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COREL-07976; No of Pages 18

Journal of Controlled Release xxx (2015) xxx–xxx

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Journal of Controlled Release

journal homepage: www.elsevier.com/locate/jconrel

Nanogels: An overview of properties, biomedical applications and


obstacles to clinical translation
Kruti S. Soni, Swapnil S. Desale, Tatiana K. Bronich ⁎
Department of Pharmaceutical Sciences and Center for Drug Delivery and Nanomedicine, College of Pharmacy, University of Nebraska Medical Center, 985830 Nebraska Medical Center, Omaha,
NE 68198-5830, USA

a r t i c l e i n f o a b s t r a c t

Article history: Nanogels have emerged as a versatile hydrophilic platform for encapsulation of guest molecules with a capability
Received 31 August 2015 to respond to external stimuli that can be used for a multitude of applications. These are soft materials capable of
Received in revised form 1 November 2015 holding small molecular therapeutics, biomacromolecules, and inorganic nanoparticles within their crosslinked
Accepted 9 November 2015
networks, which allows them to find applications for therapy as well as imaging of a variety of disease conditions.
Available online xxxx
Their stimuli-responsive behavior can be easily controlled by selection of constituent polymer and crosslinker
Keywords:
components to achieve a desired response at the site of action, which imparts nanogels the ability to participate
Nanogels actively in the intended function of the carrier system rather than being passive carriers of their cargo. These
Therapeutic carrier properties not only enhance the functionality of the carrier system but also help in overcoming many of the
Drug delivery challenges associated with the delivery of cargo molecules, and this review aims to highlight the distinct and
Imaging unique capabilities of nanogels as carrier systems for the delivery of an array of cargo molecules over other
Clinical nanomaterials. Despite their obvious usefulness, nanogels are still not a commonplace occurrence in clinical prac-
Translation tice. We have also made an attempt to highlight some of the major challenges that need to be overcome to ad-
vance nanogels further in the field of biomedical applications.
© 2015 Elsevier B.V. All rights reserved.

1. Introduction for incorporation of a plethora of guest molecules ranging from inorgan-


ic nanoparticles to biomacromolecules like proteins and DNA with suit-
Nanogels are three-dimensional hydrogel materials in the nanoscale able modifications of the materials used for their construction, without
size range formed by crosslinked swellable polymer networks with a compromising their gel-like behavior [10–16]. This multifunctionality
high capacity to hold water, without actually dissolving into the and stability is hard to find in other types of nanoparticulate systems
aqueous medium. Nanogels can be composed of a variety of naturally [17,18]; especially the ability to incorporate entities with very different
occurring polymers, synthetic polymers or a combination thereof. physical properties within the same carrier. Inorganic nanomaterials
Their characteristics such as size, charge, porosity, amphiphilicity, soft- have distinct material properties like optical activity, electrical conduc-
ness, and degradability can be fine-tuned by varying the chemical com- tivity and magnetic properties that make them suitable for in vivo diag-
position of the nanogels. They are mostly spherical particles but the nostic and imaging applications, but they suffer from limitations of poor
current advancement in synthetic strategies allow for the fabrication colloidal stability, low aqueous solubility and rapid elimination by the
of nanogels of different shapes [1,2]. They can be also designed to mononuclear phagocytic system (MPS). Polymeric nanogels can be
have either a core–shell or a core–shell–corona structure, with at least used as carriers for such imaging probes by imparting stability and in-
one of the layers crosslinked for structural integrity. Being mostly hy- creasing their utility. This leads to the evolution of a new class of agents
drophilic in nature, nanogels are highly biocompatible with a high load- termed ‘nanohybrids’ which are nanogels incorporating inorganic ma-
ing capacity for guest molecules and their unique physical properties terials [19,20]. Such nanohybrids can contain a wide variety of diagnos-
offer them distinct advantages over other types of nanomaterials for tic and imaging agents for different types of medical conditions.
biomedical applications. Nanogels not only protect the cargo from deg- Nanogels prevent biomolecules like enzymes and genetic material
radation and elimination but also participate actively in the delivery from degradation while their own macromolecular properties help in-
process due to their characteristic properties like stimuli-responsive be- crease the circulation half-lives of small molecules, and serve as a highly
havior, softness and swelling to help achieve a controlled, triggered re- convenient platform for combination delivery of therapeutic molecules.
sponse at the target site [3–9]. The versatility of their architecture allows They can be targeted specifically to the site of interest by conjugation
with a targeting ligand or due to the passive targeting that is a charac-
⁎ Corresponding author. teristic feature of their nanoscale size. Despite such diversity in their
E-mail address: tbronich@unmc.edu (T.K. Bronich). applications, nanogels are not yet a part of clinical use. Many

http://dx.doi.org/10.1016/j.jconrel.2015.11.009
0168-3659/© 2015 Elsevier B.V. All rights reserved.

Please cite this article as: K.S. Soni, et al., Nanogels: An overview of properties, biomedical applications and obstacles to clinical translation, J. Con-
trol. Release (2015), http://dx.doi.org/10.1016/j.jconrel.2015.11.009
2 K.S. Soni et al. / Journal of Controlled Release xxx (2015) xxx–xxx

comprehensive and more specialized review articles on synthesis and structural change in the nanogel, which is mediated by various factors in-
application of nanogels were recently published. For that reason, in cluding, but not limited to, transition in the temperature below or above
the present paper we attempted to briefly consider characteristic fea- its lower critical solution temperature (LCST) or the ionization of acidic
tures of nanogels and to demonstrate representative examples for or basic functional groups on the polymer chains. These changes also
major directions in their applications in the biomedical field. We also alter the hydrophilicity and/or hydrophobicity of the nanogels or, in
highlight some of the key hurdles that need to be overcome to make other words, the extent of interaction of the system with water molecules
nanogels a part of routine clinical practice. [34–36] and is mostly manifested in the form of swelling or deswelling of
the nanogel network, which in turn causes responses like release of the
2. Nanogels: characteristic features entrapped cargo [37,38]. One of the considerable advantages of nanogels
over the macroscopic gels is their very rapid response to a change in en-
2.1. Synthesis of nanogels vironmental conditions [39]. The responsiveness of nanogels to the exter-
nal physical or chemical signals can be tightly regulated by controlling the
Nanogels can be synthesized by a number of techniques. Since an in- structure of the materials used for preparation of the nanogels [3,7]. For
depth discussion of all the available techniques is beyond the scope of example, in the case of polyelectrolyte nanogels pH-dependent ionization
this review, a brief overview of the techniques is given, along with of functional groups results in an increase of osmotic pressure inside the
reference to more detailed sources. Traditionally, nanogels have been nanogel due to entrapped counterions and ultimately results in the swell-
classified based on the method of crosslinking as either physically or ing of the nanogels [7,40,41]. It is well recognized that a balance between
chemically (covalently) crosslinked nanogels. Chemical crosslinking in- the osmotic pressure and the polymer elasticity sets the physical dimen-
volves formation of covalent bonds between the polymer chains during sions of a hydrogel particle [42]. Thus, the extent of swelling also depends
polymerization of low molecular weight monomers or crosslinking of on structural characteristics of the nanogel like the chemical composition,
polymer precursors. The most extensively employed methods for hydrophilicity of crosslinkers, and the degree of crosslinking of the
preparing chemically crosslinked nanogels utilize heterogeneous poly- nanogel network, which controls the freedom of conformational mobility
merization reactions in the presence of either bifunctional or multifunc- of the polymer chains [43,44]. Tan et al. studied pH-responsive
tional crosslinkers [21,22]. Conventional and controlled/living radical polyampholyte nanogels with different fractions of methacrylic acid
polymerization techniques allow for preparation of nanogels with dif- (MAA) and 2-diethylaminoethyl methacrylate (DEAM) units in the
ferent composition, dimensions, and architectures including core–shell crosslinked polymer cores that were sterically stabilized by poly(ethylene
and hollow nanogel particles [23,24]. The use of functional initiators glycol), PEG, shell [45]. The MAA segment in this nanogel is a weak acid
and macroinitiators further allows the incorporation of functionalities and DEAM is a weak base (pKa = 5.4 for MAA and pKa = 7.3 for
in the interior or on the surface of nanogels, which facilitate multivalent DEAM). The DEAM segments could be protonated at low pH, imparting
bioconjugation [25]. A variety of other crosslinking approaches includ- a positive charge to this segment, whereas the MAA units are negatively
ing click chemistry, Schiff-base reactions, thiol-disulfide exchange, charged at high pH. As a result, these nanogels exhibited marked swelling
amide crosslinking, photo-induced crosslinking, enzyme-mediated at both high and low pH values, but shrunk in the vicinity of isoelectric
crosslinking etc., have been developed for the synthesis of nanogels point due to overall charge neutralization. It was demonstrated that
from the polymer precursors. In addition, the crosslinking reactions car- swelling–deswelling transition can be tuned by varying the composition
ried out on preformed core–shell self-assemblies such as polymer mi- of MAA and DEAM in the nanogels. The unique charge-switching proper-
celles allow introducing a high degree of spatial organization into the ties of amphoteric nanogels based on poly(N-isopropylacrylamide) func-
nanogels [26,27]. For a comprehensive look into crosslinking strategies, tionalized with aminophenylboronic acid (PBA) were explored by Hoare
the reader is directed to excellent review written by Haag and col- and Pelton to regulate the release of preloaded insulin in glucose-
leagues [28]. Recent advances in nanoscale fabrication methods provid- dependent manner [46]. The binding of glucose to the PBA residues shifts
ed further unique opportunities to fabricate well-defined nanogels with the boronic acid ionization equilibrium, increases the anionic charge den-
precise control over size, shape, deformability and surface chemistry in sity on the gel and drives a gel swelling response. These nanogels were
a high-throughput manner [29–31]. Physically crosslinked systems designed to both swell and deswell in response to glucose according to
though formed under the mild conditions, tend to be more fragile the pH of the medium, the concentration of PBA groups grafted to the
than their covalently crosslinked counterparts since they are stabilized nanogel, and the relative concentrations of the cationic and anionic
by relatively weak interactions between polymer chains such as hydro- functional groups in the platform nanogel. Such PBA-nanogels had a
gen bonding, hydrophobic interactions or ionic interactions. The use of high capacity for insulin uptake and selectively released insulin under
hydrophobically-modified polymers and other associating polymers physiological conditions in an “on–off” manner with fluctuating glucose
for preparing functional nanogels has recently been reviewed by Sasaki level. Similar approach was adapted by Wu et al. for the design of a
and Akiyoshi [32]. One of the challenges in the formation of nanogels by nanogel-based glucose sensor, which comprised of a core of Ag nanopar-
such polymers is a control over the particle size, which requires fine- ticle encapsulated within a glucose-recognizing crosslinked shell contain-
tuning of the polymer concentrations or environmental parameters ing PBA residues. The swelling of the gel in response to glucose binding
such as temperature, pH, and ionic strength. A study by Nielsen and causes a change in the refractive index of the medium around Ag nano-
co-workers has demonstrated that these challenges can be addressed particle that leads to change in its fluorescence properties. The response
by utilizing a microfluidics-based approach [33]. Overall, the advances rate is reported to be on the order of 100 ns, and the detection sensitivity
in polymer chemistry led to the exceptional diversity and control over for change in glucose concentration was reported to be ±0.1 mM
the composition, architecture and functionality of crosslinked nanogels, [47].Thus, both sensitivity and rate of response of the nanogels can be pro-
which in turn provide more flexibility to tune their properties to comply grammed and fine-tuned by alterations in the structure of the building
with targeted biomedical applications. blocks [3,7]. Accordingly, the cargo (mostly drugs) can either be conjugat-
ed chemically to the nanogels or merely entrapped physically into its core
2.2. Stimuli-responsive behavior [15], depending on the stimulus that is most feasible for utilization at the
targeted site of disease.
Stimuli-responsive behavior of nanogels is a sequence of events initi- Often multifunctional nanogels, which respond to a combination of
ated by an external cue that comes either from the specific environment more than one external signal, can also be fabricated for more site-
within the body like change in pH, temperature, redox conditions or en- specific response [48–51]. These stimuli are frequently disease- or
zyme concentration, or a stimulus that can be applied externally such as organ-specific, since different pathological conditions are associated
light, magnetic field, etc. This stimulus then causes a conformational or with changes in pH, temperature and redox balance or expression levels

