreover, the higher positively charged
NPs under mildly acidic conditions (pH 5.5) could facilitate the
cellular uptake of their protein-cargo. Clustered regularly inter-
spaced short palindromic repeats (CRISPR) and the CRISPR-
associated protein9 (Cas9) system associated technologies have
been considered as promising approach for the treatment of
genetic diseases, which could be guided to specific genomic
locations and cleave a target DNA sequence by a short RNA
search string, enabling precise genome editing and cell engi-
neering for treating genetic disorders.[36] There are two delivery
ways of genome editing. One way is the delivery of DNA or
mRNA that can generate Cas9 proteins alongside accompa-
nying single guide RNA (sgRNA) inside cells, and the other
way is the direct delivery of CRISPR/Cas9 ribonucleoproteins
(RNPs) into live cells and animals. Compared with the former,
the latter has more advantages include reduced off-target
effects, low toxicity, high editing efficacy, etc. Recently, Qiao
et al. utilized biodegradable materials CS and red fluorescent
protein (RFP) to prepare NPs for delivering RNPs alongside
DNA donors (ssDNA).[37] In order to enhance the electrostatic
interaction between positively charged RFP@CS and nega-
tively charged Cas9 RNPs and facilitate Cas9 enzyme to enter
the nucleus for efficient genome editing, the Cas9 enzyme
was inserted twenty glutamate residues in the N terminus
and three repeating nuclear localization signals (NLS) at the
C terminus. Finally, the constructed Cas9 RNP-ssDNA-RFP@
CS NPs were internalized into the cytoplasm via caveolae- and
micropinocytosis-mediated endocytosis followed by endosomal
escape, releasing Cas9 RNPs and ssDNA donors, and eventu-
ally entering the nucleus for efficient genome editing.
Natural CS is soluble in the presence of acid solution due
to free protonatable amino groups at the glucosamine units.
However, CS is not soluble in aqueous solution at neutral pH,
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which is a big obstacle for its application in protein delivery. To
enhance its solubility in aqueous solution, various CS deriva-
tives have been designed by chemical modifications including
alkylation, thiolation, carboxylation, quaternization, PEGyla-
tion and acylation.[38–41] The side amino and hydroxyl groups of
CS could be modified with diverse array of ligands, functional
groups and moieties. On the one hand, this kind of modification
could enhance its aqueous solubility in different pH solution;
on the other hand, it could also improve stability, mucoadhe-
sivity, and TJs abilities by coupling with the targeted moieties.
Yao and co-workers recently reported CMCS-based multifunc-
tional NPs by modification of carboxymethyl chitosan (CMCS)
with l-valine (LV) and phenylboronic acid (PBA). As shown in
Figure 2, PBA was served as hydrophobic and glucose-sensitive
unit, which could combine with the 1,2-diols of glucose to form
soluble phenyl borates and result in the swelling of the NPs,
allowing “triggered release” of >92% of insulin with concen-
tration of glucose at 20 × 10−3 m. LV was used to facilitate the
absorption of the small intestine. Both in vitro and in vivo assay
indicated that the multifunctional NPs could internalize the
loaded insulin into HT-29 cells and protect insulin from deg-
radation in the simulated gastric fluid and simulated intestinal
fluid environments. Furthermore, this delivery system exhib-
ited improved hypoglycemic effect after oral administration to
the diabetic rats than that of free insulin.[42]
CS derivatives could conserve their positive charge at a neu-
tral pH value by quaternization of CS. Among them, trimethyl
CS (TMC) is well studied derivative as it possesses mucoadhe-
sive and TJs abilities. Compared with other quaternized CS-
based NPs, TMC-based NPs exhibited strongest effectiveness on
the opening of TJs, thus could deliver insulin across Caco-2 cell
monolayers.[43] To prove the enhancement of the TJs opening
ability and the transepithelial transport of insulin-loaded TMC
NPs, Liu et al. designed a dissociable “mucus-inert” hydrophilic
coating of N-(2-hydroxypropyl) methacrylamide copolymer
(pHPMA) derivative on the surface of TMC NPs. Experiments
displayed that the novel self-assembled NPs permeated effi-
ciently through both epithelium and blanket of mucus gel,
enhanced the cellular uptake and exhibited higher hypogly-
cemic effect on diabetic rats than uncoated TMC NPs.[44]
Besides the above chemical modifications of CS-based NPs,
thiolation, PEGylation acylation, as well as amino acid coupling
in the construction of functional nanocarriers have been devel-
oped for protein delivery. Recently, Gan and co-workers reported
CS-based deoxycholic acid modified NPs (DANPs) to overcome
multiple barriers of the intestinal epithelium by exploiting the
bile acid pathway.[45] It is noted that DANPs were internalized
into the intestinal epithelium through apical sodium-dependent
bile acid transporter (ASBT)-mediated endocytosis. Due to the
interaction with cytosolic ileal bile acid-binding protein, DANPs
could escape rapidly from endolysosome, thus protecting the
insulin from degradation and facilitating the intracellular traf-
ficking. Finally, DANPs accessed the basolateral membrane and
promoted the basolateral release of insulin. Further pharmaco-
dynamic and pharmacokinetic studies in diabetic rats proved
that the oral administration of insulin-loaded DANPs with
enteric capsules coating led to slower but prolonged reduction
in blood glucose levels and exerted a stronger hypoglycemic
effect.
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Figure 2.  a) Schematical illustration of the CMCS-PBA-LV-based multifunctional nanocarriers to overcome multiple barriers for oral delivery of insulin.
b) TEM images of CMCS-PBA-LV-based multifunctional nanocarriers. c) CLSM images of HT-29 cells treated with FITC-insulin loaded NPs after
incubation for 6 h. d) Insulin in vitro release profiles of insulin-loaded NPs against SGF (pH = 1.2) and SIF (pH = 6.8) in PBS solution with different
concentration of glucose. e) Blood glucose levels in diabetic rats followed oral administration of insulin-loaded nanocarriers, saline and insulin solu-
tion. INCs: insulin-loaded nanocarriers. Adapted with permission.[42] Copyright 2016, Elsevier B.V.

Thiolated CSs are synthesized by chemically modification
of sulfhydryl bearing agents such as glutathione, cysteine and
thioglycolic. Obviously, the major advantage of thiolated CS
derivatives is the thiol functional group, which could bind
with cysteine-rich subdomains of glycoproteins in the mucus
layer, thus promoting efficient mucoadhesion in the intestinal
wall. Special modification of CS derivatives with amino acids
contain various ability in anticoagulation, cell penetrating func-
tion and enhanced TJs opening with positive charges in neutral
conditions, thus can be used as promising materials for protein
delivery. Recently, a series of amino acid modified CS deriva-
tives (Lys-CS, Arg-CS, and Phe-CS) were synthesized to shield
nano droplet of linoleic acid for the codelivery of caspase-3 pro-
tein and paclitaxel.[46] The positively charged caspase-3 could
adsorb on the surface of nanocapsules to enhance the stability,
and hydrophobic drug paclitaxel was encapsulated into the oil
core. Due to possess smaller size and higher storage stability,
the Arg-CS-NPs was selected for the further study. Subsequent
results showed that this codelivery nanosystem was taken-up
into HeLa cells and exhibited synergetic effects to enhance the
apoptosis-inducing activity.
3.1.2. Synthetic Polymer–Based Nanoparticles
Poly(dl-lactide-co-glycolide) (PLGA): PLGA is one of the most
important synthetic polymers, which has been developed as a
promising delivery material. It is a biocompatible and biode-
gradable synthetic polymer that has been proved for safe drug
delivery without causing tissue damage.[47,48] PLGA is a polymer
ester that is composed of two α hydroxyacids lactic and glycolic
acids. Nguyen and co-workers systematically characterized

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physiochemical properties of six polymeric NPs including gela-
tion, alginate, CS, PLGA, CMCS-coated PLGA, and PLGA/PEG-
based NPs.[49] By comparison, PLGA-based NPs possessed the
best performance, such as the smallest size (≈160 nm), con-
sistent particles sizes in PBS, water and simulated lung fluid,
the most favorable profiles for effective delivery to lung tissue,
as well as the highest cytocompatibility in human alveolar
type-1 epithelial cells. These results further demonstrated that
PLGA-based NPs are promising carriers for pulmonary pro-
tein/DNA delivery. Moreover, another approach was applied to
improve the mucoadhesion and cell uptake of PLGA-based NPs
for mucosal delivery proteins by coating positive charged poly-
mers or proteins on their surface[50–53] For example, To improve
proteins stability and their enzymatic activities, Amoozgar and
Goldberg synthesized a delivery system with proteins coating
on the particles surface PLGA NPs.[53] In this study, various
proteins including lysozyme, deoxyribonuclease (DNase), anti-
CD62E antibody, and collagenase I were immobilized on the
surface of NPs by Michael addition or Schiff-base formation
with polydopamine. Doxorubicin (Dox) was encapsulated into
the NPs that could sustainedly release to immunosuppressive
M2 macrophages. In order to increase the transcellular uptake
of PLGA-based NPs and enhance the protein absorption, var-
ious ligands were conjugated for specific binding with the cel-
lular receptors. For example, folic acid (FA) is often used as a
target for many epithelial cancer cells as those cells could over-
express the folate receptor, which could promote endocytosis
of FA-attached nanocarriers via the folate receptor. Recently,
Jiang and co-workers fabricated the FA-CS-coated NPs based
PLGA for oral delivery of insulin.[54] Experimental data showed
that this system protected insulin against digestive enzyme
solution and improved the cellular uptake of HT-29 cells and
intracellular delivery. In vivo studies further revealed that
these insulin-loaded nanocarriers could prolong the hypogly-
cemic effect in the rats. Due to possess galactose residues in
molecule, lactose acid is the ligand for the hepatic asialoglyco
protein receptor, which can be conjugated with PLGA for liver
target. For example, Zhou et al. reported lactose acid-PLGA/ε-
polylysine NPs for BSA delivery.[55] In this system, lactose acid
was selected as liver-targeted ligand. ε-Polylysine was used as
protein protectant and antiacidic agent to enhance the stability
of BSA and cellular uptake in HepG2 cells. In vitro and in vivo
experiments exhibited the significant decreased initial burst
release and enhanced liver distribution in mice. In addition,
the same approaches were applied for synthesizing PLGA-l-va-
line,[56] PLGA-gambogic acid,[57] PLGA-PEG-concanavalin A,[58]
and PLGA-PEG-FA conjugates.[59] Upon emulsification, insulin-
loaded NPs were formed with surface expressing functional
ligands for specific receptors binding and mediated transport in
vitro and in vivo. Moreover, the PLGA-based NPs could be also
coated with functional proteins (e.g., transferrin and Con A),
which are used to bind to the receptors for enhancing oral
insulin delivery.[60,61] For instance, poly(ester amide)s (PEAs)
and PLGA were selected to form PLGA-PEA NPs with trans-
ferrin coating as functional target.[61] In this system, PEAs were
prepared with various Phe to Arg ratios to adjust the NPs with a
broad range of properties, thereby effecting the zeta potential of
NPs from +11.8 to 31.3 mV and insulin release kinetics. Experi-
mental results showed that all PLGA-PEA NPs with small size
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of 80–110 nm possessed loading efficiency of insulin as high
as 90% and exhibited fivefold higher cellular uptake than that
of BSA-coated NPs. Obviously, the transferrin coated NPs could
enhance their transepithelial transport via receptor-mediated
transcytosis and elicit a remarkable hypoglycemic response at
the dose of 50 U k−1. It is noted that cell-penetrating peptides
(CPPs) could also be utilized in PLGA-based nanocarrier for
increasing its cellular uptake and transportation. For example,
Wei and co-workers prepared poly(arginine)8 (L-R8 and D-R8)
functionalized PLGA NPs.[62] Due to a group of cationic amino
acid sequences in CPP, significant ability for membrane trans-
location was achieved. In addition, comparing with unmodified
NPs, the L-R8 and D-R8-modified NPs could improve hypogly-
cemic effects by 2.5- and 3.7-times and enhanced bioavailabili-
ties of insulin by 3.2- and 4.4-times, respectively.
Poly(allylamine)s: Poly(allyamine)s could be synthesized by
Michael-type polyaddition of primary or bis-secondary amines
to bis(acrylamide)s, which are water-soluble, biocompatible and
biodegradable cationic polymers. Poly(allyamine)s have some
advantages such as low cytotoxicity and easy degrade into oli-
gomeric products in aqueous media within days or weeks, thus
make them to be potential in drug delivery application. Previous
studies were used to designed bioreducible poly(allyamine)s
based nanocarriers for intracellular protein delivery. By Michael-
type polyaddition of 4-amino-1-butanol (ABOL) to cystamine
bisacrylamide (CBA), the functionalized poly(allyamine)s
were synthesized. Next, the positively charged (CBA-ABOL)/
proteins PECs were developed via the self-assembly of negative
proteins with positive disulfide-containing poly(allyamine)s.
Experimental results demonstrated that the bioreducible
poly(allyamine)s NPs showed higher mucoadhesive properties
than poly(allyamine)s NPs without disulfide bonds. Moreover,
this system exhibited fast disintegration in reductive solution,
which could promote the release of the proteins in the intracel-
lular environment.[63,64] In order to efficiently deliver the posi-
tively charged proteins, Engbersen and co-workers synthesized
poly(allyamine)s polymers with negatively charged citraconate
groups in the side chains.[65] The citraconamide carboxylate
derivatives in the side chains become hydrolyzed with the for-
mation of protonated amino moieties, which could promote
polymer–endosomal membrane interactions and enhance the
cationic protein lysozyme delivery into the cytosol. In addition,
the side chains were linked by disulfide, so that the intracellular
cleavage of disulfide linkages of poly(allyamine)-based NPs
would further improve the protein unpacking in the cytosol.
Further results showed that the loading lysozyme was released
in its active form from the nanocarriers and successfully inter-
nalized into human umbilical vein endothelial cells.
Cell-Penetrating Poly(disulfide)s: The structures of cell-pene-
trating poly(disulfide)s (CPDs) is similar as polyarginine except
the polypeptides backbone is replaced with poly(disulfide)s.[66,67]
The CPDs were initially developed by Matile’s group in 2013,
which could be grown directly on the substrates by surface-
initiated ring-opening disulfide-exchange polymerization
method.[68,69] The polymer size could be adjusted by changing
the polymerization time, initiator and monomer concentra-
tion. Interestingly, the changes of side chain moieties would
further affect their intracellular destination. In comparison
with CPPs, CPDs could efficiently enter the cells and deliver
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Figure 3.  a) Structure of AO-SH and Tz-SH initiators. b) Schematic illustration of CPD-based intracellular delivery of native proteins with PTM-based
tagging and traceless tagging approaches. a,b) Adapted with permission.[72] Copyright 2018, Wiley-VCH. c) Schematic illustration of thiol-mediated
uptake of CPDs with no endosomal trapping. Reproduced with permission.[67] Copyright 2015, Royal Society of Chemistry.

the cargos into the cytosol via thiol-mediated pathways without
being trapped by endocytic vesicles. In addition, CPDs could be
rapidly degraded in the cytosol by glutathione (GSH)-assisted
depolymerization (<5 min) and showed minimal cytotoxicity,
thus can be used for intracellular delivery of other thiol-
containing or modified cargos.[70] For example, in order to effi-
ciently and directly deliver proteins into cell cytosol, Yao and
co-workers designed CPDs with three types of thiol-containing
initiators that comprise biotin, nitrilotriacetic acid (NTA) and
tetrazine (Tz), respectively.[71] These structures of AO-SH and
Tz-SH initiators are shown in Figure 3a. Four of recombinant
proteins including avidin, trans-cyclooctyne-BSA (TCO-BSA),
TCO-anti-rabbit immunoglobulin G (IgG) and BRD-4, either
covalently or noncovalently conjugated with CPDs were suc-
cessfully delivered into different mammalian cells via endo-
cytosis-independent pathways. In addition, CPD-assisted
delivery of functional proteins recombinant (His)6-tagged cas-
pase-3 into HeLa cells could retain its biological activity. More-
over, to avoid genetical or chemical modification of proteins
which might affect their activities, recently, the same group
reported two complementary CPD-facilitated approaches
(post-translation modification (PTM)-based tagging and trace-
less tagging) for delivery of “native” proteins (Figure 3b).[72] In
this study, glycoproteins (including all antibodies) were chemi-
cally or chemoenzymatically modified with biorthogonal tag
and subsequently conjugated with corresponding oxime-con-
taining CPD/TzCPD. On the other hand, nonglycosylated pro-
teins (e.g., BSA) with primary amines react with two cleavable
linkers NBL and TCOL which contain p-nitrophenylcarbonate.
Experimental data showed that “native” proteins could be suc-
cessfully released in live cells undergo GSH-catalyzed disulfide
cleavage followed by spontaneous cleavage of the self-immo-
late linker. Furthermore, functional glycoprotein horseradish
peroxidase (HRP) and nonglycosylated protein ribonuclease A
(RNase A) were selected to investigate their biological activi-
ties. The subsequent results displayed that the delivered HRP
remained activity of enzyme by catalyzing oxidization of a suit-
able substrate in the presence of H2O2 and RNase A. These
results indicated that PTM-based tagging/traceless tagging
CPDs conjugated delivery strategy is an effective approach for
intracellular delivery and release of native proteins with imme-
diate bioavailability.

