Article - Cancer-Targeting Nanoparticles For Combinatorial Nucleic Acid Delivery

HHS Public Access
Author manuscript
Adv Mater. Author manuscript; available in PMC 2021 April 01.
Author Manuscript
Published in final edited form as:

Adv Mater. 2020 April ; 32(13): e1901081. doi:10.1002/adma.201901081.
Cancer-Targeting Nanoparticles for Combinatorial Nucleic Acid

Delivery
Hannah J. Vaughan, Jordan J. Green, Stephany Y. Tzeng
Department of Biomedical Engineering, Translational Tissue Engineering Center, and Institute for
NanoBioTechnology, Johns Hopkins University School of Medicine, 400 North Broadway, Smith
Building 5001, Baltimore, MD 21231
Author Manuscript
Abstract
Nucleic acids are a promising type of therapeutic for the treatment of a wide range of conditions,
including cancer, but they also pose many delivery challenges. For efficient and safe delivery to
cancer cells, nucleic acids must generally be packaged into a vehicle, such as a nanoparticle, that
will allow them to be taken up by the target cells and then released in the appropriate cellular
compartment to function. As with other types of therapeutics, delivery vehicles for nucleic acids
must also be designed to avoid unwanted side effects; thus, the ability of such carriers to target
their cargo to cancer cells is crucial. This review will discuss classes of nucleic acids, hurdles that
must be overcome for effective intracellular delivery, types of non-viral nanomaterials used as
delivery vehicles, and the different strategies that can be employed to target nucleic acid delivery
specifically to tumor cells. Moreover, nanoparticle designs that facilitate multiplexed delivery of
Author Manuscript
combinations of nucleic acids will be reviewed.
Graphical Abstract
Author Manuscript
*
green@jhu.edu; stzeng1@jhmi.edu.
Vaughan et al. Page 2
Author Manuscript
Author Manuscript
Synthetic nanoparticles can be used for delivery of combinations of nucleic acid, with different
materials conferring different advantages for safe and effective delivery. Targeting strategies can
also be employed for cancer-specific delivery, including passive targeting based on nanoparticle
physical properties; active targeting using cancer-specific ligands; transcriptional targeting using
cancer-specific promoters; and targeting of cancer-specific proteins or signaling pathways.
Author Manuscript
Keywords
nanoparticles; targeted delivery; gene delivery; cancer therapy; nucleic acid
1. Introduction
The delivery of DNA and other types of nucleic acid allows cells to be altered at the genetic
level. By changing the gene expression profile of target cells, researchers can directly
address the cause of diseases that have a genetic component, including not only inherited
diseases but also acquired genetic diseases like cancers. For instance, malignant cells whose
gene expression patterns are incorrectly regulated could potentially be treated by inducing
Author Manuscript
overexpression of genes that induce apoptosis or by decreasing the expression of genes that
promote cancer cell survival. However, delivery of nucleic acids to cancer cells remains a
challenge. The ability to target cancer cells is crucial to maximize the efficiency of anti-
cancer treatment as well as to avoid causing unwanted side effects on healthy tissues.
Various types of nanoparticles have been studied for their ability to package nucleic acids by
simultaneously protecting the payload from degradation and improving delivery precision.

In this review, we will discuss nucleic acids—largely DNA, mRNA, siRNA, miRNA, and
Author Manuscript
immunostimulatory nucleic acids but also including a few examples of other classes—that
have been studied for cancer therapy, as well as hurdles that must be overcome to deliver
them to cells. We also cover types of nanomaterials used in the field and the properties that
are important for gene delivery. Then, different methods of achieving cancer-specific
delivery of nucleic acids will be reviewed, and finally, we will cover the promise and
challenges of delivering multiple nucleic acids or combinations of nucleic acids with other
therapeutic agents for cancer treatment.
2. Nucleic acids as cargo