Please cite this article as: K.S. Soni, et al., Nanogels: An overview of properties, biomedical applications and obstacles to clinical translation, J. Con-
trol. Release (2015), http://dx.doi.org/10.1016/j.jconrel.2015.11.009
K.S. Soni et al. / Journal of Controlled Release xxx (2015) xxx–xxx 3

of certain biomolecules as compared to the normal physiological condi- molecular weight and surface density of the PEG used [61,62]. While
tions [52]. The reversible crosslinks containing disulfide bonds that are PEGylation endows nanosystems with long circulation properties and
stable in extracellular milieu but are cleaved in the reductive intracellu- reduces MPS uptake, eventually opsonization and macrophage clear-
lar environments due to the differences in the reductive potential ance still occurs [63]. A number of studies have demonstrated that
between the extracellular and intracellular compartments, are success- PEGylation of nanoparticles tends to shift their accumulation towards
fully utilized in design of redox-responsive nanogels [12,49,53,54]. The the spleen instead of the liver as compared to their non-PEGylated
nanogels with disulfide links are stable during circulation in the blood counterparts [64]. A unique feature that helps nanogels partially escape
but labile while internalized in cells, thus, facilitating the release of the splenic filtration process is their softness and deformability. This is can
drug. Matyjaszewski et al. have prepared biodegradable nanogels with be explained by an example in nature, namely erythrocytes, which in
disulfide-functionalized dimethacrylate crosslinkers that degraded spite of having a size range in microns are easily able to pass through
into individual polymeric chains in the presence of glutathione and en- the splenic filtration bed that has a pore size of few hundred nanome-
abled both the release of encapsulated cargos and the removal of the ters, due to their flexibility and deformability [62,65]. In fact, old RBCs
carriers [55]. Thayumanavan and coworkers reported the preparation are cleared from circulation mainly because they lose their flexibility.
of redox-sensitive nanogels based on random amphiphilic copolymer This biomimetic property of the nanogels can be highly advantageous
that contains hydrophilic oligoethyleneglycol (OEG) and hydrophobic for their in vivo application. For example, Lyon and colleagues have re-
pyridyldisulfide (PDS) units as side-chain functionalities [56]. Self- cently reported that soft spherical acrylamide-based nanogels deform
assembled nanostructures formed by this polymer were crosslinked and pass through membrane pores several times smaller than their
by initiating a thiol-disulfide exchange reaction among the PDS groups hydrodynamic diameter under physiological pressures [66]. Also, vary-
using dithiothreitol (DTT). The size of the resulting nanogels can be ing the moduli of similar nanogels has been shown to affect their
tuned by varying the copolymer molecular weight, composition and mechanism and rate of uptake in macrophages [67]. Merkel et al. re-
concentration, while the extent of crosslinking can be controlled by ported that decreasing the modulus of microgel particles altered their
the amount of added DTT. The release of the encapsulated doxorubicin biodistribution properties, allowing them to bypass several organs,
(DOX) was triggered by glutathione treatment and the release rate such as the lung, that entrapped their more rigid counterparts, resulting
can be tuned by adjusting the crosslinking density. in increasingly longer circulation times [68]. Convincing evidence for
The group of Wang took advantage of the activity of bacterial lipases, the prolonged circulation time of soft PEG-based hydrogel nanoparticles
which are abundant in microbial flora, to construct a nanogel for the on- compared to hard nanogels of the same size was recently provided by
demand release of antibiotics [52]. In this approach, the triple-layered Mitragori and coworkers [69]. The deformability of the nanogels can
nanogel contains a hydrophobic lipase-sensitive poly(ε-caprolactone) be modulated by varying the crosslink density within the particle ma-
(PCL) interlayer between the crosslinked polyphosphoester core and trix as well as by varying the size of the crosslinking moiety [70]. Incor-
the PEG shell. Prior to reaching sites of bacterial infection, the antibiotics poration of electrolyte moieties into the polymer network of hydrogel
are protected inside the polyphosphoester core and are not released due particles to increase the swelling ratio is another straightforward and
to the compacted PCL molecular fence. However, rapid drug release was quite efficient way to decrease the modulus [2]. However, the distribu-
observed in the presence of lipase or lipase-secreting bacteria. tion of charged groups on the surface of a particle can accelerate the
In another scenario, the changes in the properties of nanogels can be clearance of particle. To address this drawback, DeSimone's group has
caused by external signals in the form of radiation or magnetic field de- recently developed a strategy to generate highly-swollen polyelectro-
pending on the accessibility of the targeted site in the body [57,58]. An lyte gel particle with near-neutral charge while retaining charged
elegant design of near-infrared (NIR) light-responsive core–shell group in the interior [71]. Fraction of nanogels that escapes clearance
nanogels was recently reported by Kang et al. [59]. Au–Ag-based nano- by the mechanisms discussed above is then distributed into various or-
rods were used as templates for the synthesis of double-stranded oligo- gans by the circulating blood. Nanogels are usually too big to pass
nucleotide crosslinked polyacrylamide shell. When exposed to NIR- through the tight junctions of the normal endothelium but can efficient-
radiation, the heating generated by Au–Ag rods through photothermal ly accumulate in solid tumors or inflamed tissues that have unique
conversion induced efficient thermal dehybridization of the linker oli- structural features such as defective leaky and loosely compacted
gonucleotides from their complementary sequences and led to a rapid vasculature and impaired lymphatic drainage leading to the well-
gel-to-sol transition of the gel shell and effective payload release. characterized enhanced permeability and retention effect (EPR) [72,
73]. In addition to EPR, bioconjugation of nanogels to targeting ligands
2.3. In vivo behavior allows directing them to specific receptors or molecules differentially
overexpressed on the diseased cells/tissues thus improving their reten-
Nanogels are macromolecular systems specifically designed to tion at the targeted site as well as facilitating their cellular uptake [74,
achieve long circulation half-lives of their cargo in vivo, along with 75]. Various small molecules, peptides, aptamers, antibodies or anti-
their ability to deliver this cargo at the desired site (Fig. 1). To realize body fragments have been explored for targeted delivery of nanogels
this, a nanogel, or any nanoparticulate system has to overcome many and other nanomedicines in tissue- or cell-specific manner [76].
barriers, especially when administered via routes other than intrave- Ligand-mediated targeting influences the overall biodistribution profile
nous, like oral, intradermal, pulmonary, intraocular, etc. Depending on of the nanogels as compared to their nontargeted counterparts and the
the route of administration, nanogels are designed specifically to over- bias of distribution is towards to those tissues that have a high expres-
come associated barriers and reach the circulation intact. Nanogels sion of the receptor. This can help avoid excess accumulation of the
prolong circulation half-life of their cargo by 1) preventing their fast nanogels at off target sites and reduce the associated side effects.
clearance especially in the case of small molecules and 2) prevent After extravasation from the blood compartment, nanogels have to
quick degradation or metabolism which is more relevant for biomole- diffuse through the tissue matrix in the interstitial space [77] and
cules. One of the most important obstacles to achieving prolonged circu- reach the targeted cells, where they are internalized by a number of dif-
lation is opsonization of the nanogels followed by their clearance via ferent endocytotic mechanisms, depending on the size, shape, softness,
organs of the MPS like liver and spleen, where they are taken up by charge and other surface properties of the nanogels and the type of cells
the resident monocytes and macrophages [60]. PEGylation of the and receptor being targeted. Internalization of the nanogels can occur
nanogel surface imparts them with ‘stealth’ properties by making the via more than one pathway, making it a highly complex process. But
surface more hydrophilic, shielding a charge that the core might carry in general, endocytosis eventually confines the particles into intracellu-
and establishing a steric hindrance for interaction with serum proteins, lar vesicles, from where they are trafficked into endosomes and ulti-
although this is highly dependent on the size of nanogel, its shape, mately lysosomes. At each of these stages, nanogels are exposed to

Please cite this article as: K.S. Soni, et al., Nanogels: An overview of properties, biomedical applications and obstacles to clinical translation, J. Con-
trol. Release (2015), http://dx.doi.org/10.1016/j.jconrel.2015.11.009
4 K.S. Soni et al. / Journal of Controlled Release xxx (2015) xxx–xxx

Fig. 1. In vivo behavior of nanogels.