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Copolymer-Based Nanoformulations: Block copolymers gener-
ally embrace two or more distinct polymer chains. Until now,
various approaches have been applied to synthesize block
copolymers, such as anionic, radical, cationic, ring-opening,
photo, and group-transfer polymerization methods. These
block polymers could be designed with tailored functionality,
variable molecular weight (Mw), and desired chemical com-
position. In this way, block polymers could form numerous
nanostructures such as NPs, micelles, polymersomes and
nanogels. Thus, copolymer-based nanocarriers have been
widely used for drugs delivery, wound dressing and tissue
engineering.[73,74]
Micelles (polymers) are organized auto-assembly formed
in a liquid and composed of amphiphilic macromolecules, in
general amphiphilic di or tri-block copolymers made of solvo-
philic and solvophobic blocks. These micelles could be used as
the platforms for cargos delivery. For example, previous studies
used cationic block polymers PEG-poly[N-{N′-(2-aminoethyl)-2-
aminoethyl}aspartamide] (PEG-pAsp(DET)) to form a pH-trig-
gered charge-conversion polymeric micelles.[75,76] The charged
density of proteins cytochrome c (CytC) and IgG were tempo-
rarily increased by conjugating with charge-conversional moie-
ties, citraconic acid amide (Cit) or cis-aconitic acid (Aco). Results
showed that the micelles were stable at the normal physiological
pH value of 7.4, however, dissociated and released the native
proteins at the endosomal pH of 5.5. In addition, the dissocia-
tion of micelles led to the fast endosomal escape of proteins and
diffused them into the cytoplasm. Gao et al. developed another
pH-sensitive positively charged polymeric micelles based on
methoxy-PEG-b-poly(β-amino ester). In this system, the pro-
teins were bonded with piperidine and imidazole rings by elec-
trostatic and hydrogen bonding interactions.[77] Apart from the
pH-triggered release in proteins encapsulated micelles, light or
sugar-responsive polyion complex micelles were also fabricated
for efficient protein delivery.[78,79] For example, Ji and co-workers
synthesized poly(N,N-dimethyl-N-(2-(methacryloyloxy)ethyl)-
N-((2-nitrobenzyl)oxy)-2-oxoethanaminium bromide)-block-
poly(carboxybetaine methacrylate) (PDMNBMA-b-PCBMA)
cationic block copolymer that could self-assemble as micelles
with negative charged BSA.[78] In vitro assay demonstrated that
the micelles disassembled into zwitterionic carboxybetaine units
under UV irradiation. In addition, micelles showed fast cellular
internalizations by A549 cells and controllable BSA release in
the presence of UV irradiation. Shi and co-workers reported
two kinds of micelles based on PEG-b-poly(glutamic acid-co-glu-
tamicamidophenylboronic acid) (PEG-b-P(Glu-co-GluPBA)) and
PEG-b-poly(L-lysine-co-ε-3,4-dihydroxyphenylcarboxyl-l-lysine)
(PEG-b-P(Lys-co-LysCA)) copolymers for intracellular protein
delivery, respectively.[79] Either negatively or positively charged
proteins (insulin or CytC) could be encapsulated into the
micelles under mild conditions. The micelles were quite stable
under physiological condition but disassembled in the pres-
ence of excess fructose or endosomal pH. Recently, Nie and co-
workers designed poly(etherimide)-poly(lactic-co-glycolic acid)2
(PEI-(PLGA)2) based micelles with lipid-peptide shell coating.
These micelles could codeliver both antibody R300 and anti-
tumor agent doxorubicin into tumor tissues (Figure  4).[80] In
this multifunctional nanocarrier, the lipid-peptide shell could
be cleaved by metalloproteinase 2 (MMP2) which overexpressed
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in tumor vascular endothelia and stroma. R300 was utilized
to deplete platelets, which could promote the diffusion of NPs
into solid tumors. Thus, the R300 and Dox were specifically
and selectively released into tumor tissues, subsequently sup-
pressed tumor growth and metastasis formation in an MCF-7
mouse tumor model.
Polymersomes are polymeric vesicles made of amphi-
philic block copolymers, which contain an aqueous interior
surrounded by a bilayer membrane for loading hydrophobic
and hydrophilic drugs. In comparison with liposomes, poly-
mersomes are more stable, easier adjusted with their stim-
uli-responsiveness, membrane thickness, and size.[73] In
addition, their surface could be modified with functional
moieties, such as CPP for efficient cellular uptake and pro-
teins delivery into different cell lines.[81] In the same way as
micelles, polymersomes could also be prepared for proteins
delivery system with thermos/pH/reduction-sensitive copoly-
mers.[82–85] For example, using reversible addition-fragmen-
tation chain transfer polymerization (RAFT) method, Cheng
et al. successfully synthesized thermo-sensitive poly(ethylene
glycol)-b-poly(acrylic acid)-b-poly(N-isopropylacrylamide)
(PEG-PAA-PNIPAM) triblock copolymers.[82] After incuba-
tion with crosslinking agent cystamine, the polymersomes
were formed with stable structure in PBS at 37 °C, but were
rapid disassembled in the presence of dithiothreitol. In vitro
assay showed that various proteins including BSA, OVA,
CytC, and lysozyme could be encapsulated into the poly-
mersomes and released under an intracellular mimicking
reductive environment. In addition, the CytC released from
the poly­mersomes into the cytosol of MCF-7 cells showed
higher apoptosis as comparing with free CytC. Recently,
dual-responsive polymersomes were developed by Zhong
and co-workers, which was based on poly(ethylene glycol)-
SS-poly(2-(diethyl amino)ethyl methacrylate) (PEG-SS-PDEA)
diblock copolymers.[83] After loading model proteins BSA and
CytC, this dual-bioresponsive polymersomes could efficiently
release the protein under acidic and/or reductive conditions.
Cell-based experimental results showed that the released
CytC could enhance apoptosis of MCF-7 cells. In order to
achieve targeted tumor treatment with therapeutic proteins,
Zhong’s group synthesized two copolymers, poly(ethylene
glycol)-b-poly(2,4,6-trimethoxybenzylidene-pentaerythritol
carbonate)-b-poly(succinic acid carbonate) (PEG-PTMBPEC-
PSAC) and poly(ethylene glycol)-b-poly(2,4,6- trimethoxy-
benzylidene-1,1,1-tris(hydroxymethyl)ethane methacrylate)-
b-poly(acrylic acid) (PEG-PTTMA-PAA).[84,85] The function-
alized polymersomes with tumor homing ligands were
fabricated by modifying PEG-PTMBPEC-PSAC with pros-
tate cancer-targeting ligand 2-[3-[5-amino-1-carboxypentyl]-
ureido]-pentanedioic acid (Acupa) and PEG-PTTMA-PAA
with lung cancer-targeting ligand anisamide, respectively.
Decorated pH-responsive poly­ mersomes dissociated under
tumors environment (acidic condition), resulting in prompt
protein granzume B release and potent tumor cell apop-
tosis. The in vivo results showed that both cancer-targeting
ligands improved the accumulation within tumor, further
indicating the polymersomes could be served as promising
nanocarriers for intracellular delivery of protein drugs.
Recently, the same group developed smart polymersomes
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Figure 4.  a) Schematic configuration of the PEI-(PLGA)2 based Dox-loaded micelles (P-D), the antibody 300 was absorbed onto the surface to form
P-D-R. Lecithins, PEGylated phospholipids and MMP2 cleavable peptides were layered onto the surface of the P-D-R micelles to form multifunctional
PLP-D-R micelles. b) MMP2 is overexpressed on the surface of tumor endothelial and stroma cells, the encapsulated R300 is released once the shell
layer of PLP-D-R is cleaved by MMP2. R300 could bind to platelet-surface receptors and deplete platelets by inducing microaggregation of 3–5 plate-
lets. c) The Dox-loaded micelles could enter to the tumor tissues as the absence of platelets in tumor induces openings in the vessel walls. d) Release
kinetics of Dox and the MMP2 accelerated the release of Dox. PLP-D-R and other drug formulations were administered into e) MCF-7 tumor-bearing
mice, and tumor volume was measured every 3 days. Reproduced with permission.[80] Copyright 2017, Springer Nature.

dually functionalized with cRGD and fusogenic GALA
peptides for targeting to ανβ3-positive A549 lung cancer
cells and enhancing cytosolic release of CytC.[86] The poly­
mersomes were assembled with PEG-b-poly(trimethylene
carbonate-co-dithiolane trimethylene carbonate)-spermine
(PEG-b-P(TMC-co-DTC)-spermine), cRGD-PEG-b-P(TMC-co-
DTC), and maleimide-PEG-bP(TMC-co-DTC) which postmod-
ified with GALA-SH. Apoptosis assays showed that cRGD/
GALA decorated polymersomes induced better apoptosis of
A549 cells than polymersomes without GALA.
Apart from the micelles and polymersomes, copolymers
could also bind with proteins by electrostatic interaction or
hydrogen bond to form nanocarriers.[87,88] Due to possess
specific properties, heparin sulfate proteoglycans are well
studied for storage and protection of numerous growth fac-
tors in the extracellular matrix. For example, the heparin-
block-PEG copolymer based NPs were synthesized by Silva
et al. to load fibroblast growth factor (FGF-2).[88] In this
system, protein-loaded NPs with uniform size and high
loading efficiency were formed by polyelectrolyte compl-
exation. In vivo experimental results showed that the FGF-2
could be protected by PEG stealth corona and the released
proteins kept their biological activity in bone marrow mesen-
chymal stem cells.
As one of the most promising nanocarriers for intracellular
protein delivery, diverse polymeric delivery systems have been
extensively designed for protein delivery and achieved much
progress. However, owing to the complexity of polymer-based
nanocarriers with multicomponent 3D structures, careful
designing and construction are needed to make sure reproduc-
ible formulations in the large-scale manufacturing processes.
Furthermore, the smart polymers with multicomponent and
multifunction, as well as controlled release on demand and
high efficient diffusion of therapeutic protein through a stim-
ulus property of the disease microenvironment are urgently
needed.[89,90]

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Figure 5.  a) Schematic illustration of TRAIL/Dox–gel–liposome (Gelipo) for site-specific drug delivery. Using R8H3 modified liposome as the nanocar-
riers sequentially deliver TRAIL to the plasma membrane and Dox to the nuclei for combination cancer treatment. b) The diameter of TRAIL/Dox-Gelipo
measured by DLS and TEM. c) HAase degrade the m-HA shell and stimulate the extracellular release of TRAIL d) In vitro cytotoxicity of TRAIL-Gelipo,
Dox-Gelipo and TRAIL/Dox-Gelipo after 30 min of HAase pre-treatment toward MDA-MB-231 cells for 24 h. e) The MDA-MB-231 tumor growth curves
after intravenous injection of different formulations of Dox at a dose of 2 mg kg−1. f) Representative images of MDA-MB-231 xenograft tumors of the
mice after treatment with saline, the Dox solution, Dox-Gelipo and TRAIL/Dox-Gelipo respectively. MBA: a crosslinker, N,N′-methylenebisacrylamide,
EPC: Egg phosphatidylcholine. Adapted with permission.[94] Copyright 2014, Wiley-VCH.

3.2. Lipid-Based Nanoparticles cholesterol-incorporated dicetyl phosphate. Because of their

Lipid-based NPs are made of natural, semi-synthetic or syn-
thetic lipids, such as fatty acid, fatty alcohols, phospholipids,
long and medium chain monoglycerides, diglycerides and tri-
glycerides. Based on their structures and compositions, they
can be classified as solid lipid nanoparticles (SLNs), liposomes,
nanoemulsions and nanocapsules. The majority of lipids
excipients are derived from dietary oil or fats. Due to their
biocompatibility, biodegradability, low toxicity, and the ability
of overcoming the multiple biological barriers (e.g., intestinal
fluids, mucus layer, and intestinal epithelium), lipid-based
nanoparticles are considered as promising protein delivery
system. Until now, commercial success has been achieved with
some lipid-based NPs for oral administration of protein or
peptide-like drugs, which encourages researchers to design
new developed lipid-based nanocarriers.[91]
3.2.1. Liposomes
Liposomes as delivery vesicles are first discovered in 1961 by
Bangham and Horne.[92] This kind of drug loaded system is
most extensively studied. Until now, there are several liposome-
based products, which contain small molecules drugs, that have
been approved by FDA for human use.[93] Liposomes are bilay-
ered vesicles with an internal aqueous cavity. Their structure
components are composed of amphiphilic lipids or phospho-
lipids including diplamitoylphosphatidylcholine (DPPC), dis-
tearoyl-phosphatidylcholine (DSPC), lecithin, stearylamine, and
special structure, various hydrophobic or hydrophilic drugs
could be filled into the liposomes and then delivered into cells
via endocytosis or liposome-cell fusion.[16,91,93] For example,
to enhance therapeutic efficacy, Jiang et al. designed gel–lipo-
some complex to codeliver two kinds of anticancer drugs,
namely tumor necrosis factor-related apoptosis-inducing ligand
(TRAIL) and Dox (Figure 5).[94] Herein, the Dox was first encap-
sulated into CPP (R8H3) modified liposome. A crosslinked gel
composed of enzymatically degradable hyaluronic acid (HA)
was attached on the surface of liposome. In the meantime,
the protein TRAIL was encapsulated in the outer shell. The
hyaluronidase (HAase) is highly overexpressed at the tumor
microenvironment, which could degrade the HA shell, thereby
promoting the released TRAIL to bind with the death recep-
tors of the plasma membrane and facilitate the tumor cellular
uptake of Dox-R8H3-liposomes. In vitro and in vivo experiment
demonstrated that this multifunctional codelivery gel–liposome
system showed significant inhibition of the tumor growth in
the MDA-MB-231 xenograft tumor animal model.
Liguori et al. reported two different protein-loaded liposomes
by using cell-free protein synthesis technologies.[95] Both of
voltage dependent anion channel protein and Bak protein were
successfully delivered into living cells with bioactivity. One
of the major drawbacks for liposomal delivery system is that
they are taken up by endocytosis and finally degraded in lys-
osomes, which could induce the side effect. To overcome this
issue, Kube et al. synthesized fusogenic liposomes, which could
deliver intracellular proteins with different size ranged from
2.3 kDa (fluorescently tagged LifeAct) over 27 kDa (EGFP) to