This review will focus mainly on nucleic acids that are used to achieve one of three
overarching goals: direct overexpression of a gene (e.g., using DNA or mRNA); knockdown
of a gene using RNA interference (RNAi) by delivering small interfering RNA (siRNA) or
Author Manuscript
microRNA (miRNA); and stimulation of the immune system by delivering nucleic acids that
trigger pattern-recognition receptors (PRRs)[1]. While these are the most commonly studied
in the field, examples of other types of nucleic acids will also be mentioned briefly, and their
properties and delivery challenges are summarized in Table 1.
DNA for overexpression of a gene is normally delivered as a double-stranded (ds) circular

plasmid consisting of, at minimum, a promoter and a gene of interest.[2] DNA elements will
be described in more detail below, as the choice of particular promoters and other design
parameters are an important method by which to achieve cell-specific gene expression. In
order to be expressed, the DNA must pass through not only the plasma membrane of the cell
but also the nuclear envelope, and once in the nucleus, it can be transcribed into mRNA for
export into the cytoplasm and translation into the protein of interest. Alternatively, single-
Author Manuscript
stranded (ss) mRNA itself can be delivered. mRNA is chemically less stable than plasmid
DNA and is more costly to produce at large scale; however, because it acts in the cytoplasm,
it does not have to enter the nucleus as does DNA, simplifying its delivery path.[3, 4]
Additionally, unlike plasmid DNA, mRNA carries no risk of undesired insertion into the
genome, which could result in mutagenesis.
Translation of mRNA can be interrupted by RNAi, leading to decreased expression of a

target gene.[5] The RNAi pathway has been studied for its uses as a tool for basic research[6]
and for disease treatment,[7, 8] and its molecular mechanisms have been reviewed in detail
elsewhere.[9] The pathway can be targeted by short lengths (~20 bp) of dsRNA. including
siRNA, which is derived from cleavage of longer dsRNA strands by the enzyme Dicer, and
miRNA, derived from cleavage of dsRNA with a hairpin loop structure. For both of these,
one ssRNA strand can be incorporated into the RNA-induced silencing complex (RISC) in
Author Manuscript
the cytoplasm. In the case of miRNA, the RNA-RISC structure then binds to mRNAs that
have partial complementarity to the miRNA sequence. This incomplete sequence match
allows miRNA to act on more than one mRNA sequence, leading to a range of potential
effects, including repression or degradation of the mRNA molecule. The ability of miRNA
to regulate pools of genes confers potentially powerful and far-reaching effects. By contrast,
siRNA-RISC binds to an mRNA with complete complementary to the siRNA sequence,
causing mRNA degradation and therefore decreased expression of a specific protein.[10]