varying pH of endosomal/lysosomal lumen, degrading enzymes or re- designing a broad range of drug formulations and inclusion of multiple
ducing environments, which are often utilized as stimuli for the release therapeutic cargos within the same nanogel carrier [27,83]. Stimuli-
of cargo held within the nanogels. Nanogel carriers can also be designed responsive drug release via temperature or pH-induced volume col-
to target specific intracellular organelles or escape them, depending on lapse can also be very attractive for drug delivery applications. The
the type of cargo that they carry. For example, it is essential for the functionalization of the nanogel surface can further facilitate their selec-
nanogels to undergo endosomal/lysosomal escape so that the encapsu- tive accumulation in the target tissue or cells [84–86]. Development of
lated siRNA or oligonucleotides can be released in their active form in nanogels that can carry, protect, target and release therapeutic agents
the cytosol where they are supposed to show their therapeutic effect in spatially and temporally controlled manner is actively ongoing and
[11]. This has brought growing interest in the use of nanogels made of their rational design can provide a platform for multiple applications.
bioresponsive polymers to promote escape by osmotic effects, mem-
brane binding or membrane fusion or using pH-sensitive or reducible 3.1. Nanogels for small therapeutic molecule delivery
crosslinkers to facilitate nanogel destabilization following internaliza-
tion and enhance delivery efficiency [78–81]. Degradability of the Over the past few years, significant progress has been achieved in
nanogels is also essential to minimize toxicities associated with the ac- application of nanogels as a delivery carrier for small biologically active
cumulation of the carrier in the body. molecules. Nanogels can be a versatile platform for the incorporation of
various small drug molecules through the combination of electrostatic
3. Nanogels as a therapeutic drug carrier and hydrophobic interactions as well as hydrogen-bond formation
[87]. The swelling of nanogels in an aqueous environment allows for
Nanogels are highly swollen and can incorporate 30% wt. or more of easy permeation of the cargos. The rational design of the nanogels
biological molecules and drugs through electrostatic, van der Waals might be an effective tool to tune the drug release rates, to affect
and/or hydrophobic interactions or covalent bonding with the polymer carrier-cell interactions, and achieve desirable therapeutic effect of
chains. These loading capacities are unusually high and exceed those of the drugs. One of the most important features of weakly-crosslinked
liposomes and polymeric micelles [3,82]. As a result of drug loading, the polyelectrolyte nanogels is their ability to incorporate molecules of
nanogels collapse forming stable nanoparticles, in which biological the opposite charge. For example, cationic crosslinked PEG-
agent becomes entrapped. Introducing dispersing hydrophilic polymers polyethyleneimine (PEG-PEI) nanogels were explored for immobiliza-
(e.g., PEG) in a nanogel structure can prevent their aggregation. During tion of negatively charged biologically active compounds such as
the collapse of the drug-nanogel complex hydrophilic polymer chains retinoic acid, indomethacin or valproic acid [87,88]. These drug formu-
become exposed at the surface and form a protective layer around lations formed stable colloidal dispersions at physiological pH and
the nanogel. The control and versatility of polymer chemistry allows ionic strength, could be lyophilized and then redispersed. Similar

Please cite this article as: K.S. Soni, et al., Nanogels: An overview of properties, biomedical applications and obstacles to clinical translation, J. Con-
trol. Release (2015), http://dx.doi.org/10.1016/j.jconrel.2015.11.009
K.S. Soni et al. / Journal of Controlled Release xxx (2015) xxx–xxx 5

nanogels and complexation strategy has been successfully utilized for cores influences solubilization capacity and release characteristics of
incorporation of various nucleoside analog 5′-triphosphates [89,90]. It the nanogels [99]. Diblock copolymer, PEG-b-poly(L-glutamic acid),
was reported that these drug-loaded nanogels could improve the deliv- hydrophobically-modified with L-phenylalanine methyl ester moieties
ery of the active triphosphates of therapeutic nucleoside analogs into was used for the synthesis of nanogels with small size (ca. 70 nm in di-
cancer cells and inhibited tumor growth in the mammary carcinoma an- ameter) and narrow particle size distribution. Stable DOX-loaded hy-
imal model [90]. Recently, the same group demonstrated the significant brid nanogels were prepared at high DOX capacity (30% wt.). It was
advantage of active 5′-triphosphates of nucleoside reverse transcriptase shown that the release rates of DOX from hydrophobically-modified
inhibitors encapsulated in cationic nanogels over free drugs in the anti- carriers were substantially less compared to nonmodified nanogels: a
viral therapy of HIV-1 infection in the central nervous system (CNS) burst release of over 85% of the incorporated drug within 8 h for
[91]. Our group utilized a controlled template synthesis of nanogels by nonmodified nanogels was observed while only ~20% of the incorporat-
polyion complexation and crosslinking of doubly hydrophilic block ed DOX was released from hydrophobically-modified nanogels during
ionomers, such as PEG-b-poly(methacrylic acid) (PEG-b-PMA) [21,40, the same period of time. These results suggested that intermolecular
92]. The resulting nanogels have swollen cores of a crosslinked PMA interactions in combination with more compact crosslinked core
network surrounded by a shell composed of PEG chains. This synthetic could account for the delayed and controlled release of DOX from
approach allows versatile control of the macroscopic properties of hydrophobically-modified polyelectrolyte nanogels. It was also found
nanogels (size, degree of swelling, drug loading) by changing the num- that these DOX-loaded nanogels exhibited an improved antitumor effi-
ber and the chemical structure of the crosslinks [92]. Notably, in con- cacy compared to free DOX in an ovarian tumor xenograft mouse model.
trast to many nanoparticles these anionic nanogels exhibited very low Nanogels have also been explored as a carrier for poorly water-soluble
nonspecific adhesion to nontargeted surfaces, which can minimize drugs. Wang et al. introduced thermoresponsive nanogels based on
their off target effect and facilitate target-specific delivery [93]. Such chitosan–poly(NIPAAm-co-acrylamide) for the delivery of paclitaxel
core–shell nanogels can incorporate very large amounts (up to 50% (PTX) [100]. Loading capacity of these nanogels for PTX was around 9%
wt.) of weakly basic drug DOX through electrostatic coupling with car- wt. and drug-nanogel formulation showed very good colloidal stability.
boxylic groups in the cores. DOX-loaded nanogels were stable for a This nanogel released PTX in a temperature-dependent manner wherein
prolonged period of time, exhibited noticeable pH-sensitive behavior significantly faster drug release was achieved at higher temperature.
with accelerated release of DOX in acidic environment due to the pro- Moreover, PTX-loaded nanogels demonstrated improved antitumor effi-
tonation and swelling of the nanogel crosslinked cores, and demonstrat- cacy in mice bearing HT-29 colon carcinoma tumors after intravenous ad-
ed cytotoxic activities in cancer cell lines [94]. Introduction of reversible ministration. In another study by Gref and coworkers, hydrophobic
crosslinks with disulfide bonds in the PMA ionic cores (cystamine was molecule benzophenone (widely used as sunscreen agent) was solubi-
used as a biodegradable crosslinker) allowed developing nanogels that lized into nanogels formed spontaneously upon the association of a
are degradable in the presence of the reducing agent (glutathione, cys- lauryl-modified dextran and a β-cyclodextrin polymer in aqueous
teine), which in turn facilitated the release of the incorporated DOX media [101]. The highest benzophenone loadings (about 2.5% wt.) were
[94]. This difference in the release kinetics led to considerable increase obtained by solubilizing it in both polymer solutions before mixing
of cytotoxicity: degradable DOX-loaded nanogels displayed nearly six- them to form nanogels. Such nanogel-based formulations present a com-
fold higher cytotoxic activity than non-degradable nanogels. Shi et al. pelling future opportunity for the application in the cosmetic field as sun
used PEG-poly(L-glutamic acid-co-L-cystine) copolymers to prepare screen carriers prepared by a simple “green” technology. The use of
nanogels stabilized by disulfide bridges and also demonstrated nanogels for the development of long-lasting formulations of local anes-
that DOX release was accelerated in intracellular reductive and acidic thetics has recently been reviewed by Tan et al. [102]. Eckmann and col-
conditions [95]. DOX was loaded into these polypeptide-based leagues explored biocompatible physically crosslinked hybrid nanogels
nanogels, and an accelerated release was observed in glutathione consisting of a partially denaturated lysozyme cores and dextran shells
monoester pretreated HeLa cells. for the local delivery of dexamethasone to alleviate acute pulmonary in-
Anionic PEG-b-PMA nanogels were further used to encapsulate hy- flammation [103]. To target the pulmonary vasculature, nanogels were
drophilic drug cisplatin through coordination interactions with COOH coated with antibodies directed to endothelial determinant, Intercellular
functionalities [40]. Cisplatin-loaded nanogels displayed pH-sensitive Adhesion Molecule-1 (ICAM). The synthesized ICAM-targeted nanogels
release of Pt(II) species in sustained manner that can be effectively con- were loaded with dexamethasone at 5% wt. In vivo studies in animal
trolled by adjusting the degree of crosslinking of the crosslinked cores. model of LPS-induced lung injury showed that nanogels targeted to
The released platinum species retained their activity and were able to lungs succeeded in delivering encapsulated dexamethasone, as was indi-
form Pt adducts with nuclear DNA in the cancer cells [40]. It was cated by drastic reduction in pulmonary vasculature inflammation to
shown that loading of cisplatin into the nanogels greatly improved levels found in naïve mice. In other work, similar dextran-lysozyme
drug therapeutic index by improving PK, enhancing tumor delivery, in- nanogels were utilized as scaffolds for the in situ synthesis of silver nano-
creasing antitumor efficacy, and mitigating the cisplatin-mediated particles [104,105]. Such hybrid nanogels exhibited bactericidal proper-
nephrotoxicity in a mouse model of ovarian cancer [96]. When the ties towards Escherichia coli and bacteriostatic properties towards
same cisplatin-loaded nanogels were also decorated with targeting li- Staphylococcus aureus, and their antibacterial activity can be tuned by
gands (e.g. folate or LHRH peptide), tumor growth inhibition could be varying lysozyme content. The tunability of the hybrid nanogels makes
even further enhanced [85,86,97]. Peng et al. described dual pH- and it possible to optimize release of the bactericidal agent for specific clinical
temperature-responsive nanogels based on N-isopropylacrylamide, use and type of infection. Nagasaki's group used the protonated
MAA, and PEG methylether methacrylate (NIPAAm-MAA-PEGMA) for crosslinked poly(2-[N,N-diethylamino]ethyl methacrylate) (PEAMA)
the entrapment and release of cisplatin. It was shown that cisplatin re- cores of the stimuli-responsive PEGylated nanogels for the synthesis of
lease can be accelerated at acidic pH, and could be additionally con- gold nanoparticles without any reducing agents (“gold nanogels”) [106].
trolled by the temperature change due to the deswelling behavior of It was shown that the tertiary amino groups in the PEAMA gel core play
these nanogels at the body temperature [98]. a crucial role in the reduction of the Au(III) ions (nanoreactor) as well
Small molecules usually contain only limited number of ionic as the immobilization of the resulting gold nanoparticles (nanomatrix)
groups, which are able to interact with oppositely charged nanogels, to attain large payloads of gold nanoparticles. The resulting gold-
and complementary hydrophobic, hydrogen or coordination bonding containing nanogels showed a remarkable photothermal efficacy in re-
between drug molecules and nanogels can further stabilize the electro- sponse to laser-irradiation resulting in selective cytotoxicity in cancer
static pairing [87]. Recently, our laboratory demonstrated that microen- cells. In other studies, a potential of the similar constructs to enhance can-
vironment formed by the hydrophobic domains in the nanogel ionic cer cell radiosensitivity was reported [107].