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Figure 6.  Schematic illustration of bioreducible lipid-like materials and negatively supercharged proteins for effective protein delivery and genome
editing. Reproduced with permission.[102] Copyright 2016, PNAS.
240 kDa (R-phycoerythrin).[96] In general, the encapsulated effi-
ciency and release rate of proteins or peptides within liposomes
are greatly influenced by their interaction with liposomal com-
ponents such as lipids, surfactants, the polymers (e.g., chi-
tosan) or inorganic materials (e.g., silica). These components,
which are coupled on the liposomal surface, could enhance
the stability of nanocarriers, control the release time of drugs
and promote proteins disposition at the specific site.[91,97,98] For
instance, Opanasopit and co-workers synthesized methylated
N-(4-N,N-dimethylaminobenzyl) CS, which is water soluble at
physiological pH. After being coated on the surface of liposome
by electrostatic interaction, this system showed an enhanced
loading efficiency of BSA and protein permeability across the
Caco-2 cell monolayers with low cytotoxicity.[99] Recently, Sal-
maso and co-workers designed a pH-sensitive stearoyl-PEG-
poly(methacryloyl sulfadimethoxine)-decorated liposome
system with BSA loading for bladder cancer treatment.[97]
Results revealed that the BSA-loaded liposomes were colloi-
dally stable at neural pH but showed remarkably high adhe-
sion with MB49 cells and more effective administration to mice
via bladder instillation under acidic environment. In addition,
Matsumura and co-workers fabricated polyampholyte-modified
liposomes in conjunction with the freeze concentration strategy
to increase lysozyme protein internalization and the efficiency
of endosomal escape in living cells.[98] The results showed that
uptaken capacity of lysozyme was increased fourfold in com-
parison with that of unfrozen cells. The temperature could be
designed as a factor that affects the protein release rate when
the liposomes incorporate with thermosensitive materials.
Lysolipid is a kind of widely studied materials which could
form the thermal sensitive liposomes. For example, Xu and
co-workers prepared the lysolipid liposome with the diameter
of 145 nm.[100] This drug loaded system showed good stability
at 37.5 °C but rapid released albumin at 42 and 44.5 °C. The
genome-editing Cas9 is natively cationic, which could not com-
plex with cationic lipids. To efficiently deliver those proteins
into mammalian cells by cationic lipids, Zuris et al. changed
the proteins to be highly anionic either by complexation with
polyanionic single-guided RNA (Cas9:sgRNA) or by fusion
with the supernegatively charged (−30) green fluorescent pro-
tein (GFP).[101] In this way, the stable nanocarriers were formed
through electrostatic self-assembly between cationic lipids
and anionic protein complex. Subsequent in vitro and in vivo
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results suggested that cationic lipids could establish a powerful
delivery system for in vivo genome editing. Furthermore, Xu
and co-workers synthesized a bioreducible lipid with disulfide
bond as linkage to the hydrophobic tail.[102] After complexing
with negatively supercharged GFP-Cre or Cas9/sgRNA, these
nanocarriers could be structurally breakable under the reduc-
tive intracellular environment and promote the cargo proteins
to escape from endosome (Figure 6).
3.2.2. Solid Lipid Nanoparticles
Solid lipid nanoparticles possess excellent biodegradability and
biocompatibility, specific function by the suitable chemical
modification and rapid uptake by cells, which have been con-
sidered as a promising developed submicron gene/protein
delivery system.[103,104] Like the above liposomes, SLNs are typi-
cally colloidal particles with an average diameter between 10
and 1000 nm. The difference with liposomes is, generally, SLNs
possess a solid lipid core matrix that can load lipophilic mole-
cules. To improve the stability, the lipid core is next stabilized by
surfactants.[105] Thus, in comparison with liposomes, SLNs are
more stable for biological applications. Over the past 25 years,
there are many techniques (e.g., water/oil/water emulsions
method and hydrophobic ion pairing technique) have been used
for achieving loadings of proteins within SLN.[91,106,107] In order
to improve the proteins loading efficiency, the hydrophobic ion
pairing technique is applied to enhance the lipophilic parti-
tion coefficient of hydrophilic peptide leuprolide. Amphipathic
molecule sodium stearate was selected to prepare hydrophobic
ion pairing of leuprolide. This technique combined with O/O
emulsion–evaporation that can not only increased the protein
entrapment, but enhanced the stability of the whole system.[107]
Furthermore, Yang et al. fabricated a novel Gel-Core-SLNs by
double emulsion and thermal sensitive gel technology.[108] In
this system, the loading efficiency of insulin and thymopentin
in the Gel-Core-SLNs were 57.36% and 61.97%, respectively.
Furthermore, the functional peptide ligands are usually utilized
to be modified with lipids to enhance cellular internalization
and drug absorption. Epithelium targeting peptides and CPPs
are two main functional peptides.[109,110] For example, Lv and co-
workers designed SLNs modified with stearic acid–octaarginine
(SA–R8) to deliver the insulin.[109] Due to the positively charged
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SA–R8, the zeta potential of the SLNs dramatically changed
from −32.13 to +29.87 mV, which could increase the Caco cells
internalization of SLNs and bioavailability of insulin. Recently,
Huang and co-worker used goblet cells affinity peptide CSKSS-
DYQC (CSK) and CPP IRQRRRR (IRQ) to conjugate with
polyoxyethylene (40) stearate, by which the salmon calcitonin–
loaded SLNs are prepared and characterized.[110] In vitro and in
vivo studies showed the peptide modified SLNs facilitated the
internalization of protein drugs on Caco-2/HT29-MTX co-cul-
tured cells and permeation in excised rat duodenum mucosa.
Besides modification of the lipids surface for improving the tar-
geted protein delivery, the protein drug can also be modified
with functional ligands to enhance the treatment of diseases.
For instance, Huang and co-workers synthesized the mem-
brane permeable sequence (MPS)-conjugated proteins CytC
(MPS-CytC), which subsequently complexed with PEGylated,
anisamide modified SLNs to enable specific targeting to the
tumor site in H460 lung carcinoma.[111] In this system, both
anisamide ligand and MPS could drive protein drugs to the
specific tumor site in a xenograft model and exhibited effi-
ciency anticancer treatment. Zwitterions are electrically neutral
compounds that consist of cation and anion group. NPs with
zwitterion lipids coating exhibit excellent mucus penetrating
ability as possessing neutral and superhydrophilic surface. In
addition, their special property that similar to the membrane of
cells makes them easily to be uptaken by living cells.[112]
In conclusion, due to their relative simplicity and safety, lipid-
based delivery systems for protein delivery in live cells and tis-
sues have been extremely studied. However, the low efficient
permeability to tissue is still the main limitation for this strategy
in clinical trials. In addition, some issues, such as easy accumu-
lation in the phagocytes, the high cost of raw material still hinder
their clinical use. Thus, other alternative materials are needed to
offer unique advantages, as well as for specific disease.
3.3. Inorganic Nanoparticles
Inorganic nanoparticles have generated some considerable
interest, such as easily tunable size and modified surface, which
enable them to efficiently enter into targeted cells, as well as
contain minimal toxicity and long circulation time. Either by
noncovalent interaction or covalent attachment, proteins could
be encapsulated or stabilized by these NPs. Typically, these
NPs are taken up by cells via endocytosis or membrane fusion
mechanism. The loaded proteins next escape from endosomal
or directly go into cytosol or other organelle.[18]
3.3.1. Silica Nanoparticles
Nowadays, silica is an important element that has been broadly
used as an additive in food, oral drugs, and cosmetics, which is
also recommended as “generally recognized as safe” by FDA.
Due to their impressive features, such as low cost, biocom-
patible, good physiochemical stability, versatile surface func-
tionalization, and advanced stages of clinical development,
silica-based nanoparticles (SiNPs) have been applied for storage
and delivery of drugs including protein molecules and enzymes
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that used for pharmaceuticals, therapeutics and diagnosis.[113]
The SiNPs with different structure and morphologies could be
fabricated by using sol–gel chemistry combined with different
structure directing agents. According to the topography, those
SiNPs could be divided into mesoporous silica nanoparticles
(MSNs), dendritic silica nanoparticles, hollow silica nanoparti-
cles (HSNs) and solid silica nanoparticles.[114] Figure  7 shows
silica-based NPs with different structure and morphologies that
used for protein delivery.
Mesoporous Silica Nanoparticles: The MCM-41 is one clas-
sical type of MSNs, which is prepared using modified Stöber
method. In this method, cationic surfactant cetyltrimethylam-
monium bromide (CTAB) is usually used as a soft template.[125]
This MSN possesses the uniform pore size of ≈2 nm, which
has been extensively studied for delivering the small drug
molecules.[113,126] However, it is difficult to encapsulate large
proteins into such small pores. In order to load the large pro-
teins, larger molecular weight template or swelling agents were
used to expand the pore size.[114,116,127] For example, Slowing
et al. synthesized the MCM-41 with large pore (diameter,
5.4 nm) by using the mesitylene as a swelling agent.[127] The
membrane-impermeable protein CytC could be encapsulated
into the MSNs and delivered into the cytoplasm of HeLa cell.
Gu et al. designed a facile strategy for preparing the large pore
MSNs with the N,N-dimethylhexadecylamine (DMHA) as a
pore size mediator.[116] In addition, the pore size could be tuned
from 2.7 to 4.6 nm by adjusting the amount of DMHA. Obvi-
ously, the loading amount of CytC was correlated with their
pore size. By adding isopropanol as a coswelling agent to adjust
the pore size (4.6–7.6 nm), large-pore and well-dispersed ben-
zene bridged mesoporous organosilica nanoparticles (MOSNs)
with only ≈50 nm diameter were synthesized by using biphasic
synthesis method.[128] In this system, the MOSNs with a larger
pore size (7.6 nm) contained a higher loading amount of
RNase A and more sustained release profile of RNase A into
the cells. SBA-15 is another classical type of MSNs, which has
large pore size (5–30 nm) and could be utilized to delivery large
therapeutic proteins. However, the particle size are in range
of 800 nm to 2 µm, which limit their usage for hardly cel-
lular uptake.[126] Recently, small SBA-15 rods with 80–200 nm
in length and 18 ± 8 nm in width were synthesized.[129] This
type SBA-15 rods showed better BSA and lysozyme absorption
and cellular uptake than that of conventional MCM-41. To con-
trol the release profiles of proteins, MSN pore surface could
be engineered with citraconic amide which could control the
CytC release by adjusting the pH of the releasing medium. The
positive protein CytC, which was entrapped in the pore sur-
face functionalized MSNs could be released at endosomal pH
and maintain the same enzymatic activity as native CytC.[130]
Sun et al. recently fabricated pH- and glucose-sensitive nano-
carriers through coating dextran-maleic acid and 3-amidophe-
nylboronic acid (APBA) on the surface of MSNs.[131] In the
neutral or acidic pH, the shell outside the MSNs would shrink
so that the insulin in the nanopore was blocked. However, the
insulin could be released after the shell swelled at alkaline pH
and exist of glucose. Furthermore, in vitro and in vivo studies
demonstrated that the nanocarriers exhibited excellent cell
internalization without cytotoxicity and produced an obvious
hypoglycemic effect. The pore size of MSNs mentioned above
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Figure 7.  Silica-based NPs with different structures for protein delivery. TEM images of a) PEGylated linker-MSNs. Adapted with permission.[115] Copyright
2011, American Chemical Society. b) MSNs using cationic surfactants as templating agent. Adapted with permission.[116] Copyright 2013, Elsevier Inc.
c) Hollow MSNs. Adapted with permission.[117] Copyright 2015, Royal Society of Chemistry. d) Silica nanorattles. Adapted with permission.[118] Copyright
2013, Wiley-VCH. e) Aldehyde-functionalized dendritic MSNs. Adapted with permission.[119] Copyright 2016, Elsevier B.V. f) GSH-responsive biodegrad-
able dendritic mesoporous organosilica nanoparticles. Adapted with permission.[120] Copyright 2016, American Chemical Society. g) Core–core-structured
MSNs. Adapted with permission.[121] Copyright 2015, Wiley-VCH. i) Amine-functionalized hollow dendritic MSNs. Adapted with permission.[122] Copyright
2016, Wiley-VCH. k) Hollow MOSNs. Adapted with permission.[123] Copyright 2016, American Chemical Society. m–p) Rough silica NPs with various
shell particles sizes. Adapted with permission.[124] Copyright 2015, Royal Society of Chemistry. Electron tomography (ET) slice of h) core–core-structured
MSNs. Reproduced with permission.[121] Copyright 2015, Wiley-VCH. j) Amine-functionalized hollow dendritic MSNs. Reproduced with permission.[122]
Copyright 2016, Wiley-VCH. l) Hollow MOSNs. Reproduced with permission.[123] Copyright 2016, American Chemical Society.

is normally below 20 nm, which could load relatively small pro-
teins such as CytC, RNase A, and insulin but difficult to encap-
sulate larger proteins (e.g., mTFP-Ferritin, ≈20 nm). Thus, the
MSNs with large pore are urgently needed for large proteins
delivery. Recently, Omar et al. reported silica–iron oxide hybrid
MSNs with large mesoporous from 20 to 60 nm, which not
only could immobilize large protein Mtfp-Ferritin, but also
could be rapid biodegraded in fetal bovine serum (FBS) and/
or at a magnetic responsiveness.[132] As showed in Figure 8, the
controllable release of the protein in vitro and in live cells was
achieved with the magnetic stimuli or at acidic environment.
This strategy has been considered as a promising design for the
next generation of biomedical applications.
Besides the proteins being encapsulated in large pore of
MSNs, another method for protein delivery is to attach the tar-
geted proteins on the surface of SiNPs through the electrostatic
interaction or chemical modification. For example, Kane and co-
workers prepared the SiNPs (≈20 nm) with the hydrophobic long
chains on the surface. The targeted proteins could be attached
on the particle surface through electrostatic and hydrophobic

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Figure 8.  a) Schematic illustration of the loading of large proteins into silica–iron oxide–propylamine nanocomposites, and hypothesis interaction
between NPs and mTFP-Ferritin. b) TEM micrographs of the NPs. c) High-magnification TEM micrograph of the NPs supporting the Sherrer’s equation
finding of 4.5 nm iron oxide nanophases incorporated into the silica framework. d) The degradability of NPs in FBS. Release profile of proteins from
the NPs via e) different pH and f) magnetic actuation. CLSM images of the protein loaded NPs in HeLa cells. g) Nucleus were stained in blue with
DAPI dyes, h) mTFP-Ferritin protein appears with the green fluorescence, i) cells membranes in red with CellMask and j) merged images. Adapted
with permission.[132] Copyright 2016, Elsevier. B.V.

interaction. Subsequent biological results proved that intracel-
lular delivery of specific proteins with this system could keep
the protein bioactivity and influence signaling pathways.[133,134]
In addition, Sun et al. designed the gold nanoparticle (AuNP)-
capped MSNs with luciferase and luciferin coimmobiliza-
tion.[115] The AuNPs were connected by disulfide linkage, which
could be cleaved by intracellular disulfide-reducing antioxidant
and subsequent resulted in the release of the luciferase and
luciferin. This system could be designed as tumor growth and
metastasis monitor. Recently, Li et al. prepared the SiNPs with
ultrasmall luminescent crystals embedding for BSA protein
delivery.[135] In this system, the BSA bonded with the amino
groups on the surface of SiNPs through the electrostatic interac-
tion and hydrophilic absorption. The protein releasing progress
was monitored by the upconversion emission intensity of lumi-
nescent crystals. To enhance the loading efficiency of proteins
on the surface of silica particles and promote the cellular uptake,
a kind of novel SiNPs with controllable surface roughness was
fabricated (Figure 7m–p). The proteins could be immobilized in
the void spaces of rough surface. Results showed that the hydro-
phobic modification of the roughness surface enhanced the pro-
tein adsorption capacity and improved the cellular uptake per-
formance.[124,136,137] Using the chemical modification method
to connect the proteins with the silica nanoparticles is another
powerful way to deliver the proteins to the target site. In order to
control the protein release at specific environment (e.g., acidic
pH and glucose) or avoid the loss of biomolecules over time,
those proteins could be modified on the surface with specific
linkage.[138–140] To an extent, the highly sensitive proteins could
also maintain their bioactivity. For example, Lin and co-workers
synthesized the MSN-based nanocarriers to allow efficient intra-
cellular protein delivery.[138] In this system, the cyclic adeno-
sine monophosphase (cAMP) was first filled in the nanopore,
followed by the modification of insulin with gluconic acid as a
linkage. In the presence of saccharides, the linkage was cleaved,
subsequently resulted in the release of insulin and cAMP. Tra-
ditional way to bind the proteins on the surface of nanoparti-
cles often leads to the immediate formation of dense monolayer,
which would not affect the bioactivity of the proteins with the
robust tertiary structure (e.g., GFP, myoglobin, or HRP), but
not suitable for the highly sensitive proteins.[140,141] To keep
the bioactivity of these sensitive proteins, the zwitterion-stabi-
lized silica nanoparticles have been synthesized by Niemeyer’s
group.[140] In this system, Schiff base was used to immobilize
the sensitive proteins, which could effectively load the very deli-
cate functional proteins to generate active biohybrid system.

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Figure 9. a) Schematic illustration of the preparation of bisorthosilicate-containing azo monomer based; CPD coated; fluorescent dye and black
hole quencher doped; antibody encapsulated biodegradable HSNs for intracellular delivery. b) The CPD-Cetuximab@BS-qNP were incubated under
normoxic and hypoxic conditions and protein was released under hypoxic condition with fluorescence turn on imaging. c) XTT assay of different cells
treated with CPD-Cetuximab@BS-NP under normoxic and hypoxic conditions. Reproduced with permission.[148] Copyright 2017, Wiley-VCH.

Dendritic Silica Nanoparticles: Owning to the large
mesoporous channels and special dendritic superstructures,
the dendritic mesoporous silica nanoparticles (DMSNs) have
recently attracted a growing interest in protein delivery applica-
tion.[142] Comparing with conventional MSNs, this kind of novel
porous materials possess larger pore size from ≈10 to ≈45 nm
that can enhance large protein loading capacity.[121,123] For
example, Yu and co-workers reported a anion assisted approach
to synthesize DMSNs with the pore size of 29.5 nm. By further
alkaline etching with Na2CO3 solution, the dendritic hollow
mesoporous silica nanoparticles (DHMSNs) could be fabri-
cated.[123] Results demonstrated that the DHMSNs possessed
higher loading capacity of BSA and lower hemolytic activity
than DMSNs, showing great potential in delivering large bio-
molecules. In addition, Zhu and co-workers prepared aldehyde
functionalized DMSNs which could form the pH labile imine
bonds between aldehyde groups on the DMSNs surface and
primary amines of BSA.[119] Due to the linkage of labile imine
bonds, it could control the BSA release by changing the pH
value. By using bis[3-(triethoxysilyl)propyl]tetrasulfide (BTES)
as silica source, Yang et al. synthesized the disulfide bond-
linked biodegradable DMSNs with different pore sizes.[120]
Results revealed that the DMSNs could load a mass of RNase A.
Furthermore, the degradation of the DMSNs at high intracel-
lular GSH level resulted in the fast release of loading RNase A,
indicating this system could deliver protein effectively in living
cells but reduce the cytotoxicity.
Hollow Silica Nanoparticles: Hollow silica nanoparticles pos-
sess a series of advantages including large surface area, low
density and tunable void volume that could offer more storage
spaces for loading proteins. Moreover, due to the external
shell protection, the proteins encapsulated into the internal
spaces could preserve the bioactivity from the enzymatic
degradation.[143,144] For example, Mou and co-workers have
successfully confined HRP in the cavity of HSNs.[145] In vitro
studies showed that this system could keep the enzyme stability
in the presence of the denaturant and urea. Furthermore, the
catalytic activity was also preserved as the prodrug indole-3-
acetic acid was oxidized by HRP to produce toxic free radicals
for killing HeLa cells. Another study showed that the mag-
nesium HSN, which was prepared using a modified classical
Stöber method, displayed highly amount of hemoglobin and
anticancer drugs absorption.[146] Next cell-based experiments
exhibited that this codelivery of HSNs system had efficient
anticancer activity. In order to improve the loading efficiency of
proteins, a new kind of HSNs, hierarchically structured HSNs
which possess shell-in-shell structure, was reported by Cao
et al.[147] In this system, glucose oxidase could be immobilized
not only onto the external surface, but also inside the inner
channel. Comparing with normal HSNs, hierarchically HSNs
exhibited higher mechanical stability and enzyme loading
capacity, lower enzyme leaching. By adding breakable orga-
nosilica matrices, such as bispropyldisulfide derivatives in the
preparation of hierarchically HSNs, the native proteins CytC,
TRAIL Apo2 ligand, and onconase could be encapsulated in
the HSNs and released in their functional conformations once
against external stimuli.[144] Recently, Yuan et al. successfully
designed biodegradable HSNs for RNaseA or native antibody
Cetuximab intracellular delivering.[148] Schematic of prepa-
ration of HSNs and the intracellular delivery of delivery are
shown in Figure  9a. In this nanosystem, the out shell of bio-
degradable HSNs are synthesized by bisorthosilicate-containing
azo monomer, which could be reduced by flavoproteins under
hypoxic conditions. In addition, the surface of HSNs were
coated with CPD that promoted the HSNs transported into
cells via endocytosis-independent uptake. Moreover, the HSNs