Because the effect of siRNA is generally limited to a single gene, unlike that of miRNA, its
Author Manuscript
use could avoid off-targeted and unexpected side effects. Although siRNA is derived from
long dsRNA strands under natural circumstances, researchers more commonly use synthetic
~20-bp siRNA directly as their cargo, bypassing cleavage by Dicer, as long dsRNA (>30 bp)
has been shown to cause unwanted immune responses.[11]
However, in some cases, an immune response is the goal: there are several types of nucleic
acids that have immunostimulatory properties; in fact, the RNAi pathway itself may have
evolved at least in part as a way to combat dsRNA viruses.[12, 13] Some immune cells have
receptors that recognize and are activated in response to certain types of nucleic acid as an
innate defense mechanism against pathogens that carry foreign nucleic acids. Several
examples of these exist, including DNA with unmethylated CpG sequences,[14] ssRNA in
endosomes,[15, 16] and dsRNA,[17] all which are recognized by one or more of the well-
studied Toll-like receptors (TLRs), whose signaling can lead to innate immune responses.[18]
Author Manuscript
Cyclic dinucleotides (CDNs)[19] can induce a type I interferon (IFN) innate immune
response by activating STING (stimulator of IFN genes). Even short siRNAs have been
found to induce an innate immune response,[20] and thus, precautions must be taken to
minimize off-target immune activation through engineering of the sequence or delivery
vehicle; alternatively, if care is taken, this immune response can be leveraged as an
additional deliberate function of siRNAs in addition to sequence-specific RNAi.
Finally, mRNA can also be cleaved by some types of DNAzymes, which are composed of a
15-nucleotide catalytic DNA sequence flanked by two binding regions.[21] The binding
regions recognize a specific RNA sequence via Watson-Crick base complementarity, while
the central catalytic region of this class of DNAzymes cleaves the bound mRNA. This
molecule requires the presence of Mg2+ as a co-factor for activity, and it acts in a catalytic
Author Manuscript
fashion: similarly to classical enzymes, it can dissociate from its mRNA substrate after
cleavage and act on other mRNA molecules.
Common to all nucleic acids are their strong negative charge, their hydrophilic nature, and
their relatively large size, with the molecular weight of siRNAs and miRNAs being on the
order of 10 kDa and that of large DNA plasmids being on the order of 103-105 kDa. They
are therefore difficult to deliver, as they cannot easily pass through cellular membranes and
tissue spaces, and some types of nucleic acids, particularly RNAs and certain patterns of
DNA, are easily recognized by the immune system and cleared. In addition, they are
sensitive to degradation in the presence of nucleases. Even after successful entry into a cell,
they must still overcome other intracellular barriers, including escape from the degrading
conditions of the endosomal pathway, trafficking within the cytoplasm to specific
Author Manuscript
intracellular locations, and/or entry into the nucleus.
Although the sequence of each nucleic acid can affect its function and its intended biological
target, many of the physical and chemical considerations important to encapsulation in
nanoparticles for delivery do not depend heavily on nucleic acid sequence. Thus, while
differences in chemical properties and geometry among different nucleic acid cargos must
still be considered, the co-delivery of multiple nucleic acids of the same type with differing
sequences in the same delivery vehicle follow the same design principles as delivery of a

single sequence of that same type. Co-delivery of different types of nucleic acids, on the
Author Manuscript
other hand, can necessitate changes to nanocarrier design to efficiently deliver and direct
different cargos to distinct cellular and subcellular locations where they can carry out their
function. Combination therapy has been a major focus of study for cancer treatment, as, due
to the heterogeneity of cancers among patients and even within a single patient, treatment
with a single drug is often insufficient to destroy all of a patient’s malignant cells.[22] The
intrinsic resistance of cancer cells to a drug, as well as well as acquired resistance through
mutation, can lead to the selective survival of a population of malignant cells that does not
respond to traditional treatment.[23] The delivery of multiple therapeutic agents in
combination can target different cellular pathways at once, preventing the survival of a drug-
resistant cell population, and the use of nanoparticles as delivery vectors for multiple drugs
in combination has been extensively reviewed elsewhere.[24, 25]
Here, we will discuss the promise of combinatorial nucleic acid delivery for cancer therapy.
Author Manuscript
Non-viral, synthetic nanoparticles, including polymeric or lipid-based nanoparticles and

inorganic nanoparticles, can be designed for encapsulation or complexation of more than
one nucleic acid, although, in every case, the advantages of co-delivering more than one
gene or genetic sequence to achieve additive or synergistic effects must be balanced against
the potential disadvantages of diluting the payload of each sequence. While research is being
conducted on making viral gene delivery vectors safer and effective for delivery of multiple
genes, they are still hampered by several challenges, including intrinsic toxicity or
immunogenicity of viruses, which may be dose-limiting or prevent repeated administration,
often needed for cancer treatment,[26–30] and cargo capacity that is limited by the viral
capsid size, which makes the co-delivery of multiple genes difficult.[31] In this review,
therefore, we will focus on non-viral nanoparticles for their versatility as combinatorial
nucleic acid delivery vehicles.
Author Manuscript
3. Nanoparticles as nucleic acid delivery vehicles

Because of broad similarities in physical and chemical properties of nucleic acids and
overlap in their extra- and intracellular trafficking routes, some major delivery challenges are
applicable for many different types of nucleic acids. However, the specific delivery barriers
that must be overcome may differ from one type of nucleic acid to another and should
therefore influence the design parameters of nanocarriers. Some of these generally
applicable considerations for nanoparticle-based nucleic acid delivery are discussed below
and are also illustrated in Figure 1.
3.1. Nucleic acid binding or encapsulation