Please cite this article as: K.S. Soni, et al., Nanogels: An overview of properties, biomedical applications and obstacles to clinical translation, J. Con-
trol. Release (2015), http://dx.doi.org/10.1016/j.jconrel.2015.11.009
6 K.S. Soni et al. / Journal of Controlled Release xxx (2015) xxx–xxx

3.2. Nanogels for oligonucleotide delivery been also widely explored for the delivery of proteins and peptides. In
their pioneering work, Akiyoshi et al. reported that the nanogel of
Therapeutic oligonucleotides (ONs) including antisense self-assembled cholesterol-modified pullulan (CHP) forms a complex
oligodeoxynucleotides (ODNs), small interfering RNAs (siRNAs), with various kinds of proteins spontaneously, primarily through hydro-
and the more recently discovered micro RNAs (miRNAs) designed for phobic interactions [131,132]. The amount of protein complexed by
targeted inhibition of specific mRNA sequences are of emerging interest such nanogels depends on the molecular weight and hydrophobicity
for the treatment and diagnosis of cancer [108–110], neurodegenera- of the protein. Complexation drastically suppressed the thermal dena-
tive disorders [111–113] and lethal viral infections [114–116]. Some turation and subsequent aggregation of proteins as well as protected
oligonucleotide-based therapies have already achieved significant suc- them from enzymatic degradation. This platform was further extended
cess in clinical trials [117,118]. However, the delivery of ONs into by cationization of the polysaccharide-based nanogels to utilize both
targeted cells remains a key challenge to realizing their full therapeutic hydrophobic and electrostatic interactions for effective protein trapping
potential because ONs are negatively charged, hydrophilic molecules and to enhance cellular internalization of the protein-loaded carriers
that are unable to penetrate cell membranes on their own, can be de- [10]. However, one of the drawbacks of such delivery systems is the de-
graded by endogenous nucleases and can stimulate innate immune sys- stabilization of the nanogel–protein complex in vivo in the presence of
tem. Thus, ONs require a delivery vehicle to bring them to site of action high protein concentrations. To preserve the long-term stability of
without adverse effects. Cationic nanogels have emerged as promising such complexes, the same group developed the raspberry-like assembly
new class of nanomaterials to address the challenges of in vivo ONs de- of nanogels with narrow size distribution (40–120 nm size) by
livery [84]. Composed of weakly crosslinked hydrophilic polymer crosslinking acrylate-group-modified CHP with thiol-modified tetra-
chains, nanogels have a high degree of porosity that permits the effec- armed PEG [133]. These nanogel assemblies showed high encapsulation
tive encapsulation of macromolecular therapeutics, which usually can- efficiency for interleukin-12 (IL-12) and were able to maintain steady
not be achieved with conventional nanocarriers. plasma IL-12 level in mice up to 72 h following subcutaneous adminis-
In one of the first reports, Vinogradov et al. exploited cationic PEG-PEI tration. Nagahama et al. demonstrated that hybrid self-assembled
nanogels as potential carriers for antisense phosphorothioate ODN specif- nanogels based on poly-(L-lactide)-grafted dextran enable sustained re-
ic to human mdr1 gene [119]. Significant enhancement of antisense inhi- lease of the entrapped lysozyme in its active form for a week without
bition of Pgp as a result of incorporation of ODN in the nanogel was initial burst release at physiological conditions [134]. Thienen et al. de-
observed. Modification of ODN-loaded nanogels with transferrin veloped biodegradable lipid-coated dextran nanogels by UV polymeri-
targeting moieties additionally increased the mdr1-inhibitory effects zation of dextran derivatized with 2-hydroxyethyl methacrylate
and was shown to facilitate transport of the loaded nanogels across the moieties using liposomes as a nanoscaled reactor [135]. Proteins can
blood–brain barrier [84]. Subsequently, DeSimone and coworkers used be loaded into the nanogel cores with high efficiency during the poly-
inverse miniemulsion polymerization of 2-acryloxyethyltrimethyl merization process and their release can be controlled by crosslinking
ammonium chloride and 2-hydroxyethylacrylate with PEG-diacrylate density of the gel matrix. The application of polysaccharide-based
crosslinker to synthesize biocompatible nanogels with controlled size, nanogels for delivering proteins and other macromolecular therapeutics
morphology, and composition capable of forming stable ODN complexes has recently been summarized in a review article [136]. Matyjaszewski
and enhancing cellular delivery of ODNs in vitro [16]. Nowadays, a variety and coworkers demonstrated that application of atom transfer radical
of cationic nanogel particles have been actively adapted to deliver siRNA polymerization (ATRP) enabled the synthesis of functionalized nanogels
molecule [81,120–128]. For example, Anderson and coworkers synthe- with a uniform network that is capable of efficient encapsulation of pro-
sized a library of 1536 structurally distinct core–shell nanogels for teins in situ [137]. Similarly, the addition of cytochrome C during the in-
siRNA complexation and delivery with great variability in the chemical verse emulsion reversible addition fragmentation chain transfer (RAFT)
nature of the protonizable amine-based core and a shell with variation polymerization of N-(2-hydroxypropyl)methacrylamide and N,N′-
in polymer length and chemical properties [129]. The evaluation of this li- bis(acryloyl)cystamine also showed high loading efficiency (73% wt.)
brary revealed that internalization and/or complexation alone is not suf- of the protein in the resulting nanogels, which were shown to release
ficient for silencing and that certain chemical functionalities with their payload under reductive conditions [138]. In another interesting
potential buffering capacity may be advantageous for nanogel-based de- work reported by Chen et al. RAFT polymerization was used to prepare
livery. Chemical modifications of siRNA may also be used to enhance disulfide crosslinked nanogels based on PEG-b-poly(2-(hydroxyethyl)
the transfection efficiency of these nanocarriers and to impart greater methacrylate-co-acryloyl carbonate) for loading (~ 50% wt.) and trig-
stability to their nucleic acid cargoes. The recent report by Zentel group gered intracellular release of proteins [139]. The in vitro release studies
[78] also emphasized that size-dependent uptake and intracellular distri- showed that release of fluorescently-labeled cytochrome C was minimal
bution mechanism of siRNA-loaded cationic nanogels may be essential under physiological conditions while complete release of the protein
for tuning their knockdown potential. The authors used two sets of from nanogel was observed in the presence of 10 mM dithiothreitol
well-defined nanogels based on amphiphilic block copolymers of over 22 h. Cytochrome C-loaded reduction-sensitive nanogels demon-
pentafluorophenyl methacrylate and tri(ethylene glycol) methyl ether strated apparently better apoptotic activity than free cytochrome C
methacrylate crosslinked by spermine with diameters of 40 nm and and reduction-insensitive controls. Shi et al. developed acid-labile
100 nm. Both formulations show similar physicochemical properties, nanogels via inverse emulsion polymerization of N-vinylformamide in
loading efficiencies and release capabilities of siRNA. However, only the the presence of a ketal-containing crosslinker. The loading capacity of
small sized anti-luciferase siRNA-loaded nanogels with diameters of these nanogels for lysozyme was ∼60% wt. and approximately 95% of ly-
about 40 nm were able to avoid acidic compartments of endolysosomal sozyme encapsulated in nanogels released over 3 h at pH 5.8 compared
uptake pathway and induce gene knockdown, while the 100 nm-sized to only ∼ 15% released at pH 7.4. Notably, released lysozyme retained
nanogels did not affect the gene expression of luciferase at all. The reader about 50% of the original activity. Although the better loading efficiency
is referred to more detailed review articles that summarize the key prop- is a significant advantage of the aforementioned in situ loading technol-
erties of the cationic nanogels that enable successful in vivo delivery of ogies, the retention of activity, structural identity, and stability of
various nucleic acids [130]. protein after encapsulation are basic concerns in development of bio-
pharmaceutical products.
3.3. Nanogels for delivery of protein therapeutics Polymeric nanogels have also found an application in the formula-
tion of a new generation of therapeutic and preventive vaccines. Intrin-
Owing to the ability of nanogels to encapsulate high amounts of sic properties of polymeric nanogels, such as material chemistry, size
biomacromolecules and prevent them from degradation, they have and shape, surface charge, and hydrophobicity or hydrophilicity, may

Please cite this article as: K.S. Soni, et al., Nanogels: An overview of properties, biomedical applications and obstacles to clinical translation, J. Con-
trol. Release (2015), http://dx.doi.org/10.1016/j.jconrel.2015.11.009
K.S. Soni et al. / Journal of Controlled Release xxx (2015) xxx–xxx 7

be determining factors in designing the induced immune response. out of fifteen vaccinated with MAGE-4A protein encapsulated in CHP
These materials can thus work as synthetic adjuvants, which can also nanogel [147]. These patients showed prolonged overall survival, signif-
be conjugated with immunostimulants. Furthermore, nanogels having icantly longer than that of patients without a MAGE-A4 antibody re-
a size of 50 nm or less were found to be quite effective in delivery across sponse after vaccination. We encourage the readers to refer to a
the hydrogel-like network of mucus [140] and it has been reported that review by Ferreira et al. [141] and the latest review by Akiyoshi [148]
in the case of nanogels for vaccine delivery, the type and intensity of im- for the details about the application of nanogels as vaccine carriers.
mune response can be modulated by altering the size of the nanogels
[141–143]. In a very interesting report published by Akiyoshi and 3.4. Nanogels for combination drug delivery
Kiyono group, cationic cholesterol-bearing pullulan (cCHP) nanogels
were explored as protein-delivery system for adjuvant-free intranasal Drug combinations directed simultaneously at multiple pharmaco-
vaccines [14]. As shown in Fig. 2, they demonstrated that intranasally logical targets have the potential to dramatically improve the response
administered cCHP nanogel loaded with a non-toxic subunit fragment to treatment and are already a standard clinical practice in the treat-
of Clostridium botulinum type-A neurotoxin BoHc/A (cCHP–BoHc/A) ment of cancer and infectious diseases [149]. However, the difference
adhered to the nasal epithelium and BoHc/A was effectively taken up in pharmacokinetic and pharmacodynamic profiles among diverse
by mucosal dendritic cells after its release from the cCHP nanogel. Im- drug molecules makes dosing and scheduling optimization very chal-
portantly, intranasally-administered cCHP–BoHc/A did not accumulate lenging. Combining drugs in one delivery carrier is a well-suited strate-
in the olfactory bulbs or brain, diminishing safety concerns about the gy for controlling the pharmacokinetics and co-delivery of the desired
potential dissemination of vaccine antigens to the CNS. Moreover, intra- drug ratio in vivo, and a variety of nanoscale carriers, including nanogels,
nasally immunized tetanus toxoid with cCHP nanogel induced strong have been investigated in terms of their ability to deliver multiple drugs
tetanus-toxoid-specific systemic and mucosal immune responses. The [27,83,150,151]. As highlighted in previous sections, the nanogel struc-
CHP platform has also been tested in Phase 1 clinical trials as carrier ture can be readily adjusted to integrate features of different materials
for HER-2 antigen in patients with therapy-refractory HER-2 positive and, thus, offer advantages for combinatorial encapsulation of drugs
cancers [144] as well as for delivery of NY-ESO-1 antigen in esophageal with varying physicochemical properties such as small molecules, pro-
cancer and malignant melanoma patients [145,146]. Collectively, the re- teins and nucleic acids. Fahmy and coworkers developed liposomal
sults from several clinical trials performed using the CHP-based antitu- nanogels of drug-complexed cyclodextrins and cytokine-encapsulating
mor vaccines demonstrated the safety of the vaccines after repeated biodegradable polymers that can deliver small hydrophobic molecular
subcutaneous administration as well as document their success in in- TGF-β inhibitor and water-soluble protein cytokine (IL-2) in a sustained
ducing both antigen-specific CD4 + and CD8 + T-cell response along fashion to the tumor microenvironment [150]. They demonstrated that
with humoral immunity. According to a more recent report, a MAGE- synergistic effects of simultaneously delivered IL-2 and TGF-β inhibitor
A4-specific humoral immune response was observed in four patients on activation of the innate arm of the immune system led to delayed