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were doped with fluorescent dye and black hole quencher, thus
could simultaneous image and monitor the intracellular pro-
tein release and cell condition under hypoxic conditions. The
results showed that Cetuximab encapsulated biodegradable
nanocapsules (CPD-Cetuximab@BS-NP) successfully uptaken
and released in hypoxic A431 cells. Importantly, the released
Cetuximab inhibited pEGFR expression, and subsequently
leading to time-dependent cell death (Figure 9b,c).
Over the years, many antibodies/proteins loaded nanocar-
riers with desired functions have been reported for intracel-
lular delivery. The delivery of native antibody/proteins into
subcellular targets is regarded as promising strategy for poten-
tial clinical application but rarely reported. In this regard,
Yao and co-workers first designed a TPP-modified biodegrad-
able HSNs for intracellular mitochondria delivery of different
kinds of proteins and antibodies, such as BSA, Cetuximab,
monoamine oxidase A (MAO-A), and anticytochrome c oxi-
dase subunit 2 (anti-MTCO2).[149] In this system, two types of
monomers, a disulfide monomer bis[3(triethoxysilyl)propyl]-
disulfide (BTEPDS) and tetraethylorthosilicate (TEOS), were
used to make HSNs, which could slowly degraded around
24–48 h under the intracellular GSH. CPDs were conjugated
on the surface of HSNs via a two-step biorthogonal reaction.
The TPP/CPDs facilitated the HSNs rapid internalization into
the mitochondria before premature degradation by cytosolic
GSH. Finally, a mitochondria enzyme MAO-A and functional
native antibody anti-MTCO2 were successfully delivered into
mitochondria and led to detectable antibody-target binding,
thus providing novel methods of subcellular antibodies/pro-
teins delivery for improving protein-based therapies.
3.3.2. Metal-Based Nanoparticles
Metal-based nanoparticles have been considered as promising
delivery systems due to their small size, nondeformable shape
and diversity, etc. In addition, the intrinsic physiochemical
properties, such as photoacoustic and photothermal effect
make them as excellent candidates in the cancer treatment.
Among them, the gold nanoparticles have been well studied for
protein carrier construction.
Gold Nanoparticles: The AuNPs contain highly tunable sur-
face area that can be engineered with various biomolecules
such as DNA/RNA (e.g., aptamers),[150] proteins (e.g., TAT
peptide, aspartic acid, and human transferrin),[151–154] carbo-
hydrate, polymer (e.g., chitosan)[155] and lipid.[156] Fabrication
of AuNPs surfaces with functional biomolecules could make
the particles high binding affinity and long-term stability, and
promote drugs efficient transport into the cells or organs.
Recently, Lee and co-workers developed a multiple proteins
delivery system based on AuNPs functionalized with His- and
GST-tag aptamers.[157,158] With this system, any recombinant
protein labelled a commonly used tag (e.g., His and GST) could
be purified and loaded onto the aptamer-conjugated AuNPs.
It is noted that the specific apoptosis-inducing BIM protein
was largely localized in the mitochondria of HeLa cells and
exhibited efficient antitumor activity. Furthermore, epidermal
growth factor (EGF), a target protein could be loaded onto the
AuNP–(His-GST)–aptamer system with BIM protein together,
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showing significantly uptake in mice and growth inhibition of
xenograft tumors without detectable systemic toxicity.[157] In
order to promote the proteins release from gold nanoparticles,
Reich and co-workers designed hollow gold nanoshells that
coated by a DNA layer.[159] The DNA layer provided terminal
amine groups used to conjugate the Ni2+ and protein chelator.
Under the irradiation of NIR laser pulses, this system could
be thermalized and release the loading proteins GFP into the
cytosol. Recently, AuNPs were functionalized with arginate
that could bind oligo-glutamic acid tagged protein.[160] This
coengineering system successfully transported proteins with
different size, charge and function into the cytosol or nucleus.
In addition, the engineered proteins Cre recombinase and
granzyme A could reserve bioactivity after cytosolic delivery.
The drug proteins could be trapped and degraded inside
endosomes and lysosomes, which has been considered as one
of the main obstacles in protein delivery. To improve cellular
uptaken efficiency and avoid endosomal sequestration, Rotello
and co-workers designed a serious AuNP-stabilized capsule for
effective protein delivery.[161,162] The AuNPs were first bonded
with a tetrapeptide, His-Lys-Arg-Lys (HKRK), which interacted
with carboxylates of the oil to pin the AuNPs on the capsule
surface. After that, the negative proteins were attached on the
AuNPs through the electrostatic interaction. In this system,
the capsule surface could fuse with cell membrane and then
release the proteins directly into the cytosol. By using caspase-3
as model, the delivered proteins effectively induced apoptosis
of HeLa cells, indicating it is a powerful strategy for efficient
delivery of functional proteins in their native forms.[163,164] In
order to further optimize the AuNPs stabilized capsules system
for delivering large proteins into the cytosol, the same group
designed AuNPs with 1-guanidino-2-(4-imidazole)propionic
acid (GIPA) as the terminal group. the guanidine group could
interact with both protein payload and the oil core to stabilize
the capsules structure. Final results showed that the large pro-
teins dsRed and β-gal were effectively transduced into cytosol
and remained its enzymatic activity by GIPA AuNPs stabilized
capsules (Figure 10).[165]
Recently, AuNPs have been utilized as Cas9 RNP loaded
nanocarriers for direct cytosolic delivery and efficient gene
editing. The AuNPs were conjugated with arginine or DNA,
which could complex with donor DNA and Cas9 RNP to pre-
pare stabilized nanocarriers.[166,167] The arginine AuNPs can
self-assemble with glutamate residues/NLS modified Cas9 RNP
to form nanoassemblies through the charge interaction.[166]
In addition, the DNA could also bind with Cas9 RNP-loaded
AuNPs to form stable nanocarriers. After conjugation with
polymer PAsp(DET), this system could trigger endosomal dis-
ruption.[167] Both kinds of AuNPs could successfully deliver
Cas9 RNP into the cytoplasm and induce gene editing in vitro
or in vivo.
Magnetic Nanoparticles (MNPs): The magnetic nanoparticles
have been developed for several technological applications such
as medical application, biosensing, magnetic storage media
and contrast agents in magnetic resonance imaging (MRI).
In order to stabilize the MNPs, their surface could be coated
with polymer (e.g., alginate and chitosan), inorganic materials
(e.g., silica and gold) or modified with chemical compounds
for binding drugs (e.g., proteins, antibodies, enzymes, or
© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.advancedsciencenews.com
Figure 10.  a) Schematic illustration of GIPA AuNPs stabilized capsules are utilized for dsRed and β-galactosidase delivery. LSCM images showing
b) dsRed and c) FITC-β-gal delivery into HeLa cells by GIPA AuNPs stabilized capsules. Reproduced with permission.[165] Copyright 2016, Royal Society
of Chemistry.
nucleotides). In addition, the external magnetic field is often
used to promote MNPs to target to the specific organ, tissue
or tumor.[168,169] For instance, Yang and co-workers constructed
long-circulating heparin functionalized MNPs for protamine
protein delivering.[170] The MNPs were coated with starch and
subsequently modified with a mount of 20 kDa PEG chains to
favor MNPs with passive target and long circulation. Moreover,
this system also showed the high capability for MRI and effi-
ciency for subcutaneous tumor magnetic targeting. Recently,
Lee and co-workers fabricated a MNPs system for proteins
transporting and trigger release with redox response as stimuli
(Figure 11).[171] In this system, the Cy5.5-labeled human serum
albumin (Cy5.5-HSA) was complexed with MNPs through the
electrostatic interaction, which was served as MRI and NIR
imaging probe in animal and tumor models (Figure 11c,d).
The disulfide-linked methoxy-poly-(ethylenelycol)-block-
poly(dopamine-diethylenetriamine-l-glutamate) ligands were
synthesized and the functional groups (dopamine-diethylen-
etriamine) facilitated the self-assembly of MNPs. In the intra-
cellular environment, the Cy5.5-HSA could be rapidly released
under high GSH conditions, and remained the bioactivity in
the living cells. By combining different nanoparticles, Chung
and co-workers developed a multifunctional gold coated MNPs/
engineered protein G system to deliver antibodies into cells.[172]
The protein G was first engineered with an affinity tag which
could bind with NTA-Ni(II) on the surface of gold-coated MNPs.
Next, this system was constructed by fusing the cell penetration
peptides with the N- and C-terminals of protein G. Cell-based
experiments demonstrated that this system could deliver the
antibodies into host cells with targeting, imaging, and manipu-
lation of mitochondria.
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Metal Organic Framework–Based Nanoparticles: Metal-organic
frameworks, which were first discovered in 1989 by Robson,[173]
are class of porous crystalline materials that are synthesized
with metal containing nodes and organic ligands.[174,175] Their
structure, crystal morphology, pore shape and size, physico-
chemical functionality (e.g., stability, hydrophobicity, and sur-
face charge) can be determined by monodentate ligands.[176]
Moreover, the surface modification of MOFs can enhance
their biomedical properties like improving their stability under
physiological conditions, introducing additional biological func-
tionalities and providing the MOFs with bioadhesive or tar-
geting properties.[177] Due to their mutable properties, MOFs
show great promise in many potential applications such as
catalysis, separation, chemical sensing and drug delivery.[178–181]
MOFs are of interest as promising drug delivery carriers
mainly due to their high drug loading capability. However,
most studies are focus on delivering small molecular drugs,
indeed, many poor solubility and/or stability drugs (e.g.,
busulfan, Dox, cidofovir, nimesulide, cisplatin, and siRNAs)
have been successfully loaded into MOFs which not only pro-
tect drugs from biodegradation but also enhance their thera-
peutic efficacy.[178,182] On the other hand, MOFs can also be
utilized as carriers for enzyme immobilization and delivering
because of their high surface area, large pore volume, easily
pore size tuning and mild fabricated conditions.[183] Generally,
the preparation strategies of MOF–enzyme composites can
be categorized into for main types: surface attachment, cova-
lent linkage, pore entrapment, and coprecipitation.[184] Those
enzymes could interact with MOFs either by electrostatic attrac-
tion, hydrogen bonding interaction or covalent conjugations.
It is noteworthy that MOF-immobilized enzymes offer many
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Figure 11.  a) Schematic illustration of the Cy5.5-HSA-loaded MNP for intracellular protein delivery. b) In vitro release of Cy5.5-HSA from redox sensi-
tive RMNs in PBS. c) In vivo NIR imaging and d) in vivo T2-weighted MR imaging of breast cancer tumor-bearing mice after intravenous injection of
Cy5.5-HSA-loaded MNP. Reproduced with permission.[171] Copyright 2017, American Chemical Society.

advantages such as improving the reusability of immobilized
enzymes, enhancing the catalytic activities and stability under
denaturing conditions.[185] For example, Zhou and co-workers
fabricated a series of highly stable MOFs and found that one
of the MOFs, PCN-333(Al), exhibits largest cage (5.5 nm) and
highest void volumes (3.84 cm3 g−1), and could encapsulate
high loading amounts of HRP, CytC and microperoxidase-11
(MP-11). Further experiments proved that those immobilized
enzymes showed better performance under harsh conditions
and exhibited smaller Km than free enzymes.[186] The conven-
tional fabrication methods of enzyme–MOF composites were
used organic solvent, which would easily denature enzymes. It
is urgent to pursue a facile and mild reaction conditions for the
synthesis of composites to preserve enzymes’ activities. Shieh
et al. developed a de novo approach under aqueous conditions to
embed catalase into zeolitic imidazolate framework 90 (ZIF-90)
supports of pore sizes smaller than the size of the enzyme.
The composites not only enhanced the stability and recycla-
bility of the enzyme, but also retained the peroxidase activity
of catalase.[187] In order to improve the therapeutic effect, Ge
and co-workers prepared a multiple enzyme-embedded ZIF-8
within 30 min at 25 °C by simply mixing aqueous solutions
containing zinc ions, 2-methylimidazole, glucose oxidase and
HRP. The confinement of the MOF scaffolds greatly shielded
the enzymes from proteolysis and chelating.[188] Li et al. suc-
cessfully encapsulated a nerve agent detoxifying enzyme,
organophosphorus acid anhydrolase into a water stable zirco-
nium MOF PCN-128y (4.4 nm) with a high loading. Moreover,
the immobilized enzyme into PCN-128y displayed long-term
stabilities at high temperature (70 °C) and in dry form.[189]
Recently, some novel methods are developed to synthesize
protein-loaded MOFs and their enzymatic activity are evaluated
in vitro and/or in vivo experiments.[190–192] Zhang et al. used
facile synthesis protocols to prepare protein embedded MOFs
vaccines. Herein, OVA was selected as model antigen that was
encapsulated into ZIF-8. In addition, the unmethylated cyto-
sine–phosphate–guanine oligodeoxynucleotides (CpG ODNs)
which used as vaccine adjuvants were attached on the surface of
the OVA@ZIF-8 nanoparticles to improve the biocompatibility
and immunogenicity of the nanoparticle. Both in vitro and in
vivo experiments proved that MOF-based vaccine platform had
good biocompatibility and pH stimulated release properties to
delivery antigens and CpG ODNs into targeted cells, inducing
strong cellular and humoral immune response.[190] Lian et al.
recently developed antioxidant enzymes (SOD) and catalase-
loaded MOFs and found those nano-MOFs could maintain
the enzymatic activity and protect human cells from toxic reac-
tive oxygen species for up to a week. Interestingly, although

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Figure 12.  a) Schematic illustration of MOF NPs for intracellular protein delivery. b) TEM image of caspase 3/HSA@ZIF-8. c) Long term activity of
β-Gal and β-Gal in ZIF-8. d) Fluorescent images of Hela cells treated with caspase 3/HSA@ZIF-8 for 4h. e) Cell viability after treatment with HSA@
ZIF-8 or caspase 3/HSA@ZIF-8 of different concentration for 4h. Reproduced with permission.[194] Copyright 2018, American Chemical Society.

the nano-MOFs were localized inside lysosomes, they still keep
their chemical stability and enzymatic activity against the pro-
tease degradation and low pH environment.[191] As mentioned
above, surface modification of MOFs could enhance their
biomedical abilities, previous studies proved that MOF con-
jugated with DNA could stabilize them in high concentration
NaCl (0.4 m) media and ensure facile cellular entry.[193] Based
on previous studies, Wang et al. fabricated nucleic acid-MOF
based delivery carriers, the results showed that the insulin
was encapsulated into MOF with high loading and enzymatic
activity and a tenfold enhancement of cellular uptake were
achieved.[192] Using the biomimetic mineralization method,
Chen et al. synthesized a biocompatible polyvinylpylrolidoe
(PVP) coated with protein-encapsulating ZIF-8 NPs for effi-
cient intracellular delivery.[194] In this systems, not only single
protein such as BSA, ferritin, EGFP, or β-Gal, but also multiple
proteins caspase 3/HSA or BSA/RFP/β-Gal could be success-
fully coencapsulated into single ZIF-8 NPs with good monodis-
persity. The coated PVP enhanced the stability of NPs and the
encapsulated proteins retained their activities throughout the
encapsulation and release. Finally, the protein-encapsulated
NPs could enter cells through a lipid-raft-mediated endocytosis
pathway and preserve the activity of proteins throughout the
intracellular delivery process (Figure 12).
MOFs are also an emerging class of platform for Cas9 RNP
cytosolic delivery and CRISPR/Cas9 genome editing. The
ZIF-90 or ZIF-8/Cas9 RNP nanocarriers could be fabricated by
self-assembling with Zn2+, proteins and imidazole-2-carboxal-
dehyde (2-ICA) or 2-methylimidazole.[195,196] Yang et al. found
that the ZIF-90 could be disintegrated by high concentration of
ATP (10 × 10−3 m) and released the protein inside cells due to
the competitive coordination between Zn2+ and ATP that dis-
assembles ZIF-90. In the ZIF-90 system, various proteins with
different molecular weights, including BSA, SOD, GFP, RNase
A-NBC and Cas9 RNP are encapsulated into the ZIF-90. In
addition, the functional proteins RNase A-NBC and Cas9 RNP
were successfully delivered into cells for enhancing cytotoxicity
against HeLa cells or inducing genome editing.[195]
3.3.3. Carbon-Based Nanomaterials
Due to possess various attractive properties, carbon-based
nanomaterials have been shown to extremely useful in energy

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Figure 13.  Schematic illustration of nuclear protein LB1-loaded SWCNTs enter into HeLa cells and quantification of SWCNTs localize to the nucleus of
cells by using fluorescence lifetime imaging microscopy. Reproduced with permission.[204] Copyright 2016, American Chemical Society.
storage, electricity, renewable energy, biosensing, and biomedi-
cine. Based on their different structures, carbon nanomaterials
have been divided into different forms, including carbon nano-
tubes (CNTs), nanodiamonds (NDs), fullerenes, carbon dots,
and carbon nanospheres (CNs).
Carbon Nanotubes: Carbon nanotubes can be designed as a
graphite sheet rolled up into a nanoscale tube. There are two
kinds of CNTs according to the number of walls around the
nanotubes, namely, single-wall carbon nanotubes (SWCNTs)
and multiwall CNTs. Due to the excellent electrical, mechanical,
electromechanical, and chemical properties, CNTs have been
used for a number of potential applications in various research
area, including reinforced materials, hydrogen storage, elec-
tronic devices, field-emission materials, biomedical imaging,
and drug delivery.[197] CNTs are receiving a great deal of atten-
tion for protein immobilization and delivery. CNTs could be
modified to generate functional groups (e.g., –COOH, –OH)
for proteins interaction via either noncovalent (e.g., electrostatic
or hydrophobic interaction) or covalent conjugations.[198–201]
For instance, Dai and co-workers reported SWCNT-based
nanocarriers, which could complex with protein through elec-
trostatic interaction. In this study, they uncovered that the
CNTs could transport proteins, such as streptavidin, protein
A (SpA), BSA, and CytC, into living cells by energy-dependent
endocytosis.[202,203] In addition, Boyer et al. recent developed
a noncovalent recombinantly engineered lamin B1 (LB1)-
based SWCNTs.[204] In this system, the LB1 contains a nuclear
localization sequence which could promote the functionalized
SWCNTs to localize in the nucleus. Moreover, the uptaken
process of SWCNTs in cells can be precisely determined and
directly visualized by using confocal Raman and fluorescence
lifetime imaging microscopes (Figure 13).
In order to stabilize protein, linkages are normally designed
for covalent immobilization of proteins onto CNTs. For example,
acid oxidized SWCNTs was incubated with 1-ethyl-3-(3-dimeth-
ylaminopropyl)carbodiimide (EDC) and biotin-LC-PEO-amine
to afford biotin functionalized SWCNTs.[205] The SWCNTs
could bind with the specific protein streptavidin and enter into
human promyelocytic leukemia cells and human T cells via the
endocytosis pathway. In addition, by a typical glutaraldehyde
crosslinking reaction between 1-aminopyrene-modified CNTs
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and proteins, the laccase could be immobilized on the CNTs and
showed high electrocatalytic activity.[206] Another important and
feasible route to enhance the proteins immobilization is the
modification of CNTs with polymers. Until now, many poly­
merization techniques (e.g., anionic polymerization and radical
polymerization) have been used to prepare polymer function-
alized CNTs, their physicochemical and biological properties
could be affected after polymer-functionalization.[198,207] The
most studies of polymer functionalization CNTs are focus on
biocatalysts, gas detector, and biosensor; however, few studies
are applied for protein delivery and disease therapy. Thus, this
research area will attract an increasing interest in future.
Nanodiamonds: Nanodiamonds are a type of carbon nano-
particles that possess a truncated octahedral architecture with
diameter of 2–10 nm. Beside the advantages of the superior
properties of bulk diamond, including high thermal conduc-
tivity, electrical resistivity, optical properties, chemical stability,
superior hardness and Young’s modulus, NDs also contain
more advantages than bulk diamond, such as larger surface
area, smaller size, better biocompatibility, and higher adsorp-
tion capacity.[208,209] In comparison with other carbon nanopar-
ticles, NDs showed less toxic to a variety of cell types and did
not produce significant reactive oxygen species.[210] Due to easy
surface modification of functional groups, NDs display various
medical application in drug delivery, tissue scaffolds, protein
mimics, and surgical implants.[208] For example, Lora Huang
and Chang designed carboxylation/oxidization NDs that coated
poly-l-lysine for covalent attachment of proteins.[211] The protein
CytC was successfully absorbed and immobilized on NDs. How-
ever, the biological activity was not investigated in this study.
Another study showed that hormone insulin could be nonco-
valently bound to detonated NDs and released by changing pH
value.[212] Studies showed that the adsorbed insulin remained
largely inactive but preserved function followed by desorp-
tion. Recently, Hu et al. prepared ND–protein complex for
oral delivery of proteins.[213] The complex could facilitate pro-
tein desorption in the cytosol by the functionalization of poly-
d-lysine (PDL) on the surface of NDs (Figure  14a). With this
system, RNase protein could be delivered into intestinal cells of
Drosophila, and consequently triggered regenerative divisions of
intestinal stem cells for tissue repair (Figure 14b,c).
© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Figure 14.  a) Adsorption of RFP to NDs or NDs-PDL in PBS and desorption of RFP from NDs or NDs-PDL in cell lysate. b) Viability assays of HeLa
cells after incubation with RNase, NDs-PDL, or NDs-PDL-RNase. c) CLSM imaging of oral delivery of RFP by NDs-PDL into Drosophila midgut. RFP:
Red fluorescent protein. Modified with permission.[213] Copyright 2017, American Chemical Society.