Fig. 2. Use of cCHP nanogel as a new antigen-delivery vehicle for intranasal vaccination. A) Generation of cCHP nanogel from a cationic type of cholesterol-bearing pullulan. B) PET images
showing that intranasally administered cCHP nanogels carrying [18F]-labeled BoHc/A were effectively delivered to the nasal mucosa. C) Direct quantitative study with [111In]-labeled BoHc/
A further demonstrated that BoHc/A was retained in the nasal tissues for more than two days after intranasal immunization with cCHP nanogel. In contrast, most naked BoHc/A disap-
peared from the nasal cavity within 6 h after administration. Adapted with permission from [14].

Please cite this article as: K.S. Soni, et al., Nanogels: An overview of properties, biomedical applications and obstacles to clinical translation, J. Con-
trol. Release (2015), http://dx.doi.org/10.1016/j.jconrel.2015.11.009
8 K.S. Soni et al. / Journal of Controlled Release xxx (2015) xxx–xxx

tumor growth and enhanced survival of melanoma tumor-bearing various imaging probes and contrast agents. Introduction of multiple
mice after systemic administration. In line with the challenge of de- functional groups either in the interior or on the surface of nanogels al-
livering drugs with different properties, our group developed multi- lows for incorporation/conjugation of multiple dyes, reporter molecules
compartment core–shell nanogels based on hybrid triblock copoly- or inorganic nanoparticles. Encapsulation of magnetic nanoparticles
mers, PEG-poly(L-glutamic acid)-poly(L-phenylalanine) (PEG–PGlu– such as iron oxide into crosslinked nanogels has been shown to confer
PPhe). Such nanogels have a central hydrophobic core formed by PPhe both colloidal stability and better sensitivity than when these agents
chains, a crosslinked anionic layer of PGlu chains, and an outer shell are administered as non-encapsulated entities [153,154]. Nanogels
composed of PEG chains [27]. These hybrid biodegradable nanogels allow for the encapsulation of a large cargo of the magnetic nanoparti-
can entrap considerable amount of drugs with very different physical cles, which can lead to generation of much stronger local magnetic fields
properties such as hydrophilic cisplatin (15% wt. loading) and hydro- due to the cluster effect [154–157]. Moreover, the hydrogel coating fur-
phobic paclitaxel (9% wt.). Binary drug combination in nanogels exhib- ther increases the relaxivities by lowering the diffusion coefficient of
ited synergistic cytotoxicity against human ovarian A2780 cancer cells water near the particles and prolonging the interaction between the
and exerted a superior antitumor activity in cancer xenograft models water protons and the high magnetic fields at the surface of the particle.
in vivo as compared to individual drug-loaded nanogels or free drugs. The extent of reduction in the diffusivity of the water molecules
The benefits of synchronized co-delivery of the platinum-taxane depends on the thickness of the gel coating around the magnetic parti-
drug combination via single carrier can be further enhanced by cle [156–159]. This could allow for partial control over the relaxation
targeting nanogels to folate receptor, which are overexpressed in times by manipulating the swelling/deswelling transition of the nanogel
most ovarian cancers [97]. In another study, we reported an efficient matrix [157]. Interestingly, Okada et al. reported that nanogels
co-encapsulation of DOX and 17-allylaminodemethoxygeldanamycin consisting of crosslinked PMA can function as magnetic resonance im-
(17-AAG) into PEG–PGlu nanogels with multiple hydrophobic domains aging (MRI) pH-sensors without the need of any inorganic paramagnet-
that are located within the crosslinked polyion PGlu core [83]. Dual ic material [160]. The shrinking of pH-sensitive nanogel at acidic pH
drug-loaded nanogels displayed potent cytotoxicity in a breast cancer induces more rigid configurations and slower rotational motions of
cell panel and exerted selective synergistic anticancer activity against the polymer chains than in the swollen state thereby leading to shorter
ErbB2-overexpressing breast cancer cell lines. This synergistic effect transverse relaxation time because the mobility of bound water mole-
was attributed to the action of 17-AAG, HSP90 inhibitor, which induces cules is highly restricted. Another factor that influences the relaxation
degradation of many of the proteins required for DNA-damage response time of contrast agents like Gd chelates is the freedom of tumbling mo-
as well as attenuates hyperactive ErbB2 downstream oncogenic signal- tion [155]. One of the best methods to restrict tumbling is by conjuga-
ing, thereby rendering cancer cells more vulnerable to the cytotoxic tion of the contrast agent to a macromolecular system, and nanogels
effects of DOX. Consistent with the in vitro findings, combination treat- serve as the perfect hydrophilic platform with flexible loading capacity
ment with nanogels exhibited superior antitumor efficacy, both in for entrapment for such agents, which was shown to result in a further
terms of tumor inhibition and survival, in an ErbB2-driven xenograft relaxivity enhancement. [156,161,162]. Gd3+ ion is toxic and it is clini-
model compared to the cocktail of free drugs at equivalent drug cally administered in the form of chelates. However, even the chelates
concentrations. have the potential to show toxicity due to transmetallation reactions,
Akiyoshi and colleagues evaluated nanogels based on acrylate which involve the displacement of the chelated metal ion by another
group-modified CHP for co-delivery of prostaglandin E2 receptor-spe- competing ion. It has been reported that diethylenetriaminepentaacetic
cific agonist EP4A (small molecule) in combination with bone morpho- acid (DTPA)-based nanogels are more inert to transmetallation reac-
genic proteins (BMP-2) for bone regeneration [151]. In this study, EP4A- tions than free chelates [163]. Encapsulation into nanogels also helps
containing nanogels and BMP-2-containing nanogels were crosslinked in reducing the toxicity associated with inorganic nanoparticles
with thiol-bearing PEG to obtain disk-like scaffolds for the implantation [164–168]. Furthermore, it has been shown that the electronic and
into large bone defects. Combination treatment with EP4A and low- optical properties of gold and silver nanoparticles are highly depen-
dose BMP-2 efficiently activated bone cells to regenerate calvarial dent on their size and shape [169–171] and encapsulation of such
bone by forming both outer and inner cortical plates as well as nanoparticles into nanogels becomes essential not only for their col-
bone marrow tissue and resulted in the formation of enough bone loidal stability but also for consistency of the imaging purpose. As in
to cover the defects in calvarial bone. In another study, cationic the case of contrast agents, the swelling/deswelling of the surround-
physically-crosslinked nanogels composed of a hexadecyl group- ing nanogel network in response to changes in the microenviron-
bearing cycloamylose and spermine-modified cycloamylose to com- ment can reversibly alter the interparticle distance between gold
plex and co-deliver plasmid DNA along with phospholipaseA2 (PLA2) nanoparticles leading to a corresponding change in their optical
[152]. As a lipolytic enzyme, PLA2 catalyzes hydrolysis of a variety of dif- properties. This can impart tissue specificity to the imaging function
ferent phospholipids. Thus, being delivered into endosomes by nanogel to a certain extent [171,172].
it could disrupt the lipid membrane and subsequently trigger the re-
lease of the co-encapsulated DNA into the cytoplasm. Transfection ex- 4.1. Nanogels as MR contrast agents
periments confirmed that DNA expression level was enhanced when
complexed with PLA2. Similarly, pDNA and proteins were successfully Small molecule MR contrast agents based on gadolinium (Gd) and
co-encapsulated using pH- and temperature-sensitive carbohydrate- manganese (Mn) are all rapidly cleared from the body and suffer from
based nanogels [79]. The nanogels had a core–shell structure with a toxicity issues. [173,174]. To overcome these challenges, Soleimani
crosslinked hydrophobic core that could be loaded with proteins and et al. prepared a nanogel by copolymerization of PEGMA, N-(2-
the shell contained carbohydrate residues that allowed for the com- aminoethyl)methacrylamide hydrochloride, and the crosslinker ethyl-
plexation of DNA. These nanogels were capable of loading larger- ene glycol dimethacrylate under free radical conditions [156]. The reac-
than-normal amounts of cargo by using a heating and cooling cycle. Al- tion conditions were optimized to obtain nanogels with a size on the
together, these studies may open new avenues for the development of order of 10 nm. An isothiocyanate derivative of the chelator DTPA was
the carrier-mediated combination therapies. then conjugated to nanogel for the insertion of Gd (III). The nanogel
contrast agent exhibited ~5-fold enhancement in relaxivity compared
4. Nanogels in diagnostics and imaging to clinically used Gd(III)–DTPA (Magenvist). Signal enhancement, how-
ever in lesser extent, was also observed for non-crosslinked polymer
Structural versatility of nanogels, high water content, fluid-like constructs containing similar amount of Gd-chelates. The modeling of
transport properties and biocompatibility make them ideal carriers for nuclear magnetic resonance dispersion data suggested that the