Other Carbon-Based Nanomaterials: Graphene oxide (GOx)
is derived from graphene upon the oxidative treatment. It
has been widely used as nanocarrier based on its promising
features, such as hydrophilic property, high surface area and
destabilization of lipid bilayer membranes. For example, Geest
and co-workers prepared OVA-modified GOx nanosheets and
demonstrated the nanosheets could be efficiently internalized
by dendritic cells.[214] In addition, Zhang and co-workers fabri-
cated the PEGylated GOx nanovector for protein delivery.[215] In
this study, RNase A, BSA and Kinase A could be loaded on the
nanovector via π–π stacking and other noncovalent interactions.
Cell-based experiments showed that the intracellular delivery
of RNase A kept activity and could lead to cell death. To pro-
tect Cas9/sgRNA against enzymatic cleavage, a new function-
alized GOx with PEG and PEI modification was fabricated by
Yue et al.[216] The results showed that GOx-based nanomaterials
provided an effective platform for Cas9/sgRNA delivery and
protection. In this system, the loaded Cas9/sgRNA adsorb on
the Gox-PEG-PEI via physical adsorption and π-stacking inter-
actions. The nanocarriers could deliver Cas9/sgRNA in human
cells through endocytosis and release based on the proton
sponge effect of PEI, resulting in efficient genome editing.
Carbon nanodots (C-dots) contain numbers of stacked gra-
phene layers with the size of diameter from 1 to 10 nm. Their
properties are similar as that of graphene and have been
designed for protein delivery. For instance, Liu and co-workers
discovered that the hydrophobic and van der Waals forces inter-
actions between two serum proteins (HSA and ϒ-globulins)
and C-dots by a series of photophysical measurements, which
can be used for proteins delivery.[217] Furthermore, Zhang et al.
mixed C-dots with EGFP to prepare C-dots/EGFP nanocomplex
with a size of around 200 nm. This nanosystem exhibited effec-
tively EGFP delivery into the cytoplasm of HeLa cells.[218]
Carbon nanospheres are another promising platform that
has been applied for delivering proteins. Scaffold matrix attach-
ment region binding protein 1 (SMAR1) have been found to
downregulate in advanced stages of human breast cancer.
Recently, Paknikar and co-workers prepared SMAR1 loaded
CNs to control breast tumor in mice model.[219] Interestingly,
this nanocomplex could internalize and localize in the nucleus
of MCF-7 cells. Furthermore, the delivered SMAR1 exhibited
apoptotic, antiproliferative and antiangiogenic activities and
significantly reduced the tumor volumes in metastatic breast
tumor bearing Balb/c mice.
Until now, various inorganic nanoparticle-mediated delivery
systems have been studied for intracellular delivery of protein.
These inorganic nanoparticles with unique physical/chemical
properties display great potential for development of a non-
toxic, biocompatible and nonimmunogenic delivery system. In
addition, unlike the lipid-based nanoparticles, these inorganic
nanoparticles are considered to be easier to scale up manu-
facturing. The stability of these inorganic nanoparticles ena-
bles them with long-term circulation in the body, however, in
another respect, makes them difficulty to be decomposed and
cleared from body, thus finally leading to the accumulation in
the liver, lung and kidney.[105] Therefore, development of the
biodegradable inorganic nanoparticle-based system is an effec-
tive way to solve this issue.
3.4. Cell-Penetrating Peptides
Besides the modification with nanoparticles/polymer, cell-pen-
etrating peptide itself could also be used for proteins delivery.
The first CPP, TAT peptide, has been discovered from the
human immunodeficiency virus (HIV) by Frankel and Pabo in

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1988, which arouse scientists intense interest as transporting
cargos for intracellular uptake.[220] During the past three dec-
ades, numerous CPPs have been developed and identified as
delivering the bioactive cargos in vitro and in vivo. Those CPPs
can be derived from proteins of microorganisms, plasmid dis-
play screenings, phage, antimicrobial peptides, heparin-, DNA-,
RNA-binding proteins and signal peptides.[221–223] For example,
Fu et al. proposed a brain-targeting peptide RDP derived from
the rabies virus glycoprotein, which is consisting of 39 amino
acids residues, for efficient target delivery of biological active
proteins, such as luciferase, β-Gal, brain derived neurotrophic
factor and glial cell-derived neurotrophic factor, into the cen-
tral nervous system.[224,225] The in vivo results showed that
the conjugate of RDP and proteins exhibited significant neu-
roprotective effect. Although CPPs can be used to effectively
deliver proteins into the host cells, however, due to the limita-
tion of instability in poor serum, CPPs can be trapped in the
endosomes and lysosomal degradation, induce cytotoxic at
high concentrations once transduced into cells, thus resulting
in poor achievement in their biological outcomes. To overcome
this issue, numerous strategies, such as introduction of the
endosomolytic agents used to disrupt endosomal membrane,
have been developed to improve the stability and transporting
efficiency.[226] For instance, Erazo-Oliveras et al. fabricated
tetramethylrhodamine-labeled dimer of the TAT and discovered
that this kind of TAT could deliver the EGFP into the cytosol
and nucleus in several cell lines.[227] Nischan and co-workers
synthesized another cyclic CPP to promote the GFP delivery
in the cytosol and nucleus.[228] Specifically, the chemoselec-
tive copper-catalyzed azide–alkyne cycloaddition was applied
to access a cyclic arginine rich TAT–GFP conjugate. In com-
paration with linear TAT–GFP, cyclic TAT–GFP could directly
enter into cells. Based on discovering the annexin family binds
to phosphatidylethanolamine, an annexin derived CPP that
riches in hydrophobic residues was screened and synthesized
from twelve isoforms of annexin.[223] Results showed that this
CPP could be served as a potential candidate for the successful
delivery of functional β-gal and Cre recombinase in vitro and in
vivo. Due to possess minimal toxicity to mammalian cells, anti-
microbial peptides (AMPs) have drawn an increasing attraction
to the scientists. For instance, Liu and co-workers screened and
discovered a 13-residue peptide, aurein 1.2 (GLFDIIKKIAESF),
which could also promote the endosomal escape of a variety of
cargos fused to +36 GFP.[229] Recently, a cationic, amphiphilic
peptide with a strong membrane lytic activity was selected as
template for the endosomolytic peptides by Akishiba et al.[230]
This endosomolytic peptides (L17E) were developed by intro-
ducing one or two negatively charged glutamic acid residues
into the hydrophobic face. In this system, the positive charges
of peptide were retained to facilitate the cell surface adsorption.
By the induction of micropinocytosis, this system could enable
the cytosolic release of endocytosed proteins (Cre recombi-
nase and IgG) in terms of physicochemical peptide-membrane
interactions. Furthermore, the L17E could also mediate cyto-
solic delivery of exosome-encapsulated proteins in living cells
(Figure 15a,b). The cell-based result showed that saporin-encap-
sulated exosomes in the presence of L17E led to significant cell
death. (Figure 15c).
The length of peptides is much shorter than antibodies,
and thus show a less expensive option for protein delivery. In
addition, peptides have the advantage of being nonimmuno-
genic in comparison to the antibodies. These features make
the peptides as promising delivery platform for a wide range
of protein-based therapeutics.[231] However, some limitations,

Figure 15.  a) Schematic illustration of L17E enhances the efficacy of exosome-mediated intracellular delivery. b) CLSM imaging of the ability of exog-
enous antibody to target cellular protein in the presence or absence of L17E. c) HeLa cells were treated with exosomes that contained saporin with or
without L17E. Reproduced with permission.[230] Copyright 2017, Springer Nature.

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such as the lack of cell selectivity of CPPs, are still needed to be
solved. For instance, it is essential to develop protein delivery
systems with the features of tumor targeting and these mem-
brane permeable CPPs to make the site-specific delivery and
intracellular trafficking. Such smart CPP-based delivery system
could be designed with either tumor-targeted peptides or com-
bined features in multicomponent delivery systems to enhance
the delivery/release capability of proteins. Additionally, further
development and research, particularly in vivo studies will be
essential for the novel peptide-based protein delivery systems.
3.5. Protein-Based Nanoparticles
Comparing with other nanocarriers, such as polymer, lipid,
and inorganic nanoparticles, protein-based nanoparticles have
more advantages including biocompatibility, biofunctionality,
molecular recognition in the biological environment, and abun-
dant renewable sources and biodegradability into small amino
acid. More importantly, the protein nanoparticles could be pre-
cise genetic engineered with functional sites of distinct loca-
tions for specific cells targeting and protein delivering.[232,233]
The extraordinary binding capacity of various drugs could be
achieved by different interactions between multifunctional
groups of polypeptides and therapeutic compounds.[232] Two
main approaches can be used to prepare the protein-
based nano­ particles, namely self-assembly and desolvation
methods.[232] Until now, there have been many kinds of proteins
extracted from animals or plants to be served as the substrates
to prepare the proteins nanoparticles, including collagen, gel-
atin, albumin, casein, elastin, whey proteins, soy proteins, silk
proteins, gliadin, and lectins.[234,235]
3.5.1. Protein Cages
Protein cages, such as ferritins, viral capsids, and heat shock
proteins, are attractive nanoscale cargo delivery vehicles which
have been used for protein delivery. The diameter of cages is
usually less than 30 nm which can exit the vasculature, per-
meate tissue, and enter the lymphatic system by passive dif-
fusion. In the protein cage-based NPs, most protein drugs
could be localized on the surface of cages via either covalent
bioconjugation or genetic fusion.[17] For example, Cao and co-
workers applied genetic method to generate human ferritin
H-chain protein (FTH1)-based NPs that fused with epidermal
growth factor receptor (EGF-FTH1).[236] Cell-based results
demonstrated that this EGF-FTH1 NPs could specifically bind
to breast cancer MCF-7 cells and MDA-MB-231 cells but not
normal breast epithelial MCF-10A cells. Kang and co-workers
genetically introduced the fragment crystallizable region (Fc)-
binding peptide (FcBP) into the loop of the ferritin cage. In this
cage, both human and rabbit IgGs could noncovalent bind with
FcBP-presenting ferritin to form antibody-based nanosystem
for breast cancer treatment (Figure  16a–d).[237] In addition,
the trastuzumab or rabbit antibody could be mixed with the
FcBP-representing ferritin cage without any chemical conjuga-
tion to form the complex. This complex could specific bind to

Figure 16.  a) Surface and ribbon diagram representation of ferritin from the position of the fourfold axis of symmetry and b) the subunits with the
inserted FcBP and extra glycine residues shown as connected spheres. c) Schematic illustration of the construction of a noncovalent antibody/fluo-
rescent labeled protein cage complex. d) TEM images of FcBP-ferritin. e) Fluorescent microscopic images of SKBR3 breast cancer cells treated with
Cy3-trastuzumab/FcBP-ferritin complexes. Modified with permission.[237] Copyright 2012, Elsevier Ltd.

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Figure 17.  Schematic illustration of the genetic and enzymatic manipulation of prostate cancer cell-penetrating M13 bacteriophage to generate nano-
carriers for the intracellular delivery of functional horseradish peroxidase to a human prostate cancer cell line. Adapted with permission.[239] Copyright
2014, American Chemical Society.
the SKBR3 breast cancer cell line or attach to the KB cell line,
which overexpresses target HER2 or folate receptor respectively
(Figure 16e).
3.5.2. Virus-Like Nanocarriers
Inspired by the development of commercial vaccines for human
papilloma and hepatitis B viruses, a broad multidisciplinary
approaches have been used to design the virus-like particles
(VLPs) for protein delivery.[238] Those VLPs can be morpholog-
ical diversity without the viral genome and derived from micro-
bial, mammalian, plant and insect viruses. The protein drugs
can be loaded on the surface or inside of VLPs and efficiently
internalized by cells.[238] M13 filamentous bacteriophage, which
consists of 5 coat proteins (p3, p6, p7, p8, and p9) is a well-
studied nanocarrier with a length of ≈930 nm and width of
≈6 nm. It has been used to deliver biotinylated HRP or GFP
intracellularly through genetic and enzymatic manipulation
(Figure 17).[239] The specific VLP was generated from the pros-
tate cancer CPP (Ypep 2) equipped on p3, which could selec-
tively penetrate into PC-3 cell lines. Studies showed that the
functional protein HRP delivered into the host cells could pre-
serve its biological activity. Recently, Chatterjee and co-workers
created Gag fusion based VLP using a truncated form of influ-
enza neuraminidase or hemagglutinin to fuse with murine
IFN-γ and human TNF-related apoptosis-inducing ligand.[240]
With this VLP, various proteins, including Cre recombinase,
GFP, cytotoxic enzymes Fcy::Fur or human Caspase 8 could be
delivered into the inside of cells.
3.5.3. Protein-Based Complex
Until now, numbers of proteins extracted from animals or
plants have been developed as the substrates to prepare the
protein-based nanoparticles, such as collagen, gelatin, albumin,
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whey proteins, casein, silk proteins, gliadin, elastin, soy pro-
teins, and lectins.[234] For instance, Chan and co-workers
discovered a novel platform for protein delivery based on
Cry3Aa protein crystals that from the bacterium Bacillus thur-
ingiensis.[241] The reporter proteins mCherry and GFP could
be efficiently delivered into mammalian cells and animals by
Cry3Aa fusion protein platform. Regarding the noncytotoxic
and customizable properties, the silk proteins from Bombyx
mori and recombinant spider silk proteins eADF4 are con-
sidered as promising candidates for proteins delivery.[242]
Herein, lysozyme was selected as a model protein and loaded
on eADF4 particles surface by electrostatic interactions. In
this system, lysozyme could be released as decreasing pH or
increasing ionic strength environment to weaken the electro-
static interactions. Among these protein-based nanocarriers,
the natural fibrous protein silk fibroin shows several advan-
tages of high loading capacity and controllable drug release
kinetics. However, the supernegative charge of fibroin parti-
cles limited their usage in the intracellular delivery. In order
to achieve cellular uptake and maintain proteins activities in
the cytoplasm, fibroin particles have been fabricated by coating
with cationic lipid layers, showing high stability and low tox-
icity in vitro assay. Furthermore, tyrosinase, luciferase, GFP,
and HRP could be effectively encapsulated into the particles
and led to hyper-pigmentation and tumor regression in mice,
suggesting the possibility of fibroin based particles for deliv-
ering proteins in clinical applications.[243] BSA is a nonim-
munogenic, nontoxic, and biodegradable protein material that
has been used as proteins carrier owning to their high binding
capacity of protein drugs. For example, Sun and co-workers
synthesized chitosan-stabilized BSA NPs with NEL-like mole­
cule- 1 protein (NELL-1) loading.[244] This NPs system showed
high entrapment efficiency up to 89.30% and maintained sus-
tained release during 8 days. Recently, a poly(oligo (ethylene
glycol) methyl ether methacrylate)-albumin conjugate was
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generated to encapsulate the anticancer proteins Sprouty 1.[245]
In contrast to BSA/protein complex, the PEGylated BSA/protein
complex micelle was more stable and had higher anticancer
efficacy by inhibiting the growth of 3D MCF-7 multicellular
tumor spheroids. Elastin is another protein material that has
been well studied for protein delivery. Actually, elastin and
elastin derivative-based NPs possess many unique properties,
such as biocompatibility, elasticity, dermal regeneration ability
and structure tunability in response to temperature change.
Elastin-like peptides (ELPs) are produced recombinantly from
synthetic genes and can be soluble at the inverse transition
temperatures (Tt), but self-assemble as coacervates when the
temperature above the Tt.[246] According to this property, ELPs
can be genetically fused to high keratinocyte growth factor
(KGF) and self-assembled into NPs at physiological tempera-
tures.[247] Subsequent results demonstrated that this KGF-ELPs
NPs could enhance granulation and re-epithelialization, as
well as improve would healing in diabetic mice. Recently, Cho
and co-workers designed PEGylated human α-elastin nano-
particles which could load significant amounts of insulin and
BSA, It is noted that sustained protein release for 72 h could
be achieved in this nanosystem.[248] GY(EIAALEK)3GC (E3)
and GY-(KIAALKE)3GC (K3) peptides which can be formed as
coiled-coil motifs have drawn remarkable attention for their
pH-triggered unfolding behaviors at late endosomal environ-
ment. For example, Zhong and co-workers fabricated E3/K3
coiled-coil peptide-crosslinked hyaluronic acid nanogels.[249]
Various antitumor proteins including saporin, CytC could be
encapsulated into the nanogel and released under mild acidic
conditions. Final results showed the antitumor proteins that
escaped from endosome could exhibit enhanced apoptotic
activity to MCF-7 cells. Due to the positive charge of some pro-
teins, they could bind to anionic cell surface proteoglycans and
exhibit cell-permeability. With those positively charged proteins
or peptides, the genetically fused protein drugs could possess
high levels of cytosolic delivery.[250,251] For instance, superposi-
tively charged GFP was selected and fused to mCherry or Cre
recombinase. With this system, significantly higher levels of
internalized protein and greater efficiencies of Cre-induced
recombination were achieved by comparing with transduc-
tion domains protein delivering system.[251] In fact, therapeutic
peptides are one of important classes of pharmaceutical, how-
ever, many peptides exhibit poor stability and oral bioavail-
ability, short half-life in the circulatory system and the lack of
tissue specificity. Those disadvantages would further limit their
clinical usage. Recent study showed that through embedding
the therapeutic peptides in the polypeptides backbone, the
therapeutic peptides/proteins could be delivered to the target
site without the above issues.[252] As shown in Figure  18a,b,
three independent polypeptides consisting of functional pep-
tides (Tat-NR2B9c: a neuroprotective peptide fused with CPP;
melittin (Mel): a cytolytic peptide for treatment of stroke and
cancer) self-assemble as activatable protein nanoparticles
(APNPs). The APNPs contain enzyme-responsive sequences
and tumor targeting peptide (RTIGPSV) that deliver the thera-
peutic peptides to targeted tissues and stimulated release active
peptides at proteases enriched microenvironment, enhancing
the antitumor effect of Mel (Figure 18c,d).

Figure 18.  a) Schematic illustration of polypeptides containing functional modular motifs and the formation of self-assembled NPs through pairwise
coiled-coil dimerization. b) TEM images of Melittin-NPs (scale bar: 20 nm). c) Cytotoxicity of Mel-APNPs with and without MMP-2 treatment and
free melittin peptide on MDA-MB-231 cells. d) Antitumor effect of Mel-APNPs evaluated in mice bearing MDA-MB-231 tumors. Adapted with permis-
sion.[252] Copyright 2018, Wiley-VCH.

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Figure 19.  Schematic illustration of development of an exosome-based drug delivery system. The exosomes can be modified with the targeting pep-
tide by the transfection method, then the exosomes are secreted by a various of cells which containing other functional cellular components such as
siRNA, microRNA, small molecules and proteins, the protein drugs could be encapsulated into the exosomes by various methods, then the functional
exosomes can be targeted on the specific receptors of cells and deliver the protein drugs into the cells.