Please cite this article as: K.S. Soni, et al., Nanogels: An overview of properties, biomedical applications and obstacles to clinical translation, J. Con-
trol. Release (2015), http://dx.doi.org/10.1016/j.jconrel.2015.11.009
K.S. Soni et al. / Journal of Controlled Release xxx (2015) xxx–xxx 9

enhancement in relaxivity for nanogel vs linear polymer arise from the 12 nm and high crystallinity were complexed with nanogels through
constraint on polymer chain motion imparted by the crosslinking that hydrophobic interactions with cholesterol groups in the nanogels
leads to additional slowing of the chelates' molecular tumbling rate. Al- [179]. The resulting hybrid showed high colloidal stability and
ternatively to post polymerization functionalization of nanogel with displayed higher relaxivity than existing contrast agent Resovist. In
contrast agents, Gd-chelates can be incorporated into nanogels within addition, the hybrid nanogel generated heat following irradiation
the crosslinking moieties or Gd3+ ions can be used as metal crosslinkers with alternating magnetic field, which indicates that these materials
themselves. The later strategy was utilized by Kim and coworkers can also be suitable for magnetic hyperthermia therapy.
to prepare Gd-coordinated nanogels by crosslinking of branched Recently, Wang et al. described a nanogel-based system co-loaded
PEI with Gd3 + ions in inverse microemulsion followed by surface with both T1 and T2 relaxivity contrast agents, manganese oxide and
functionalization with PEG chains in order to increase the blood circula- SPIO nanoparticles, since the use of dual contrast agents can potentially
tion time [175]. It was shown that polydentate chelation by PEI is robust allow for higher accuracy of imaging. These agents cannot be simply
enough to minimize the liberation of toxic Gd3+ ions under the physio- mixed in a single system, since in close proximity they would lead to
logical conditions. Interestingly, the determined longitudinal T1 mutual nullification of the signal, as the water molecules can interact
relaxivity values for Gd-PEI nanogels were lower than that for Gd- freely with both the agents. This problem, however, was solved by the
DTPA chelates. The reduced relaxivity was attributed to the large use of stimuli-responsive chitosan-based nanogels, which release
nanogel core size with a small surface-to-volume ratio, which hinders Mn2+ ions only in the acidic pH inside the tumor cells while at physio-
the direct Gd3+-water contact that is prerequisite for influencing the logical pH in normal cells the system remains silenced, thus improving
longitudinal relaxation. In contrast to other T1-enhancing Gd com- specificity of imaging. This technique can be potentially useful for detec-
plexes, Gd-PEI nanogels showed the capability of enhancing negative tion of small tumors with better accuracy than the existing single agent
T2 contrast, which was assigned by high density of the Gd3 + ions in probes [180].
the cores of the nanogels. Almutairi's group reported Gd-chelating Nagasaki's group developed a unique tumor specific nanogel-based
polyacrylamide nanogels prepared by radical polymerization of acryl- probe for 19F magnetic resonance spectroscopic imaging (19F MRS/I)
amide with metal chelating crosslinking agents in an inverse emulsion [181]. 19F MRS/I has been recognized as a powerful methodology due
[163]. The resulting crosslinked nanogels had a size in the range of to high MR sensitivity of 19F and low background noise which is attrib-
50–85 nm, were stable against transmetallation, and exhibited a sub- uted to the absence of endogenous 19F in the body. The 19F MRS/I of tu-
stantial (3 to 4-fold) increase in relaxivity. To further improve nanogels mors can be further improved using a probe that attenuates the 19F MR
biocompatibility, the same group utilized nanogels formulated from signal outside of the tumor and switch on the signal inside the tumor. To
cholesterol- and acryloyl-modified pullulan (CHPOA). The surface this end, a pH-responsive nanogel consisting of a crosslinked PEAMA-
acryloyl groups of CHPOA were chemically crosslinked with Gd- co-poly(2,2,2-trifluoroethyl methacrylate) gel core and PEG shells was
chelating 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid synthesized. These nanogels showed remarkable activation of 19F MR
(DOTA) crosslinker. At a magnetic field of 1.41 T, T1 relaxivity of these signals upon change of pH from 7.4 to pH 6.5, which corresponds to
Gd-chelating pullulan nanogels was measured to be 24.1 ± the extracellular pH of tumor tissues (7.0–6.5). Such enhancement
0.6 mM−1 s−1, which corresponds to 6-fold enhancement as compared was attributed to the volume phase transition of the gel core at acidic
to low-molecular weight chelates, Magnevist and Dotarem. The report- pH that leads to an increase in molecular motion of the 19F atoms and
ed value was also the highest relaxivity among all other nanogel-based longer T2 relaxation times.
contrast agents. MRI of tumor-bearing mice showed that Gd-CHPOA
provides great contrast in tumors with exceptionally high signal-to-
background ratio. This allowed for discriminative and accurate tumor 4.2. Nanogels for PET imaging
identification along with determination of the surgical margin. Impor-
tantly, the long-term accumulation of Gd-CHPOA in organs of the MPS Polyacrylamide-based nanogel crosslinked with polydentate che-
like liver and spleen did not cause any damage or toxicity up to three lating ligands that were developed by Allmutairi and coworkers can
months after injection. be also used as a scaffold for metal radionuclides to obtain PET radio-
Superparamagnetic iron oxide (SPIO) nanoparticles function as tracers [182]. Various crosslinkers based on DTPA, DOTA or 1,4,7-
T 2-weighted MR contrast agents and have exerted a large impact triazacyclononane-1,4,7-triacetic acid (NOTA) were synthesized to
on the field of molecular and bioimaging. Their nonspecific uptake optimize the chelation stability of nanogels. Experiments in mouse
by MPS cells found clinical applications for imaging liver tumors serum indicated that NOTA-based nanogels retained 64Cu most sta-
[176] and lymph nodes [177]. SPIO nanoparticles' magnetic proper- bly, with very little transchelation in comparison with the other
ties can be manipulated by changes in size and surface chemistry. two crosslinkers. 64Cu-DOTA-crosslinked nanogels showed high ac-
Specifically, the local T2 proton relaxivity of SPIO nanoparticles can cumulation in the tumor as well as lower signal in the liver and
be modified via interfacial surface chemistry to induce aggregation spleen compared to DOTA-based nanogels. In some cases, the accu-
or dispersion in the colloid, thus providing control of the ultimate mulation of 64Cu-DOTA in metastases was even higher than in the
nanoparticle aggregate size and magnetic properties [178]. Katagiri primary subcutaneous tumor. These promising data suggest that
et al. reported a synthetic approach for the in situ formation and im- nanogels incorporating metal-chelating crosslinks can be useful as PET
mobilization of SPIO nanoparticle aggregates in nanogels [153]. Iron agents in cancer diagnosis and therapy monitoring. An advantage of
ions were complexed within the pre-heated network of physically such systems is that the radioisotope can be easily incorporated into
crosslinked CHP nanogels and oxidized by addition of ammonia preformed nanogels immediately before their clinical application.
water solutions. This strategy minimized homogeneous nucleation Singh et al. designed reduction-sensitive crosslinked nanogels con-
outside of nanogels, and the resulting hybrid nanogels had a size taining chelating groups for 68Ga and other trivalent metals [183].
similar to that of the empty nanogels (~ 30 nm). Essentially, the Self-assembly of amphiphilic, partially thiolated star-shaped poly(eth-
empty nanogels acted as nanoreactors wherein the volume in ylene oxide-stat-polypropylene oxide) copolymers was used for the
which the reaction could occur was confined by the nanogel net- synthesis of the nanogels. The thiol chemistry was used for both
work. However, the crystallinity of iron oxide particles synthesized crosslinking and derivatization for radiolabeling. It was shown that
68
in nanogels was relatively low, and will not translate into high satu- Ga can be chelated very efficiently with high radiochemical yields.
ration magnetization; therefore, these hybrid nanogels are not suit- However, the size of the resulting nanogels (290 ± 50 nm) may not pro-
able for use as MR contrast agent. To overcome this drawback, the vide optimal circulation time and tumor uptake. In vivo PET imaging of
68
preformed oleate-coated iron oxide nanoparticles with size of Ga-labeled nanogels has not yet been reported.

Please cite this article as: K.S. Soni, et al., Nanogels: An overview of properties, biomedical applications and obstacles to clinical translation, J. Con-
trol. Release (2015), http://dx.doi.org/10.1016/j.jconrel.2015.11.009
10 K.S. Soni et al. / Journal of Controlled Release xxx (2015) xxx–xxx