Protein-based delivery platforms show exciting ways to sig-
nificantly improve protein delivery and therapeutic effects.
While the progress is still needed to develop the potential in
the improvement and mechanistic understanding. One of
the potential strategies is suggested to increase the efficacy of
protein-based nanoparticles through the combination of these
nanoparticles with other FDA-approved treatments. In addition,
future work will also indicate the feasibility of using protein-
based nanoparticles and their derivative in combination therapy
in the clinical field of tumor treatment in vivo.
3.6. Exosomes
Exosomes are lipoproteic nanosized vesicles which are secreted
from a variety of cells, particularly from dendritic cells, T cells,
B cells and macrophages. They can interact with cellular mem-
branes as abundance of adhesive proteins attach on their nat-
ural lipid bilayers. Exosomes play functional roles in removing
obsolete proteins. In addition, they also facilitate the transfer
of proteins, siRNA, mRNAs, microRNAs and other functional
cellular components in the cell or cell-to-cell communications.
Moreover, exosomes deliver their payload (small molecular
drugs, proteins, nucleic acid) to target cells as they could con-
tain various surface proteins and specific vector ligands such
as heat shock proteins, tetraspanins, lysosomal proteins and
fusion proteins. Until now, various approaches have been used
to develop exosomes based nanocarriers, such as chemical-
based transfection, electroporation, cell activation, transfec-
tion of exosome-producing cells, and incubation method.[253]
Figure 19 Schematic illustration of development of an exosome-
based drug delivery system.
The method of exosome-producing cells transfection is
generally very effective for loading of functional proteins into
exosomes. In this way, the transfection of donor cells is gen-
erated to overexpressed specific proteins on the surface mem-
brane of the exosome. After that, the engineered exosomes are
isolated from the donor cells and used for disease treatments.
Saydam and co-workers first developed genetic cells which
overexpressed the suicide therapeutic mRNA/protein for cyto-
sine deaminase (CDE) fused in-frame with uracil phosphoribo-
syltransferase (UPRT).[254] The CDE-UPRT can convert prodrug
5-fluorocytosine to 5-fluoro-deoxyuridine monophosphate
(5-FdUMP), which could cause cells to go under apoptosis.
Based on the conversion of CDE-UPRT to 5-fluorocytosine, the
isolated exosomes carried high amounts of CDE-UPRT mes-
sage and protein were injected into schwannomas and sub-
sequently resulted in regression of the tumor in combination
with prodrug of 5-fluorocytosine. Own to its wider tropism
and high effective membrane fusion without adverse effect,
vascular stomatitis virus protein (VSVG) is routine for viral
pseudotyping to enhance the target range and transduction
efficiency. By expressing gene-encoding VSVG fusion proteins
in the mother cells, the exosomes that encapsulated VSVG
were isolated, which could be developed as the protein delivery
nanosystem for enhancing the uptaken efficiency by recipient
cells.[255,256] Another two studies have shown that VSVG based
fusogenic exosomes could efficiently load proteins and mediate
the delivery of therapeutic membrane proteins. One example is
the generation of fusion proteins, VSVG that integrated GFP,