4.3. Nanogels for optical imaging intensity was relatively small at the injection site and limited to only
one sentinel node with no spreading to consecutive distal lymph
In vivo fluorescence-based optical imaging is most effective by using nodes. These characteristics of NIR-CHP make them useful as imaging
agents that emit in the NIR region (N 700 nm) due to minimal auto- contrast agents in sentinel node navigation surgery.
fluorescence from the tissues in this wavelength range, and deep tissue In recent years, there has also been a considerable interest in the de-
penetration of excitation light [184,185]. Among available NIR probes, velopment of optical probes based on nanogels and inorganic nanopar-
only indocyanin green (ICG) has been approved for clinical imaging ap- ticles such as quantum dots and gold nanoparticles. The reader is
plications. ICG, however, suffers from several limitations such as low referred to some excellent review articles on the design of such hybrid
quantum yield, relatively short circulation half-life, degradation in nanogels and their application for optical sensing and bioimaging
aqueous media and self-quenching above a certain concentration, and [165,192].
nonspecific interaction with various proteins in blood plasma, which
alter its fluorescence emission properties [186]. Nanogels have been 4.4. Nanogels for multimodal imaging agents
employed to overcome the above shortcomings of the dye in order to
extend its potential as a NIR imaging agent. In fact, some of these limita- While a number of imaging techniques can be employed to obtain
tions were used as advantages to design systems that would be activat- detailed information regarding various organs and tissues of the body,
ed specifically in the targeted cell population. Park et al. designed cancer each imaging modality has limitations of either sensitivity or resolution.
cell-targeted nanogels containing ICG as the reporter molecule that Therefore, the integration of several imaging agents with different prop-
were non-fluorescent in the normal cells. A pH sensitive polymer, erties into multifunctional nanoparticles may provide precise informa-
poly(β-amino ester) (PBAE) and CD44 receptor-targeting hyaluronic tion about the existing pathological conditions through synergetic
acid were used to form the nanogels which entrapped ICG. Under nor- multimodal imaging. [193]. Due to their unique multifunctionality,
mal physiological conditions, the ICG fluorescence was quenched in large surface area, and structural diversity, nanogels are capable of load-
the intact nanogels. Once ICG-loaded nanogel is internalized by ing more than one imaging/contrast agent within the same carrier that
receptor-mediated endocytosis, PBAE is degraded in the acidic environ- can help realize this objective.
ment of endosomes and lysosomes, gradually releasing ICG, thus gener- For example, some of optical imaging complications associated with
ating a fluorescence signal [187]. In a similar vein, hyaluronic acid was poor spatial resolution and lack of anatomical reference can possibly be
used to prepare nanogels that could be used for monitoring hyaluroni- mitigated when combined with other high resolution anatomical imag-
dase activity in vivo, as it is associated with tumor metastasis and angio- ing modalities such as MRI. This combination was employed to define
genesis. Shell crosslinked nanogels were prepared from hyaluronic acid the glioma margins using targeted pH/temperature sensitive nanogels
via a reducible covalent linkage, which could incorporate ICG deriva- based on poly(NIPAAm-co-acrylic acid). Magnetic SPIO nanoparticles
tives [188]. In the absence of the enzyme, HA-based nanogels had neg- were incorporated into nanogel network during emulsion polymeriza-
ligible fluorescence as compared to free ICG due to self-quenching tion followed by conjugation with Cy5.5-labled lactoferrin as an optical
effect. However, in the presence of the enzyme, the nanogel structure probe and an effective targeting ligand for glioma [194,195]. The LCST of
was disturbed and a strong fluorescence signal was generated. This the nanogels at physiological pH of 7.4 was 40 °C, making the nanogels
was illustrated both in vitro and in vivo after intradermal injection of swollen and hydrophilic, which could prolong the blood circulation
the nanogels in the forepaw of mice. Nanogels showed a strong fluores- time. In the acidic environment of tumor tissues (pH 6.8), the LCST is
cence signal 5 min after injection of hyaluronidase into the forepaw, in- lowered to 34 °C leading to collapse of the nanogels, which could in
dicating that this system could be used as hyaluronidase-specific probe. turn potentiate their accumulation in tumor tissue and internalization
NIR imaging is also useful for detecting the cancer metastasis to regional by tumor cells. The authors reported very accurate signal localization
lymph nodes, particularly the sentinel lymph node (SLN). Biopsy of SLN and correlation of in vivo results to ex vivo images of excised brain
is usually done during tumor resection surgeries to determine whether (Fig. 4) which was attributed to active targeting ability of the lactoferrin
metastasis has occurred, for which visualization of these lymph nodes is and passive targeting ability enhancement caused by the pH/tempera-
necessary during the surgical procedure. Small molecule dyes used clin- ture sensitivities of the nanogels [196].
ically for this purpose suffer from the drawbacks of rapid lymphatic Another multi-imaging study using nanogel-based dual MRI and op-
clearance and spreading to the entire lymphatic system and can there- tical imaging probe was reported by Lim et al. Core–shell nanogels con-
fore give false-positive results. Dextran–poly(acrylic acid) nanogels taining Gd+3-coordinated PEI core and PEG shell were functionalized
(DNG) were synthesized via self-assembly assisted method which in- with a NIR dye, Cy5.5 and evaluated in SCC7 tumor-bearing mice
volved hydrogen bonding between hydroxyl groups of dextran and car- [175]. Although no targeting group was attached to the nanogels, tu-
boxyl groups of poly(acrylic acid) [189]. The additional free carboxyl mors displayed strong NIR fluorescence signals up to 1 day post injec-
groups of poly(acrylic acid) served as a platform for the conjugation of tion. Notably, the amount of injected nanogels corresponded to a
amine-containing dyes. As a model agent, green 5-aminofluorescein much lower dose of contrast agent (38 μmol Gd/kg) than the standard
was conjugated to obtain fluorescent DNG, which were found to drain dose of Gd-DTPA (100 μmol Gd/kg) and no notable contrast enhance-
via the lymphatic vessels and into SLN within a minute of being injected ment was observed in T1-weighted images. However, a significant sig-
intradermally, and the fluorescence signal peaked around 12 h and nal darkening was seen in certain regions of the tumor at 2 h post-
lasted for 60 h. This extended imaging window is well-suited for surgi- injection, visualizing the intratumor structure at high spatial resolution,
cal applications wherein the visualization of lymph nodes is required. indicating that these nanogels could act as negative contrast enhancers
Another nanogel-based system designed for the same application has (see Section 4.1) for tumor imaging with the added advantage of fluo-
been described by Noh et al. [190]. IRDye800 containing nanogels rescence imaging [175].
composed of cholesterol-modified pullulan (NIR-CHP) had hydrody- Nanogels can also be designed to have self-fluorescence properties,
namic diameters of about 30 nm, which is considered optimal for as demonstrated by Kim et al. [197] using oppositely charged polyelec-
lymph node uptake (Fig. 3). NIR-CHP probes exhibited enhanced trolytes to form the gel network within which MnFe2O4 nanoparticles
photostability, and could accumulate and retain in the SLN after intra- were encapsulated. Negatively charged poly(γ-glutamic acid) was con-
dermal injection. The NIR-CHP were present at qualitatively consistent densed with polylysine (PLL) in the presence of MnFe2O4. The free
levels in the lymph node even at 48 h post injection, whereas the amino groups of PLL were then crosslinked with glutaraldehyde,
IRDye800 was not visibly present in the lymph nodes at this time which also involved formation of a self-fluorescent carbon–nitrogen
point. In another study, the utility of NIR-CHP probes for SLN mapping double bond (C_N) and carbon–carbon double bond (C_C). These
was evaluated in large animal models [191]. The NIR-CHP signal nanogels were finally surface-grafted with PEG. These dual-modality

Please cite this article as: K.S. Soni, et al., Nanogels: An overview of properties, biomedical applications and obstacles to clinical translation, J. Con-
trol. Release (2015), http://dx.doi.org/10.1016/j.jconrel.2015.11.009
K.S. Soni et al. / Journal of Controlled Release xxx (2015) xxx–xxx 11

Fig. 3. Schematic illustration of sentinel lymph node (SLN) mapping using a nanoscale imaging probe. Imaging probes on 5–10 nm size scale can flow through the SLN into adjacent nodes
in the chain; nanoprobes N300 nm in size rarely leave the injection site, while those with a size range of 10–50 nm exhibit rapid uptake into SLN and do not leave. Adapted with permission
from [190].

nanoprobes showed high performance in the labeling and monitoring of sensitive corona of crosslinked PNIPAAm. High magnetization and/or
therapeutic immune cells both in vitro and in vivo. Nanogels can also be bright luminescence was achieved due to a core of maghemite and
fabricated to have a core–shell–corona structure in which a shell of inor- CdSe(ZnS) while the silica shell provided a strong protection to the
ganic material like silica protects the core of magnetic nanoparticles core from degradation in conditions of extreme pH, thus giving these
while its permeability to water allows for the interaction of the water nanogels a potential for oral administration [198].
molecules with the magnetic core that is essential for its imaging func- Recently, Wang et al. developed a multifunctional nanogel-based
tion. Ruhland et al. reported such nanogels that had a temperature- probe for pathological responsive ultrasound and MR imaging [199].

Fig. 4. In vivo studies: representative results of rats bearing gliomas treated with saline as a control (upper row), Cy5.5-Lf-MPNA nanogels (middle row), and MPNA nanogels (lower row),
respectively (n = 9 in each group). T2-weighted MR Images of gliomas before injection (A, G, M) and at 48 h post-injection (B, H, N); normal photographs (C, I, O) and ex vivo fluorescence
images (D, J, P) of gliomas at 48 h post-injection; Adapted with permission from [196].

Please cite this article as: K.S. Soni, et al., Nanogels: An overview of properties, biomedical applications and obstacles to clinical translation, J. Con-
trol. Release (2015), http://dx.doi.org/10.1016/j.jconrel.2015.11.009
12 K.S. Soni et al. / Journal of Controlled Release xxx (2015) xxx–xxx