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YFP or luciferase in the exosomal membrane, which could
preserve their function in the targeted cells.[255] The other one
is, Kim and co-workers fabricated fusogenic exosomes by har-
boring VSVG and GFP-fused tetraspanin CD63 (CD63-GFP as
a reporter).[256] The membrane of fusogenic exosomes could
fuse with recipient cell membranes at acidic pH. In this system,
the model protein GLUT4 was incorporated into the exosomal
surface and finally retained their functionality after intracellular
delivery.
Catalase is one of the most efficient antioxidants that is
often used to treat PD. In order to effectively encapsulate the
catalase into exosomes, various approaches including soni-
cation, extrusion, freeze–thaw cycles, and permeabilization
with saponin have been developed. Studies showed that the
catalase encapsulated into exosomes could preserve enzymatic
activity. Particularly, the methods of sonication and extrusion
showed the highest catalytic activity.[257] Recently, Kabanov
and co-workers uncovered that naïve exosomes released from
macrophage (M-exosomes) could penetrate the blood-brain bar-
rier (BBB).[258] M-exosomes contained the integrin lymphocyte
function-associated antigen 1 (LFA-1) and intercellular adhe-
sion molecule 1 (ICAM-1), both of which play important role
in the uptake of M-exosomes in brain endothelial cells. Studies
showed that the inflammation processes could promote the
exosomes uptake in the BBB cells because of the overexpres-
sion of ICAM-1 receptors. The model protein brain derived
neurotrophic factor (BDNF) could bind with exosomes by the
electrostatic and polysaccharide interactions. High amount of
BDNF was successfully delivered to brain under inflammation,
indicating M-exosomes are promising nanocarriers for brain
delivery of therapeutic proteins.
Despite the numerous advantages of using exosomes as drug
carriers, several challenges are still remained. For instance, the
composition of exosomes and their functions are considered
to be the major challenge.[259] It is known that exosomes are
involved in cellular communication from the host to the recip-
ient cell. However, the transported biomolecules, their roles,
and the associated heterogeneity of the exosomes are unclear
exactly. Exosomes also suffer from the heterogeneity of the
biomolecules inside the aqueous lumen and on the surface.
In addition, the scale-up process to produce a large amount of
the vesicles is difficult to achieve. Further research is preferred
to require into the composition of exosomes and their delivery
efficacy in vivo.
4. Summary and Outlook
Various nanocarriers have been developed for intracellular pro-
teins delivery and shown great promise for enabling the cur-
rent and next generations of therapeutics. However, there still
remain many challenges that include the following points:
1) The choice of materials is used to be platform must be con-
sidered carefully. That is because those materials have been
screened in vitro and approved biocompatibility and nontox-
icity to the cells, may not accurately predict the in vivo toxic-
ity. Hence, it is necessary to investigate the in vivo results
exposure to nanomaterials before any potential therapeutic
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applications, especially for materials which are not biode-
gradable.
2) Many obstacles still need to be overcome during the prepa-
ration of proteins loaded nanocarriers, either covalent modi-
fications to protein surface or noncovalent strategies may
cause the protein structure changes and subsequent loss of
function, the development nanocarriers in organic solvents
or interaction between proteins and materials could also alter
the protein structures.
3) After entering the body, nanocarriers elimination via mecha-
nism or excretion processes should be considered serious-
ly as many of them are resistant to elimination routes, for
instance, due to the large size and higher chemical stability,
injected semiconductor quantum dots in mice remained
intact for more than 2 years in mouse tissues.[260]
4) There are still many nanocarriers entrapped into the en-
dosomes and difficult to escape into cytosol. Highly efficient
for the intracellular transduction is still a challenge for nano-
carriers.
5) Even though many proteins were successfully delivered into
cells and animal model, showing promising biological activ-
ity. However, most proteins are selected as model proteins
which may contain strong structure and keep stability in the
biological environment, whether all protein drugs showed
biological activities once delivered into targets need to further
confirm.
6) Small mouse model is frequently used to test the biological
activities of proteins loaded NPs, whether the positive results
could be reproduced in clinical trials that need to be further
verified. Thus, it is necessary to build up novel models that
represent the better human conditions.
7) Last but not least, every single person is truly unique with
clinical, genomic and environmental information, and this
uniqueness should be used to guide the disease manage-
ment, personalized nanomedicine contain multifunctional
abilities for early detection, combination of diagnostics with
therapeutics, and delivering proteins to the specific target,
however, due to the lack of sufficient patient’s information
and the communication among cells are changing all the
time, functionalized nanocarriers conjugate specific ligands
should be changed judiciously, which is still at the early stage
of development and meets various challenges.
To date, several strategies to develop protein-loaded nano-
carriers have been utilized, those nanocarriers are built with
various materials for enhancing drug encapsulation and
improving the protein delivery to the cytosol by endosomal
escape. Moreover, the symbiosis of different materials opens
up new opportunities for building novel multifunctional nano-
carriers, polymers within protein cages offer the possibility of
increasing the loading density of drug molecules and proteins.
Combination therapy provide the potential for enhanced effi-
cacy through synergies between therapeutics. Using nanocar-
riers for codelivery small molecules and proteins is regarding
as a promising therapeutic strategy which has been applied in
cancer therapy.[261] Moreover, to improve protein intracellular
delivery and active targeting, especially for the tumor cells, the
surface of nanocarriers could be incorporated with cell receptor
© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.advancedsciencenews.com
targeting ligands (e.g., folic acid and hyaluronic acid), cell pen-
etrating peptide, or antibody (e.g., Herceptin and transferrin)
to specifically bind with a receptor of the cancer cell mem-
brane. Clearly, many NP-based drug delivery systems have
been focused on targeted drug delivery, especially the surface
modification of targeted ligands for tumor-specific targeting
and enhancing the anticancer treatment. In recent years, orga-
nelle and subcellular drug targeting has gained increasingly
acceptance. For example, targeting the nuclear is a direct way to
interfere DNA transcription and damage the genetic informa-
tion. Thus, the subcellular targeting strategy could achieve max-
imum therapeutic responsive as well as avoiding cytotoxic side
effects of the drugs. However, the protein-loaded NPs for the
subcellular targeted and disease treatment are rarely reported.
The development of protein delivered NPs for targeting spe-
cific subcellular could be regarded as the new generation drug
delivery technology for the precise disease treatment.
The first nanotherapeutic Doxil (PEGylated liposomal Dox)
has been approved by FDA in 1995 and used to treat ovarian
cancer and multiple myeloma.[262] Abraxane, a paclitaxel loaded
albumin-bound nanoparticles, was available in the market in
2005 and achieved great commercial success for the treatment
of recurrent breast cancer.[263] Nowadays, at least 15 liposome
drugs or nanomedical based therapies have been approved by
FDA for commercial use. Global markets are projected to reach
177.6 billion $US by 2019.[264,265] Those successful applications
confirmed the high potential in the development of protein-
loaded nanomedicines. However, the clinical development of
protein loaded nanocarriers is still in their early stages. Most
protein drugs contain fragile tertiary structure that are sensitive
to the unfavored physicochemical conditions. The development
of multifunctional NPs to achieve the long-term stability of pro-
tein drugs for commercialization is a great challenge. Among
the numerous materials that applied for drugs delivery systems,
various types of VLPs could be manufacture at a large scale
by a large assortment of expression systems (bacterial, insect,
yeast, mammalian, and plant), and therapeutic protein could be
genetic produced with VLPs as a protein loaded nanocarriers
by expression systems. Moreover, some VLPs are exceptionally
stable biological materials that can withstand high temperature,
thus VLPs can be regarded as promising vehicles for potential
commercialization.
In conclusion, the potential for protein delivery with nano-
carriers in this field is tremendous, and more and more novel
material or delivery vehicles emerge and can be uncovered by
further scientific research.
Acknowledgements
X.Q. and C.Y. contributed equally to this work. This study was
financially supported by the National Natural Science Foundation
of China (81672508 and 61601218), Jiangsu Provincial Foundation
for Distinguished Young Scholars (BK20170041), Natural Science
Foundation of Shaanxi Province (2019JM-016), GSK-EDB Trust Fund
(R-143-000-688-592), the Synthetic Biology Research & Development
Programme (SBP) of National Research Foundation (SBP-P4 and SBP-P8)
of Singapore, China-Sweden Joint Mobility Project (51811530018),
and Fundamental Research Funds for the Central Universities and the
Natural Science Foundation of Guangdong Province (2017A030313299).
Adv. Mater. 2019, 31, 1902791 1902791  (28 of 32)
www.advmat.de
Conflict of Interest
The authors declare no conflict of interest.
Keywords
inorganic nanoparticles, lipids, polymeric nanoparticles, protein delivery
Received: May 2, 2019
Revised: July 4, 2019
Published online: September 8, 2019
[1] B. Leader, Q. J. Baca, D. E. Golan, Nat. Rev. Drug Discovery 2008,
7, 21.
[2] C. Ng, M. S. H. Guan, C. Koh, X. Ouyang, F. Yu, E. Tan,
S. P. O’Neill, X. Zhang, J. Chung, K. Lim, J. Neurosci. 2012, 32,
14311.
[3] L. Hang, J. Thundyil, K. Lim, Ann. N. Y. Acad. Sci. 2015, 1350, 37.
[4] F. Du, Q. Yu, S. Yan, G. Hu, L. Lue, D. G. Walker, L. Wu, S. F. Yan,
K. Tieu, S. S. Yan, Brain 2017, 140, 3233.
[5] M. Yu, J. Wu, J. Shi, O. C. Farokhzad, J. Controlled Release 2016,
240, 24.
[6] M. M. Pakulska, S. Miersch, M. S. Shoichet, Science 2016, 351,
aac4750.
[7] R. T. Dean, Nature 1975, 257, 414.
[8] W. Wang, Int. J. Pharm. 1999, 185, 129.
[9] M. C. Manning, K. Patel, R. T. Borchardt, Pharm. Res. 1989, 06,
903.
[10] M. C. Manning, D. K. Chou, B. M. Murphy, R. W. Payne,
D. S. Katayama, Pharm. Res. 2010, 27, 544.
[11] A. S. De Groot, W. Martin, Clin. Immunol. 2009, 131, 189.
[12] S. Frokjaer, D. E. Otzen, Nat. Rev. Drug Discovery 2005, 4, 298.
[13] A. Szlachcic, M. Zakrzewska, J. Otlewski, Biotechnol. Adv. 2011, 29,
436.
[14] T. Vermonden, R. Censi, W. E. Hennink, Chem. Rev. 2012, 112,
2853.
[15] C. D. Spicer, C. Jumeaux, B. Gupta, M. M. Stevens, Chem. Soc.
Rev. 2018, 47, 3574.
[16] Z. Gu, A. Biswas, M. Zhao, Y. Tang, Chem. Soc. Rev. 2011, 40,
3638.
[17] M. Rother, M. G. Nussbaumer, K. Renggli, N. Bruns, Chem. Soc.
Rev. 2016, 45, 6213.
[18] F. Scaletti, J. Hardie, Y.-W. Lee, D. C. Luther, M. Ray, V. M. Rotello,
Chem. Soc. Rev. 2018, 47, 3421.
[19] J. de A. Pachioni-Vasconcelos, A. M. Lopes, A. C. Apolinário,
J. K. Valenzuela-Oses, J. S. R. Costa, L. de O. Nascimento,
A. P. Jr., L. R. S. Barbosa, C. de O. Rangel-Yagui, Biomater. Sci. 2016,
4, 205.
[20] Y. Lu, W. Sun, Z. Gu, J. Controlled Release 2014, 194, 1.
[21] Y. Yun, Y. W. Cho, K. Park, Adv. Drug Delivery Rev. 2013, 65, 822.
[22] K. M. El-Say, H. S. El-Sawy, Int. J. Pharm. 2017, 528, 675.
[23] S. Gupta, A. Jain, M. Chakraborty, J. K. Sahni, J. Ali, S. Dang, Drug
Delivery. 2013, 20, 237.
[24] S. K. Samal, M. Dash, S. Van Vlierberghe, D. L. Kaplan, E. Chiellini,
C. van Blitterswijk, L. Moroni, P. Dubruel, Chem. Soc. Rev. 2012,
41, 7147.
[25] S. S. Silva, J. F. Mano, R. L. Reis, Green Chem. 2017, 19, 1208.
[26] H. Liu, C. Wang, C. Li, Y. Qin, Z. Wang, F. Yang, Z. Li, J. Wang,
RSC Adv. 2018, 8, 7533.
[27] G. Rassu, E. Soddu, M. Cossu, E. Gavini, P. Giunchedi, A. Dalpiaz,
J. Drug Delivery Sci. Technol. 2016, 32, 77.
[28] L. Hu, Y. Sun, Y. Wu, Nanoscale 2013, 5, 3103.
© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.advancedsciencenews.com
[29] P. Mukhopadhyay, R. Mishra, D. Rana, P. P. Kundu, Prog. Polym.
Sci. 2012, 37, 1457.
[30] B. prasanth Koppolu, S. G. Smith, S. Ravindranathan, S. Jayanthi,
T. K. Suresh Kumar, D. A. Zaharoff, Biomaterials 2014, 35, 4382.
[31] L. Li, L. Yang, M. Li, L. Zhang, Carbohydr. Polym. 2017, 174, 182.
[32] Y. Luo, Q. Wang, Int. J. Biol. Macromol. 2014, 64, 353.
[33] V. Bourganis, T. Karamanidou, O. Kammona, C. Kiparissides,
Eur. J. Pharm. Biopharm. 2017, 111, 44.
[34] Y. Hu, T. Yang, X. Hu, Polym. Bull. 2012, 68, 1183.
[35] M. B. Stie, H. S. Thoke, O. G. Issinger, J. Hochscherf, B. Guerra,
L. F. Olsen, Colloids Surf., B 2019, 174, 216.
[36] P. D. Hsu, E. S. Lander, F. Zhang, Cell 2014, 157, 1262.
[37] J. Qiao, W. Sun, S. Lin, R. Jin, L. Ma, Y. Liu, Chem. Commun. 2019,
55, 4707.
[38] M. Amidi, E. Mastrobattista, W. Jiskoot, W. E. Hennink, Adv. Drug
Delivery Rev. 2010, 62, 59.
[39] M.-C. Chen, F.-L. Mi, Z.-X. Liao, C.-W. Hsiao, K. Sonaje,
M.-F. Chung, L.-W. Hsu, H.-W. Sung, Adv. Drug Delivery Rev. 2013,
65, 865.
[40] C. Y. Wong, H. Al-Salami, C. R. Dass, J. Controlled Release 2017,
264, 247.
[41] Y. Yang, S. Wang, Y. Wang, X. Wang, Q. Wang, M. Chen, Biotechnol.
Adv. 2014, 32, 1301.
[42] L. Li, G. Jiang, W. Yu, D. Liu, H. Chen, Y. Liu, Z. Tong, X. Kong,
J. Yao, Mater. Sci. Eng., C 2017, 70, 278.
[43] A. M. M. Sadeghi, F. A. Dorkoosh, M. R. Avadi, M. Weinhold,
A. Bayat, F. Delie, R. Gurny, B. Larijani, M. Rafiee-Tehrani,
H. E. Junginger, Eur. J. Pharm. Biopharm. 2008, 70, 270.
[44] M. Liu, J. Zhang, X. Zhu, W. Shan, L. Li, J. Zhong, Z. Zhang,
Y. Huang, J. Controlled Release 2016, 222, 67.
[45] W. Fan, D. Xia, Q. Zhu, X. Li, S. He, C. Zhu, S. Guo, L. Hovgaard,
M. Yang, Y. Gan, Biomaterials 2018, 151, 13.
[46] D.-Y. Wu, Y. Ma, X.-S. Hou, W. Zhang, P. Wang, H. Chen, B. Li,
C. Zhang, Y. Ding, Carbohydr. Polym. 2017, 157, 1470.
[47] M. Allahyari, E. Mohit, Hum. Vaccines Immunother. 2016, 12, 806.
[48] F. Cui, K. Shi, L. Zhang, A. Tao, Y. Kawashima, J. Controlled Release
2006, 114, 242.
[49] J. U. Menon, P. Ravikumar, A. Pise, D. Gyawali, C. C. W. Hsia,
K. T. Nguyen, Acta Biomater. 2014, 10, 2643.
[50] X. Zhang, M. Sun, A. Zheng, D. Cao, Y. Bi, J. Sun, Eur. J. Pharm.
Sci. 2012, 45, 632.
[51] S. Dyawanapelly, U. Koli, V. Dharamdasani, R. Jain, P. Dandekar,
Drug Delivery Transl. Res. 2016, 6, 365.
[52] J. Sheng, L. Han, J. Qin, G. Ru, R. Li, L. Wu, D. Cui, P. Yang, Y. He,
J. Wang, ACS Appl. Mater. Interfaces 2015, 7, 15430.
[53] Z. Amoozgar, M. S. Goldberg, Nanomedicine 2017, 12, 729.
[54] B. Xu, G. Jiang, W. Yu, D. Liu, Y. Liu, X. Kong, J. Yao, Mater. Sci.
Eng., C 2017, 78, 420.
[55] P. Zhou, T. An, C. Zhao, Y. Li, R. Li, R. Yang, Y. Wang, X. Gao, Int. J.
Pharm. 2015, 478, 633.
[56] A. Jain, S. K. Jain, Acta Diabetol. 2015, 52, 663.
[57] R. Ganugula, M. Arora, M. Guada, P. Saini, M. N. V. R. Kumar,
ACS Macro Lett. 2017, 6, 161.
[58] R. Sharma, U. Gupta, N. K. Garg, R. K. Tyagi, N. K. Jain, Colloids
Surf., B 2015, 127, 172.
[59] S. Jain, V. V Rathi, A. K. Jain, M. Das, C. Godugu, Nanomedicine
2012, 7, 1311.
[60] P. Hurkat, A. Jain, A. Jain, S. Shilpi, A. Gulbake, S. K. Jain, J. Nano-
part. Res. 2012, 14, 1219.
[61] X. Zhu, J. Wu, W. Shan, W. Tao, L. Zhao, J. M. Lim, M. D’Ortenzio,
R. Karnik, Y. Huang, J. Shi, O. C. Farokhzad, Angew. Chem., Int. Ed.
2016, 55, 3309.
[62] X. Liu, C. Liu, W. Zhang, C. Xie, G. Wei, W. Lu, Int. J. Pharm. 2013,
448, 159.
[63] G. Coué, J. F. J. Engbersen, J. Controlled Release 2011, 152, 90.
Adv. Mater. 2019, 31, 1902791 1902791  (29 of 32)
www.advmat.de
[64] S. Cohen, G. Coué, D. Beno, R. Korenstein, J. F. J. Engbersen,
Biomaterials 2012, 33, 614.
[65] G. Coué, C. Freese, R. E. Unger, C. James Kirkpatrick,
J. F. J. Engbersen, Acta Biomater. 2013, 9, 6062.
[66] N. Chuard, G. Gasparini, A. Roux, N. Sakai, S. Matile, Org. Biomol.
Chem. 2015, 13, 64.
[67] G. Gasparini, E.-K. Bang, J. Montenegro, S. Matile, Chem.
Commun. 2015, 51, 10389.
[68] E.-K. Bang, G. Gasparini, G. Molinard, A. Roux, N. Sakai, S. Matile,
J. Am. Chem. Soc. 2013, 135, 2088.
[69] G. Gasparini, G. Sargsyan, E. K. Bang, N. Sakai, S. Matile, Angew.
Chem., Int. Ed. 2015, 54, 7328.
[70] G. Gasparini, E. K. Bang, G. Molinard, D. V. Tulumello, S. Ward,
S. O. Kelley, A. Roux, N. Sakai, S. Matile, J. Am. Chem. Soc. 2014,
136, 6069.
[71] J. Fu, C. Yu, L. Li, S. Q. Yao, J. Am. Chem. Soc. 2015, 137, 12153.
[72] L. Qian, J. Fu, P. Yuan, S. Du, W. Huang, L. Li, S. Q. Yao, Angew.
Chem. 2018, 130, 1548.
[73] V. Agrahari, V. Agrahari, Drug Discovery Today 2018, 23, 1139.
[74] J. Nicolas, S. Mura, D. Brambilla, N. Mackiewicz, P. Couvreur,
Chem. Soc. Rev. 2013, 42, 1147.
[75] Y. Lee, T. Ishii, H. Cabral, H. J. Kim, J. H. Seo, N. Nishiyama,
H. Oshima, K. Osada, K. Kataoka, Angew. Chem. 2009, 121, 5413.
[76] Y. Lee, T. Ishii, H. J. Kim, N. Nishiyama, Y. Hayakawa, K. Itaka,
K. Kataoka, Angew. Chem. 2010, 122, 2606.
[77] G. H. Gao, M. J. Park, Y. Li, G. H. Im, J. H. Kim, H. N. Kim,
J. W. Lee, P. Jeon, O. Y. Bang, J. H. Lee, D. S. Lee, Biomaterials
2012, 33, 9157.
[78] Q. Jin, T. Cai, Y. Wang, H. Wang, J. Ji, ACS Macro Lett. 2014, 3, 679.
[79] J. Ren, Y. Zhang, J. Zhang, H. Gao, G. Liu, R. Ma, Y. An, D. Kong,
L. Shi, Biomacromolecules 2013, 14, 3434.
[80] S. Li, Y. Zhang, J. Wang, Y. Zhao, T. Ji, X. Zhao, Y. Ding, X. Zhao,
R. Zhao, F. Li, X. Yang, S. Liu, Z. Liu, J. Lai, A. K. Whittaker,
G. J. Anderson, J. Wei, G. Nie, Nat. Biomed. Eng. 2017, 1, 667.
[81] S. F. M. Van Dongen, W. P. R. Verdurmen, R. J. R. W. Peters,
R. J. M. Nolte, R. Brock, J. C. M. Van Hest, Angew. Chem., Int. Ed.
2010, 49, 7213.
[82] R. Cheng, F. Meng, S. Ma, H. Xu, H. Liu, X. Jing, Z. Zhong,
J. Mater. Chem. 2011, 21, 19013.
[83] J. Zhang, L. Wu, F. Meng, Z. Wang, C. Deng, H. Liu, Z. Zhong,
Langmuir 2012, 28, 2056.
[84] X. Li, W. Yang, Y. Zou, F. Meng, C. Deng, Z. Zhong, J. Controlled
Release 2015, 220, 704.
[85] L. Lu, Y. Zou, W. Yang, F. Meng, C. Deng, R. Cheng, Z. Zhong,
Biomacromolecules 2015, 16, 1726.
[86] P. Yao, Y. Zhang, H. Meng, H. Sun, Z. Zhong, Biomacromolecules
2019, 20, 184.
[87] R. E. Whitmire, D. Scott Wilson, A. Singh, M. E. Levenston,
N. Murthy, A. J. García, Biomaterials 2012, 33, 7665.
[88] C. Silva, A. Carretero, D. Soares da Costa, R. L. Reis,
R. Novoa-Carballal, I. Pashkuleva, Acta Biomater. 2017, 63, 283.
[89] R. Rupaimoole, F. J. Slack, Nat. Rev. Drug Discovery 2017, 16, 203.
[90] Y. Chen, D.-Y. Gao, L. Huang, Adv. Drug Delivery Rev. 2015, 81, 128.