This was achieved by co-loading of two enzymes, catalase (CAT) and su- polypeptoids have been recently explored as antifouling agents. Poly
peroxide dismutase (SOD) along with SPIO nanoparticles into glycol (N-methyl glycine) or polysarcosine has the simplest structure in class
chitosan-based pH-sensitive nanogels. Most pathological sites have a and possesses a flexible backbone [213,214]. A comparative study of
high concentration of reactive oxygen species, which are converted block copolymer micelles, differing only in their hydrophilic block as
into molecular oxygen (O2) by SOD and CAT. O2 is released in the being either PEG or polysarcosine, revealed considerable overlap be-
form of microbubbles that act as a contrast medium for ultrasound im- tween the two systems in terms of their physical properties, solution
aging. Encapsulation of enzymes within nanogels protects them from and assembly behavior, indicating the potential of peptoids like
inactivation in circulation while the porous and hydrophilic nature of polysarcosine in serving as alternatives to PEG in nanogel systems of
the gel network allows the substrate molecules to diffuse freely within the next generation [215].
the matrix thus allowing the enzymes to perform their catalytic activity
[200,201]. Moreover, the density of cationic charges on the gel surface
5.2. Charge
increases in the acidic pathological microenvironment leading to im-
proved accumulation of nanogels in the target area. In addition, encap-
The presence of surface charge can alter the opsonization profile of
sulation of SPIO within the nanogel significantly enhances transverse
the nanogel, its recognition by cells in the organs of the MPS and its plas-
relaxation rates and generates strong MR signals. The proof-of-
ma circulation profile. Gel particles having a close to neutral surface
concept in vivo studies indicated that signals obtained from ultrasound
charge have been demonstrated to have longer circulation times [216,
imaging from tumors 1 h after injection are enhanced approximately
217]. However, the most important feature of nanogels, their stimuli-
7-fold. In the case of MR imaging, significantly darkened tumor signals
responsive behavior, is usually attributable to the charged groups incor-
with approximately 16% enhancement were detected after the injection
porated into network. Moreover, the presence of such groups is often
of nanoprobes.
desired to facilitate the binding of drug cargo. Thus it is very difficult
to achieve this delicate balance between charge-related responsive be-
5. Obstacles to clinical translation and strategies being developed to
havior and avoidance of the nonspecific interactions of the nanogels
overcome them
with other in vivo components. Cationic polymers like PEI are common-
ly used in the preparation of nanogel carriers for the delivery of nucleic
Nanogels, since the time of their discovery, have come a long way and
acids. Complexation with negatively charged DNA minimizes the
found applications in almost all biomedical fields, ranging from drug ther-
charge-related toxicity of the complexes in circulation; additionally
apy to imaging and diagnostics. While a few nanogel-based formulations
PEG can be conjugated to the nanogels to provide further charge
have reached clinical trials [144–147] for subcutaneous delivery of vac-
shielding. However, once nucleic acid is released from the carrier intra-
cine antigens, the clinical translation of nanogels for all other diverse ap-
cellularly, the polymer returns back to its native cationic state and is free
plications that they are suitable for remains to be realized. A number of
to show toxicity like shrinkage of the cells, formation of vacuoles and
parameters affect the efficacy of this delivery system and requires further
disturbance in the cell cycle [218]. This toxicity was found to be depen-
optimization. Similar to many other nanomedicines, one of the major
dent on the molecular weight of PEI used as well as its dose. To reduce
drawbacks is that no more than 5–10% of the injected dose actually
toxicity, polymers based on low molecular weight PEI conjugated via la-
reaches the target site, with maximum dose reaching organs involved in
bile linkers were used to prepare the gene carriers [210,219], but it is
clearance like kidney, liver, spleen, etc. [202,203]. The tissue distribution
hard to eliminate the charge effect completely.
of nanogels is governed by a number of parameters like their size,
shape, charge, composition, surface properties and the cargo that they
carry. We have attempted to summarize some of the key factors that 5.3. Challenges pertaining to biomolecule delivery
are proving to be roadblocks in this journey, along with the alternative ap-
proaches being explored to overcome them. However, it must be realized The most effective carriers for the delivery of genes are viral vectors;
that the final efficacy of the nanogel-based delivery system depends on a however, due to the limited loading capacity and risk of immune reac-
complex interplay of all of these factors and an ideal system would be the tions, non-viral delivery systems like those based on nanogels have
one with a perfect balance between all of them. gained popularity [220,221]. A major limitation encountered by these
systems is the low efficiency relative to viral vectors. This is mainly
5.1. Rapid clearance due to the degradation of the nucleic acids in the endosomes and late ly-
sosomes and it is a great challenge to design a delivery system that can
Spleen is one of the important organs responsible for the filtration of facilitate effective endosomal release of the carrier [222,223]. Majority
foreign substances from blood. Splenic filtration is lower for softer par- of the synthetic carriers used for the delivery of nucleic acids are cationic
ticles like nanogels having a size less than 200 nm, that can squeeze in nature in order to form stable complex with the negatively charged
through the splenic filtration bed [204,205], while extremely small nucleic acid to protect them from degradation and to improve circula-
nanogels (b 20 nm) are usually excreted via renal filtration [70,206], in- tion time. It has been shown that many of such systems do not remain
dicating that the size and flexibility of the nanogels have to be well- intact in circulation and are disrupted at the glomerular basement
defined. Shape of the nanogels is also an important parameter that de- membrane that also carries a negative charge due to the presence of
fines their circulation half-life, and filamentous or rod-shaped particles proteoglycans. Thus, the purpose of prolonging the half-life of such
are known to have a considerably longer circulation time than their agents is defeated [224,225], and the therapeutic agents are cleared by
spherical counterparts [207]. Apart from the size, opsonization or coat- the kidney quite quickly after the disruption of the carrier system
ing by serum proteins also plays a key role in the clearance of the parti- [118]. Moreover, the high positive charge density of the carriers increase
cles. Adsorption of plasma proteins leads to their uptake by the organs the propensity of hemolysis and opsonization in circulation, especially if
of MPS, such as liver and spleen, and can be partially prevented by the system is found to be unstable in circulation which can expose the
PEGylation [205,208]. However, PEG is not completely inert, and PEG- charged residues to serum components. Therapeutic proteins can also
specific IgM antibodies are known to be generated after administration be incorporated into nanogels for their delivery. Activity of proteins is
of a single dose of PEGylated nanoparticles, resulting in their accelerated highly dependent on their conformational flexibility that makes pro-
blood clearance. This compromises their efficacy on subsequent dosing teins quite sensitive to various steps involved in formulation develop-
[209–211]. Furthermore, this response is known to be dependent on the ment, both physical processes like shear processes experienced during
hydrophobicity of the core of carrier to which PEG is conjugated, as well vortexing, and chemical conjugation reactions like crosslinking of the
as the type of cargo [209,212]. N-substituted polypeptides or nanogel networks after protein incorporation [226–228]. Careful

Please cite this article as: K.S. Soni, et al., Nanogels: An overview of properties, biomedical applications and obstacles to clinical translation, J. Con-
trol. Release (2015), http://dx.doi.org/10.1016/j.jconrel.2015.11.009
K.S. Soni et al. / Journal of Controlled Release xxx (2015) xxx–xxx 13

selection of the process parameters is therefore essential to preserve 5.6. Drug release
protein function.
Nanogels are designed to be stimuli-responsive for the release of
5.4. Targeted delivery their cargo. At the same time, biodegradable systems are intended to
undergo degradation at the target site. Thus, it is difficult to control
In the case of nanogel carriers designed to deliver chemotherapeu- the rate of both stimuli-responsive drug release and degradation kinet-
tics to tumors, the probability of nanogels reaching the core of the tu- ics along with stability in circulation, and it is always a trade-off be-
mors, even if they are small enough (30–50 nm) and deformable, is tween these properties that decides the ultimate performance of the
limited which, among other factors, is due to the hampered interstitial delivery system. This may lead to a pattern of release very different
transport and the high interstitial fluid pressure [229,230]. In many hyp- from the one usually observed in the in vitro experimental settings.
oxic tumors, the vasculature is poorly developed, and almost all large Nanogels can also demonstrate burst release for their hydrophilic as
tumors have a tendency to develop a hypoxic and necrotic core, thus well as hydrophobic cargoes which can result in substantial loss of the
making it inaccessible for nanogels via the EPR effect [231]. This drug in circulation upon intravenous administration and leaving very
approach of passive targeting is therefore useful only for well- little of the drug to be delivered to the target site through the nanogel
vascularized, small tumors or can be potentiated by extravasation/ carrier, while at the same time exposing healthy organs to toxic drugs
penetration-enhancing pretreatment [77,232,233]. Active targeting of [249]. These limitations can be addressed partially by altering the com-
the nanogels by their conjugation to receptor-specific ligands, although position of the polymers used for the synthesis of nanogels, but add an-
can improve the binding of the carrier to specific cells after extravasa- other layer of complexity to an already intricate system [250,251].
tion, has its own set of challenges. It is often hard to find receptors
that are exclusively expressed only on the tissue of interest. For exam-
6. Conclusions and future perspectives
ple, folate receptor is overexpressed in a large number of malignancies,
but also has a moderate to high level of expression on other normal or-
As a carrier system, nanogels have evolved over time to be able to
gans like small intestine, placenta and kidneys [234,235]. Moreover, the
encapsulate different types of guest molecules. This is a direct result of
expression of receptors on all the cells of the malignant tissue is seldom
the advancement in their synthesis techniques as well as a deeper un-
homogenous, which eventually results in non-uniform accumulation of
derstanding of their material properties like softness and swelling be-
the delivery system in that tissue [236–238]. Active targeting approach
havior. This understanding allows us to explore their applications in
is also challenging to execute from the perspective of nanogel design.
diverse biomedical fields along with the possibility of fine-tuning
Despite of the recent advances in nanoscale fabrication methods that
these properties to our advantage. Progress in analytical techniques
allow better control over the size and particle size distribution of the
also gives us a better insight about their behavior in vivo that can give
nanogels, in majority of cases nanogel samples are heterogeneous, and
a direction to efforts being made to improve their pharmacokinetic
it is difficult to control the stoichiometry of functional biomolecules on
and degradation profiles to design nanogels of the future.
their surface. Biomolecules like antibodies or their fragments, or even
Nanogels can be designed to be compatible with small molecules
peptides are known to be very sensitive to bioconjugation reactions,
like drugs and fluorophores, proteins, peptides, nucleic acids and even
and can easily lose their binding affinity due to the attachment to
inorganic nanoparticles composed of gold, silver, or iron oxide. These
nanocarrier [239]. Reproducibility of the nanogel-antibody conjugates
tiny carriers can also hold a combination of two or more agents depend-
in a consistent manner from batch-to-batch is difficult to achieve,
ing on the purpose, and recent years have witnessed the evolution of
since majority of the conjugation reactions use free amines on the lysine
nanogels as multi-drug carriers and multi-modal imaging agents. They
side-chain or the cysteine residues obtained from reduction of disulfide
can be surface-functionalized to present targeting ligands to a receptor
bridges in the hinge region of antibodies, which not only results in a var-
of interest to home them at the desired site. Nanogels respond to envi-
iable stoichiometry of conjugation but can also affect the binding affinity
ronmental or external stimuli by undergoing volume phase transitions
of the antibody [240–245]. In recent years, various bioorthogonal chem-
that are manifested in the form of swelling of their crosslinked network.
istries have been successfully developed to achieve site-specific conju-
This allows for spatial as well as temporal control of drug release and/or
gation, but these strategies also required introduction of artificial
activation of reporter molecules, which in turn can generate signals for
chemical moieties to the ligand structure and may be less viable for pro-
the purpose of imaging and diagnosis. These properties allow nanogels
teins produced through recombinant or bioengineering strategies [246].
to surpass other nanoparticulate systems in terms of their applicability.
In all cases, the introduction of targeting ligands on the surface of the
Despite the progress made in the field of nanogel design so far, very
nanogels can also have detrimental effects on their surface characteris-
few nanogels have been explored in the clinical studies. The complexity
tics like charge and hydrophobicity, which may lead to increased
of the system and intricate structural properties demand careful engi-
opsonization, aggregation and clearance by the MPS in vivo [247].
neering of the nanogel in order to achieve the desired effect. The scal-
able production and batch-to batch reproducibility can also be hurdles
5.5. Degradation
that need to be addressed. Challenges exist in terms of delivery of the
cargo to the desired site as well as efficient clearance of the nanogels
Because the molecular weight of nanogels is far above the renal
once they have accomplished their mission in vivo. Although many
threshold (~ 40 kDa for copolymers), they cannot be removed from
studies tested the efficacy of nanogel formulations and their safety, re-
the body via the kidneys and if the polymers are nondegradable, so
ports on their long-term accumulation and degradation profiles are
they would tend to accumulate in the body. Even if the nanogels are de-
few and far between. Improvements in the design along with detailed
signed to be degraded into smaller polymer fragments to activate the
investigations regarding the in vivo behavior of the nanogels will help
release of their cargo and eventually facilitate their renal elimination,
in eventually taking them from bench to bedside.
there is a risk of cellular accumulation of polymer chains by sequestra-
tion in the lysosomal compartments. Thus, the use of biodegradable
polymers, both natural and synthetic, is most preferable. However, it Acknowledgments
should be noted that chemical functionalization can alter the pattern
and rate of polymer degradation or even render natural polymer non- The authors gratefully acknowledge the support by an Institutional
biodegradable [248]. Therefore, further investigation of the metabolism Development Award (IDeA) from the National Institute of General
and elimination profile of the polymeric nanogels is warranted before Medical Sciences of the National Institutes of Health under grant
they can be proposed for long-term clinical use. P20GM103480.

Please cite this article as: K.S. Soni, et al., Nanogels: An overview of properties, biomedical applications and obstacles to clinical translation, J. Con-
trol. Release (2015), http://dx.doi.org/10.1016/j.jconrel.2015.11.009
14 K.S. Soni et al. / Journal of Controlled Release xxx (2015) xxx–xxx

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