[91] Z. Niu, I. Conejos-Sánchez, B. T. Griffin, C. M. O’Driscoll,
M. J. Alonso, Adv. Drug Delivery Rev. 2016, 106, 337.
[92] A. D. Bangham, R. W. Horne, J. Mol. Biol. 1964, 8, 660.
[93] U. Bulbake, S. Doppalapudi, N. Kommineni, W. Khan, Pharmaceu-
tics 2017, 9, 12.
[94] T. Jiang, R. Mo, A. Bellotti, J. Zhou, Z. Gu, Adv. Funct. Mater. 2014,
24, 2295.
[95] L. Liguori, B. Marques, A. Villegas-Mendez, R. Rothe,
J.-L. Lenormand, J. Controlled Release 2008, 126, 217.
[96] S. Kube, N. Hersch, E. Naumovska, T. Gensch, J. Hendriks,
A. Franzen, L. Landvogt, J. P. Siebrasse, U. Kubitscheck,
B. Hoffmann, R. Merkel, A. Csiszár, Langmuir 2017, 33, 1051.
© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.advancedsciencenews.com
[97] M. Vila-Caballer, G. Codolo, F. Munari, A. Malfanti, M. Fassan,
M. Rugge, A. Balasso, M. de Bernard, S. Salmaso, J. Controlled
Release 2016, 238, 31.
[98] S. Ahmed, S. Fujita, K. Matsumura, Nanoscale 2016, 8, 15888.
[99] J. Kowapradit, A. Apirakaramwong, T. Ngawhirunpat,
T. Rojanarata, W. Sajomsang, P. Opanasopit, Eur. J. Pharm. Sci.
2012, 47, 359.
[100] X. Zhang, P. F. Luckham, A. D. Hughes, S. Thom, X. Y. Xu, Int. J.
Pharm. 2011, 421, 291.
[101] J. A. Zuris, D. B. Thompson, Y. Shu, J. P. Guilinger, J. L. Bessen,
J. H. Hu, M. L. Maeder, J. K. Joung, Z.-Y. Chen, D. R. Liu, Nat. Bio-
technol. 2015, 33, 73.
[102] M. Wang, J. A. Zuris, F. Meng, H. Rees, S. Sun, P. Deng, Y. Han,
X. Gao, D. Pouli, Q. Wu, I. Georgakoudi, D. R. Liu, Q. Xu, Proc.
Natl. Acad. Sci. USA 2016, 113, 2868.
[103] C. Botto, N. Mauro, E. Amore, E. Martorana, G. Giammona,
M. L. Bondì, Int. J. Pharm. 2017, 516, 334.
[104] S. A. Wissing, O. Kayser, R. H. Müller, Adv. Drug Delivery Rev. 2004,
56, 1257.
[105] Z. Bai, J. Wei, C. Yu, X. Han, X. Qin, C. Zhang, W. Liao, L. Li,
W. Huang, J. Mater. Chem. B 2019, 7, 1209.
[106] M. García-Fuentes, D. Torres, M. J. Alonso, Colloids Surf., B 2003,
27, 159.
[107] H. Yuan, S.-P. Jiang, Y.-Z. Du, J. Miao, X.-G. Zhang, F.-Q. Hu,
Colloids Surf., B 2009, 70, 248.
[108] R. Yang, R.-C. Gao, C.-F. Cai, H. Xu, F. Li, H.-B. He, X. Tang, Chem.
Pharm. Bull. 2010, 58, 1195.
[109] Z.-H. Zhang, Y.-L. Zhang, J.-P. Zhou, H.-X. Lv, Int. J. Nanomed.
2012, 7, 3333.
[110] T. Fan, C. Chen, H. Guo, J. Xu, J. Zhang, X. Zhu, Y. Yang,
Z. Zhou, L. Li, Y. Huang, Eur. J. Pharm. Biopharm. 2014, 88,
518.
[111] S. K. Kim, M. B. Foote, L. Huang, Biomaterials 2012, 33, 3959.
[112] W. Shan, X. Zhu, W. Tao, Y. Cui, M. Liu, L. Wu, L. Li, Y. Zheng,
Y. Huang, ACS Appl. Mater. Interfaces 2016, 8, 25444.
[113] Z. Li, J. C. Barnes, A. Bosoy, J. F. Stoddart, J. I. Zink, Chem. Soc.
Rev. 2012, 41, 2590.
[114] M. Yu, Z. Gu, T. Ottewell, C. Yu, J. Mater. Chem. B 2017, 5, 3241.
[115] X. Sun, Y. Zhao, V. S. Y. Lin, I. I. Slowing, B. G. Trewyn, J. Am.
Chem. Soc. 2011, 133, 18554.
[116] J. Gu, K. Huang, X. Zhu, Y. Li, J. Wei, W. Zhao, C. Liu, J. Shi, J. Col-
loid Interface Sci. 2013, 407, 236.
[117] H. Zhang, H. Xu, M. Wu, Y. Zhong, D. Wang, Z. Jiao, J. Mater.
Chem. B 2015, 3, 6480.
[118] F. Gao, L. Li, C. Fu, L. Nie, D. Chen, F. Tang, Adv. Mater. 2013, 25,
5508.
[119] Z. Tian, Y. Xu, Y. Zhu, Mater. Sci. Eng., C 2017, 71, 452.
[120] Y. Yang, J. Wan, Y. Niu, Z. Gu, J. Zhang, M. Yu, C. Yu, Chem. Mater.
2016, 28, 9008.
[121] C. Xu, M. Yu, O. Noonan, J. Zhang, H. Song, H. Zhang, C. Lei,
Y. Niu, X. Huang, Y. Yang, C. Yu, Small 2015, 11, 5949.
[122] A. K. Meka, P. L. Abbaraju, H. Song, C. Xu, J. Zhang, Small 2016,
12, 5169.
[123] Y. Yang, S. Bernardi, H. Song, J. Zhang, M. Yu, J. C. Reid,
E. Strounina, D. J. Searles, C. Yu, Chem. Mater. 2016, 28, 704.
[124] Y. Niu, M. Yu, J. Zhang, Y. Yang, C. Xu, M. Yeh, E. Taran,
J. J. C. Hou, P. P. Gray, C. Yu, J. Mater. Chem. B 2015, 3, 8477.
[125] J. S. Beck, J. C. Vartuli, W. J. Roth, M. E. Leonowicz, C. T. Kresge,
K. D. Schmitt, C. T. W. Chu, D. H. Olson, E. W. Sheppard,
S. B. McCullen, J. B. Higgins, J. L. Schlenker, J. Am. Chem. Soc.
1992, 114, 10834.
[126] F. Tang, L. Li, D. Chen, Adv. Mater. 2012, 24, 1504.
[127] I. I. Slowing, B. G. Trewyn, V. S. Y. Lin, J. Am. Chem. Soc. 2007, 129,
8845.
Adv. Mater. 2019, 31, 1902791 1902791  (30 of 32)
www.advmat.de
[128] Y. Yang, Y. Niu, J. Zhang, A. K. Meka, H. Zhang, C. Xu, C. X. C. Lin,
M. Yu, C. Yu, Small 2015, 11, 2743.
[129] Y. N. Yang, S. Karmakar, J. Zhang, M. H. Yu, N. Mitter, C. Z. Yu,
J. Mater. Chem. B 2014, 2, 4929.
[130] H. S. Park, C. W. Kim, H. J. Lee, J. H. Choi, S. G. Lee, Y. P. Yun,
I. C. Kwon, S. J. Lee, S. Y. Jeong, S. C. Lee, Nanotechnology 2010,
21, 225101.
[131] L. Sun, X. Zhang, Z. Wu, C. Zheng, C. Li, Polym. Chem. 2014, 5,
1999.
[132] H. Omar, J. G. Croissant, K. Alamoudi, S. Alsaiari, I. Alradwan,
M. A. Majrashi, D. H. Anjum, P. Martins, R. Laamarti, J. Eppinger,
B. Moosa, A. Almalik, N. M. Khashab, J. Controlled Release 2017,
259, 187.
[133] S. S. Bale, S. J. Kwon, D. a Shah, A. Banerjee, J. S. Dordick,
R. S. Kane, ACS Nano 2010, 4, 1493.
[134] D. A. Shah, S. J. Kwon, S. S. Bale, A. Banerjee, J. S. Dordick,
R. S. Kane, Biomaterials 2011, 32, 3210.
[135] Y. Li, X. Chen, H. Liu, X. Mou, Z. Ren, Z. Ahmad, X. Li, G. Han,
Chem. Eng. J. 2017, 330, 166.
[136] Y. Niu, M. Yu, S. B. Hartono, J. Yang, H. Xu, H. Zhang, J. Zhang,
J. Zou, A. Dexter, W. Gu, C. Yu, Adv. Mater. 2013, 25, 6233.
[137] Y. Niu, M. Yu, A. Meka, Y. Liu, J. Zhang, Y. Yang, C. Yu, J. Mater.
Chem. B 2016, 4, 212.
[138] Y. Zhao, B. G. Trewyn, I. I. Slowing, V. S. Lin, J. Am. Chem. Soc.
2009, 131, 8398.
[139] X. Wu, S. Wu, L. Yang, J. Han, S. Han, J. Mater. Chem. 2012, 22,
17121.
[140] X.-D. Wang, K. S. Rabe, I. Ahmed, C. M. Niemeyer, Adv. Mater.
2015, 27, 7945.
[141] P. Aggarwal, J. B. Hall, C. B. Mcleland, M. A. Dobrovolskaia,
S. E. Mcneil, Adv. Drug Delivery Rev. 2009, 61, 428.
[142] X. Du, S. Zhang Qiao, Small 2015, 11, 392.
[143] J. Zhang, S. Karmakar, M. Yu, N. Mitter, J. Zou, C. Yu, Small 2014,
10, 5068.
[144] E. A. Prasetyanto, A. Bertucci, D. Septiadi, R. Corradini,
P. Castro-Hartmann, L. De Cola, Angew. Chem., Int. Ed. 2016, 55,
3323.
[145] F. P. Chang, Y. Hung, J. H. Chang, C. H. Lin, C. Y. Mou, ACS Appl.
Mater. Interfaces 2014, 6, 6883.
[146] T.-W. Sun, Y.-J. Zhu, C. Qi, F. Chen, Y.-Y. Jiang, Y.-G. Zhang, J. Wu,
C. Wu, J. Mater. Chem. B 2016, 4, 3257.
[147] S. Cao, L. Fang, Z. Zhao, Y. Ge, S. Piletsky, A. P. F. Turner, Adv.
Funct. Mater. 2013, 23, 2162.
[148] P. Yuan, H. Zhang, L. Qian, X. Mao, S. Du, C. Yu, B. Peng,
S. Q. Yao, Angew. Chem., Int. Ed. 2017, 56, 12481.
[149] P. Yuan, X. Mao, X. Wu, S. S. Liew, L. Li, S. Q. Yao, Angew. Chem.,
Int. Ed. 2019, 58, 7657.
[150] D. A. Giljohann, D. S. Seferos, P. C. Patel, J. E. Millstone,
N. L. Rosi, C. A. Mirkin, Nano Lett. 2007, 7, 3818.
[151] P. Ghosh, X. Yang, R. Arvizo, Z.-J. Zhu, S. S. Agasti, Z. Mo,
V. M. Rotello, J. Am. Chem. Soc. 2010, 132, 2642.
[152] C. H. J. Choi, C. A. Alabi, P. Webster, M. E. Davis, Proc. Natl. Acad.
Sci. USA 2010, 107, 1235.
[153] P. Wang, L. Zhang, Y. Xie, N. Wang, R. Tang, W. Zheng, X. Jiang,
Adv. Sci. 2017, 4, 1700175.
[154] H. M. Joshi, D. R. Bhumkar, K. Joshi, V. Pokharkar, M. Sastry, Lang-
muir 2006, 22, 300.
[155] D. R. Bhumkar, H. M. Joshi, M. Sastry, V. B. Pokharkar, Pharm. Res.
2007, 24, 1415.
[156] S. Rana, A. Bajaj, R. Mout, V. M. Rotello, Adv. Drug Delivery Rev.
2012, 64, 200.
[157] S. M. Ryou, J. H. Yeom, H. J. Kang, M. Won, J. S. Kim, B. Lee,
M. J. Seong, N. C. Ha, J. Bae, K. Lee, J. Controlled Release 2014,
196, 287.
© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.advancedsciencenews.com
[158] J.-H. Yeom, M. Joo, B. Lee, K. P. Kim, N.-C. Ha, Y. Park, J. Bae,
K. Lee, J. Ind. Eng. Chem. 2017, 45, 5.
[159] D. P. Morales, G. B. Braun, A. Pallaoro, R. Chen, X. Huang,
J. A. Zasadzinski, N. O. Reich, Mol. Pharmaceutics 2015, 12, 600.
[160] R. Mout, M. Ray, T. Tay, K. Sasaki, G. Yesilbag Tonga, V. M. Rotello,
ACS Nano 2017, 11, 6416.
[161] C. S. Kim, R. Mout, Y. Zhao, Y. C. Yeh, R. Tang, Y. Jeong, B. Duncan,
J. A. Hardy, V. M. Rotello, Bioconjugate Chem. 2015, 26, 950.
[162] Y.-C. Yeh, R. Tang, R. Mout, Y. Jeong, V. M. Rotello, Angew. Chem.,
Int. Ed. 2014, 53, 5137.
[163] R. Tang, C. S. Kim, D. J. Solfiell, S. Rana, R. Mout,
E. M. Velázquez-Delgado, A. Chompoosor, Y. Jeong, B. Yan,
Z. J. Zhu, C. Kim, J. A. Hardy, V. M. Rotello, ACS Nano 2013, 7,
6667.
[164] M. Ray, R. Tang, Z. Jiang, V. M. Rotello, Bioconjugate Chem. 2015,
26, 1004.
[165] R. Tang, Z. Jiang, M. Ray, S. Hou, V. M. Rotello, Nanoscale 2016, 8,
18038.
[166] R. Mout, M. Ray, G. Yesilbag Tonga, Y. W. Lee, T. Tay, K. Sasaki,
V. M. Rotello, ACS Nano 2017, 11, 2452.
[167] K. Lee, M. Conboy, H. M. Park, F. Jiang, H. J. Kim, M. A. Dewitt,
V. A. Mackley, K. Chang, A. Rao, C. Skinner, T. Shobha,
M. Mehdipour, H. Liu, W. C. Huang, F. Lan, N. L. Bray, S. Li,
J. E. Corn, K. Kataoka, J. A. Doudna, I. Conboy, N. Murthy, Nat.
Biomed. Eng. 2017, 1, 889.
[168] J. Long, X. Yu, E. Xu, Z. Wu, X. Xu, Z. Jin, A. Jiao, Carbohydr. Polym.
2015, 131, 98.
[169] S. Laurent, D. Forge, M. Port, A. Roch, C. Robic, L. Vander Elst,
R. N. Muller, Chem. Rev. 2008, 108, 2064.
[170] J. Zhang, M. C. Shin, A. E. David, J. Zhou, K. Lee, H. He,
V. C. Yang, Mol. Pharmaceutics 2013, 10, 3892.
[171] H. Y. Yang, M. S. Jang, Y. Li, J. H. Lee, D. S. Lee, ACS Appl. Mater.
Interfaces 2017, 9, 19184.
[172] Y. T. Lim, M. Y. Cho, J. M. Lee, S. J. Chung, B. H. Chung, Biomate-
rials 2009, 30, 1197.
[173] B. F. Hoskins, R. Robson, J. Am. Chem. Soc. 1989, 111, 5962.
[174] H. Cai, Y. Huang, D. Li, Coord. Chem. Rev. 2019, 378, 207.
[175] C. Doonan, R. Ricco, K. Liang, D. Bradshaw, P. Falcaro, Acc. Chem.
Res. 2017, 50, 1423.
[176] M. Sindoro, N. Yanai, A.-Y. Jee, S. Granick, Acc. Chem. Res. 2014,
47, 459.
[177] M. Giménez-Marqués, T. Hidalgo, C. Serre, P. Horcajada, Coord.
Chem. Rev. 2016, 307, 342.
[178] P. Horcajada, R. Gref, T. Baati, P. K. Allan, G. Maurin, P. Couvreur,
G. Ferey, R. E. Morris, C. Serre, Chem. Rev. 2012, 112, 1232.
[179] V. Lykourinou, Y. Chen, X. Wang, L. Meng, T. Hoang, L. Ming,
R. L. Musselman, S. Ma, J. Am. Chem. Soc. 2011, 133,
10382.
[180] W. Liu, S. Lo, B. Singco, C. Yang, H. Huang, C. Lin, J. Mater. Chem.
B 2013, 1, 928.
[181] Y. Cao, Z. Wu, T. Wang, Y. Xiao, Q. Huo, Y. Liu, Dalton Trans. 2016,
45, 6998.
[182] C. He, K. Lu, D. Liu, W. Lin, J. Am. Chem. Soc. 2014, 136, 5181.
[183] E. Gkaniatsou, C. Sicard, R. Ricoux, J.-P. Mahy, N. Steunou,
C. Serre, Mater. Horiz. 2017, 4, 55.
[184] X. Lian, Y. Fang, E. Joseph, Q. Wang, J. Li, S. Banerjee, C. Lollar,
X. Wang, H. Zhou, Chem. Soc. Rev. 2017, 46, 3386.
[185] F. Lyu, Y. Zhang, R. N. Zare, J. Ge, Z. Liu, Nano Lett. 2014, 14,
5761.
[186] D. Feng, T. Liu, J. Su, M. Bosch, Z. Wei, W. Wan, D. Yuan, Y. Chen,
X. Wang, K. Wang, X. Lian, Z. Gu, J. Park, X. Zou, H. Zhou, Nat.
Commun. 2015, 6, 5979.
[187] F.-K. Shieh, S.-C. Wang, C.-I. Yen, C.-C. Wu, S. Dutta, L.-Y. Chou,
J. V. Morabito, P. Hu, M. H. Hsu, K. C.-W. Wu, C.-K. Tsung, J. Am.
Chem. Soc. 2015, 137, 4276.
Adv. Mater. 2019, 31, 1902791 1902791  (31 of 32)
www.advmat.de
[188] X. Wu, J. Ge, C. Yang, M. Hou, Z. Liu, Chem. Commun. 2015, 51,
13408.
[189] P. Li, S. Moon, M. A. Guelta, S. P. Harvey, J. T. Hupp, O. K. Farha,
J. Am. Chem. Soc. 2016, 138, 8052.
[190] Y. Zhang, F. Wang, E. Ju, Z. Liu, Z. Chen, J. Ren, X. Qu, Adv. Funct.
Mater. 2016, 26, 6454.
[191] X. Lian, A. Erazo-oliveras, J. Pellois, H. Zhou, Nat. Commun. 2017,
8, 2075.
[192] S. Wang, Y. Chen, S. Wang, P. Li, C. A. Mirkin, O. K. Farha, J. Am.
Chem. Soc. 2019, 141, 2215.
[193] W. Morris, W. E. Briley, E. Auyeung, M. D. Cabezas, C. A. Mirkin,
J. Am. Chem. Soc. 2014, 136, 7261.
[194] T. T. Chen, J. T. Yi, Y. Y. Zhao, X. Chu, J. Am. Chem. Soc. 2018, 140,
9912.
[195] X. Yang, Q. Tang, Y. Jiang, M. Zhang, M. Wang, L. Mao, J. Am.
Chem. Soc. 2019, 141, 3782.
[196] S. K. Alsaiari, S. Patil, M. Alyami, K. O. Alamoudi, F. A. Aleisa,
J. S. Merzaban, M. Li, N. M. Khashab, J. Am. Chem. Soc. 2018,
140, 143.
[197] M. Trojanowicz, TrAC, Trends Anal. Chem. 2006, 25, 480.
[198] N. Saifuddin, A. Z. Raziah, A. R. Junizah, J. Chem. 2013, 2013, 1.
[199] J. Liu, A. G. Rinzler, H. Dai, J. H. Hafner, R. Kelley Bradley,
P. J. Boul, A. Lu, T. Iverson, K. Shelimov, C. B. Huffman,
F. Rodriguez-Macias, Y. S. Shon, T. R. Lee, D. T. Colbert,
R. E. Smalley, Science 1998, 280, 1253.
[200] Z. Guo, P. J. Sadler, S. C. Tsang, Adv. Mater. 1998, 10, 701.
[201] B. R. Azamian, J. J. Davis, K. S. Coleman, C. B. Bagshaw,
M. L. H. Green, J. Am. Chem. Soc. 2002, 124, 12664.
[202] N. W. S. Kam, H. Dai, J. Am. Chem. Soc. 2005, 127, 6021.
[203] N. W. S. Kam, Z. Liu, H. Dai, Angew. Chem. 2006, 118, 591.
[204] P. D. Boyer, S. Ganesh, Z. Qin, B. D. Holt, M. J. Buehler,
M. F. Islam, K. N. Dahl, ACS Appl. Mater. Interfaces 2016, 8, 3524.
[205] N. W. S. Kam, T. C. Jessop, P. A. Wender, H. Dai, J. Am. Chem. Soc.
2004, 126, 6850.
[206] H. L. Pang, J. Liu, D. Hu, X. H. Zhang, J. H. Chen, Electrochim.
Acta 2010, 55, 6611.
[207] M. Adeli, R. Soleyman, Z. Beiranvand, F. Madani, Chem. Soc. Rev.
2013, 42, 5231.
[208] V. N. Mochalin, O. Shenderova, D. Ho, Y. Gogotsi, Nat. Nano-
technol. 2012, 7, 11.
[209] Y. Zhu, J. Li, W. Li, Y. Zhang, X. Yang, N. Chen, Y. Sun, Y. Zhao,
C. Fan, Q. Huang, Theranostics 2012, 2, 302.
[210] A. M. Schrand, H. Huang, C. Carlson, J. J. Schlager, E. Osawa,
S. M. Hussain, L. Dai, J. Phys. Chem. B 2007, 111, 2.
[211] L.-C. Lora Huang, H.-C. Chang, Langmuir 2004, 20, 5879.
[212] R. A. Shimkunas, E. Robinson, R. Lam, S. Lu, X. Xu, X. Q. Zhang,
H. Huang, E. Osawa, D. Ho, Biomaterials 2009, 30, 5720.
[213] X. Hu, X. Li, M. Yin, P. Li, P. Huang, L. Wang, Y. Jiang, H. Wang,
N. Chen, C. Fan, H. Song, ACS Appl. Mater. Interfaces 2017, 9,
18575.
[214] H. Li, K. Fierens, Z. Zhang, N. Vanparijs, M. J. Schuijs,
K. Van Steendam, N. Feiner Gracia, R. De Rycke, T. De Beer,
A. De Beuckelaer, S. De Koker, D. Deforce, L. Albertazzi,
J. Grooten, B. N. Lambrecht, B. G. De Geest, ACS Appl. Mater.
Interfaces 2016, 8, 1147.
[215] H. Shen, M. Liu, H. He, L. Zhang, J. Huang, Y. Chong, J. Dai,
Z. Zhang, ACS Appl. Mater. Interfaces 2012, 4, 6317.
[216] H. Yue, X. Zhou, M. Cheng, D. Xing, Nanoscale 2018, 10, 1063.
[217] Z.-Q. Xu, Q.-Q. Yang, J.-Y. Lan, J.-Q. Zhang, W. Peng, J.-C. Jin,
F.-L. Jiang, Y. Liu, J. Hazard. Mater. 2016, 301, 242.
[218] J. Zhang, M. Zheng, Z. Xie, J. Mater. Chem. B 2016, 4, 5659.
[219] P. N. Bhagat, S. H. Jadhav, S. Chattopadhyay, K. M. Paknikar,
Nanomedicine 2018, 13, 353.
[220] A. D. Frankel, C. O. Pabo, Cell 1988, 55, 1189.
[221] F. Heitz, M. C. Morris, G. Divita, Br. J. Pharmacol. 2009, 157, 195.
© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.advancedsciencenews.com
[222] C. Bechara, S. Sagan, FEBS Lett. 2013, 587, 1693.
[223] H. Y. Kim, S. Y. Yum, G. Jang, D.-R. Ahn, Sci. Rep. 2015, 5, 11719.
[224] A. Fu, Y. Wang, L. Zhan, R. Zhou, Pharm. Res. 2012, 29, 1562.
[225] A. Fu, Z. Zhao, F. Gao, M. Zhang, Pharm. Res. 2013, 30, 2108.
[226] A. Erazo-Oliveras, N. Muthukrishnan, R. Baker, T.-Y. Wang,
J.-P. Pellois, Pharmaceuticals 2012, 5, 1177.
[227] A. Erazo-Oliveras, K. Najjar, L. Dayani, T.-Y. Wang, G. A. Johnson,
J.-P. Pellois, Nat. Methods 2014, 11, 861.
[228] N. Nischan, H. D. Herce, F. Natale, N. Bohlke, N. Budisa,
M. C. Cardoso, C. P. R. Hackenberger, Angew. Chem., Int. Ed. 2015,
54, 1950.
[229] M. Li, Y. Tao, Y. Shu, J. R. LaRochelle, A. Steinauer, D. Thompson,
A. Schepartz, Z. Y. Chen, D. R. Liu, J. Am. Chem. Soc. 2015, 137,
14084.
[230] M. Akishiba, T. Takeuchi, Y. Kawaguchi, K. Sakamoto, H.-H. Yu,
I. Nakase, T. Takatani-Nakase, F. Madani, A. Gräslund, S. Futaki,
Nat. Chem. 2017, 9, 751.
[231] S. Dissanayake, W. A. Denny, S. Gamage, V. Sarojini, J. Controlled
Release 2017, 250, 62.
[232] L. P. Herrera Estrada, J. A. Champion, Biomater. Sci. 2015, 3, 787.
[233] N. M. Molino, S.-W. Wang, Curr. Opin. Biotechnol. 2014, 28, 75.
[234] A. O. Elzoghby, W. M. Samy, N. A. Elgindy, J. Controlled Release
2012, 161, 38.
[235] L. Yin, C. Yuvienco, J. K. Montclare, Biomaterials 2017, 134, 91.
[236] X. Li, L. Qiu, P. Zhu, X. Tao, T. Imanaka, J. Zhao, Y. Huang, Y. Tu,
X. Cao, Small 2012, 8, 2505.
[237] H. J. Kang, Y. J. Kang, Y.-M. Lee, H.-H. Shin, S. J. Chung, S. Kang,
Biomaterials 2012, 33, 5423.
[238] A. Zeltins, Mol. Biotechnol. 2013, 53, 92.
[239] S. M. DePorter, B. R. McNaughton, Bioconjugate Chem. 2014, 25,
1620.
[240] S. J. Kaczmarczyk, K. Sitaraman, H. a Young, S. H. Hughes,
D. K. Chatterjee, Proc. Natl. Acad. Sci. USA 2011, 108, 16998.
[241] M. S. Nair, M. M. Lee, A. Bonnegarde-Bernard, J. A. Wallace,
D. H. Dean, M. C. Ostrowski, R. W. Burry, P. N. Boyaka,
M. K. Chan, PLoS One 2015, 10, e0127669.
[242] M. Hofer, G. Winter, J. Myschik, Biomaterials 2012, 33, 1554.
[243] W.-J. Kim, B.-S. Kim, Y.-D. Cho, W.-J. Yoon, J.-H. Baek, K.-M. Woo,
H.-M. Ryoo, Biomaterials 2017, 122, 154.
[244] Y. Li, H. Song, S. Xiong, T. Tian, T. Liu, Y. Sun, Int. J. Biol. Mac-
romol. 2018, 109, 672.
[245] Y. Jiang, H. Lu, F. Chen, M. Callari, M. Pourgholami, D. L. Morris,
M. H. Stenzel, Biomacromolecules 2016, 17, 808.
Adv. Mater. 2019, 31, 1902791 1902791  (32 of 32)
www.advmat.de
[246] S. R. Macewan, A. Chilkoti, J. Controlled Release 2014, 190, 314.
[247] P. Koria, H. Yagi, Y. Kitagawa, Z. Megeed, Y. Nahmias, R. Sheridan,
M. L. Yarmush, Proc. Natl. Acad. Sci. USA 2011, 108, 1034.
[248] J. D. Kim, Y. J. Jung, C. H. Woo, Y. C. Choi, J. S. Choi, Y. W. Cho,
Colloids Surf., B 2017, 149, 122.
[249] L. Ding, Y. Jiang, J. Zhang, H. A. Klok, Z. Zhong, Biomacromol-
ecules 2018, 19, 555.
[250] T. Gaj, J. Liu, K. E. Anderson, S. J. Sirk, I. Carlos, F. Barbas, ACS
Chem. Biol. 2014, 9, 1662.
[251] J. J. Cronican, D. B. Thompson, K. T. Beier, B. R. McNaughton,
C. L. Cepko, D. R. Liu, ACS Chem. Biol. 2010, 5, 747.
[252] X. Yu, X. Gou, P. Wu, L. Han, D. Tian, F. Du, Z. Chen, F. Liu,
G. Deng, A. T. Chen, C. Ma, J. Liu, S. M. Hashmi, X. Guo, X. Wang,
H. Zhao, X. Liu, X. Zhu, K. Sheth, Q. Chen, L. Fan, J. Zhou, Adv.
Mater. 2018, 30, 1705383.
[253] K. B. Johnsen, J. M. Gudbergsson, M. N. Skov, L. Pilgaard,
T. Moos, M. Duroux, Biochim. Biophys. Acta 2014, 1846, 75.
[254] A. Mizrak, M. F. Bolukbasi, G. B. Ozdener, G. J. Brenner,
S. Madlener, E. P. Erkan, T. Ströbel, X. O. Breakefield, O. Saydam,
Mol. Ther. 2013, 21, 101.
[255] C. Meyer, J. Losacco, Z. Stickney, L. Li, G. Marriott, B. Lu, Int. J.
Nanomed. 2017, 12, 3153.
[256] Y. Yang, Y. Hong, G. H. Nam, J. H. Chung, E. Koh, I.-S. Kim, Adv.
Mater. 2017, 29, 1605604.
[257] M. J. Haney, N. L. Klyachko, Y. Zhao, R. Gupta, E. G. Plotnikova,
Z. He, T. Patel, A. Piroyan, M. Sokolsky, A. V. Kabanov,
E. V. Batrakova, J. Controlled Release 2015, 207, 18.
[258] D. Yuan, Y. Zhao, W. A. Banks, K. M. Bullock, M. Haney,
E. Batrakova, A. V Kabanov, Biomaterials 2017, 142, 1.
[259] J. E. Pullan, M. I. Confeld, J. K. Osborn, J. Kim, K. Sarkar, S. Mallik,
Mol. Pharmaceutics 2019, 16, 1789.
[260] H. Soo Choi, W. Liu, P. Misra, E. Tanaka, J. P. Zimmer, B. Itty
Ipe, M. G. Bawendi, J. V. Frangioni, Nat. Biotechnol. 2007, 25,
1165.
[261] C. He, Z. Tang, H. Tian, X. Chen, Adv. Drug Delivery Rev. 2016, 98,
64.
[262] Y. Barenholz, J. Controlled Release 2012, 160, 117.
[263] E. Miele, G. P. Spinelli, E. Miele, F. Tomao, S. Tomao, Int. J.
Nanomed. 2009, 4, 99.
[264] H. Zhong, G. Chan, Y. Hu, H. Hu, D. Ouyang, Pharmaceutics 2018,
10, 263.
[265] R. M. Kannan, E. Nance, S. Kannan, D. A. Tomalia, J. Intern. Med.
2014, 276, 579.
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