Author Manuscript
Nucleic acids on their own are vulnerable to degradation and various mechanisms of
clearance. In order to reach cells intact, they generally must be loaded or condensed into
nano-sized structures that can protect them from the environment and facilitate their
trafficking to target sites, and a range of materials and strategies can be used to accomplish
this. Many canonical delivery carriers are optimized to deliver one particular therapeutic
agent, so encapsulating different types or cargo is a significant hurdle for combinatorial
delivery. Many groups have taken advantage of the high negative charge of nucleic acids by

complexing them electrostatically with cationic materials. Positively charged polymers like
Author Manuscript
poly(L-lysine) (PLL), polyethylenimine (PEI), polyamidoamine (PAMAM), and poly(beta-

amino ester)s (PBAEs) can bind and condense nucleic acids into nanoparticles via
electrostatic interactions with amines.[32, 33] Similarly, cationic lipids have been used
recently to co-encapsulate multiple types of nucleic acids into lipidoid nanoparticles (LNPs).
[34] Liposomes contain both hydrophilic and hydrophobic moieties which can bind small
molecule therapeutic agents for co-delivery with nucleic acids. Artificial vesicles like
liposomes have an aqueous core that can be used to encapsulate the hydrophilic nucleic
acids,[35–37] and other materials, like polyesters, can physically entrap nucleic acids via
emulsification. In particular, PLGA-PEG carriers show desirable small molecule delivery,
but their chemical properties and synthesis techniques are not conducive to co-encapsulation
with nucleic acids. To improve nucleic acid loading in PLGA-PEG polymer nanoparticles,
carriers can be loaded with synthetic nucleic acid analogs click nucleic acids (CNAs), which
bind to nucleic acids in a sequence-specific manner.[38] On the other hand, to improve
Author Manuscript
loading of small molecule drug into carriers optimized for nucleic acid delivery, the drug
may be intercalated into therapeutic DNA prior to loading the agents into a delivery vehicle.
Other nanoparticles, including inorganic nanoparticles, bind nucleic acids by covalent
conjugation.[39, 40] One significant challenge that must be considered in combinatorial
delivery is that the loading of one molecule into the carrier does not affect the loading of a
second agent. The capacity of the carrier is a limiting factor in loading, but further
unfavorable charge or hydrophobic interactions should be anticipated and considered when
designing a vehicle for co-delivery. Loading the interfering agents into different zones or
layers is a potential approach to overcome this challenge.
Differences in size and physical properties among nucleic acids can also affect loading and
binding. For instance, CDNs, siRNA, and miRNA are much smaller than plasmid DNA and
Author Manuscript
thus have fewer binding sites per molecule for electrostatic complexation, and this reduced
multivalency can reduce binding affinity.[41–43] On the other hand, smaller cargos may be
more amenable to chemical conjugation to nanoparticles. mRNA is intermediate in size
between plasmid DNA and smaller RNA oligos, but, as mentioned above, ssRNA is a less
stable molecule than dsDNA,[4] though both are vulnerable to enzymatic degradation by
nucleases under certain conditions. It is thus particularly important to easily degradable
nucleic acids like mRNA that binding and encapsulation by a biomaterial can have an
important effect on the stability of the cargo in addition to condensing into a nanoscale
particle. Electrostatic complexation or chemical conjugation of nucleic acids to cationic
materials can protect them from enzymatic degradation;[44] encapsulation within a vesicle or
solid nanoparticle can also provide a physical barrier to enzymes.[45, 46] Nucleic acid
properties should be carefully considered when combining multiple types into a single
Author Manuscript
carrier. Larger nucleic acids, such as plasmid DNA and mRNA, provide a high density of
negative charge which can stabilize lipid or polymer carriers. Therefore, combination
therapy with larger nucleic acid molecules can enhance the delivery and efficacy of smaller
molecules, even if they act on distinct targets.[34] However, the relative amount of these
stabilizing nucleic acids should be optimized, as very stable carriers with strong electrostatic
interactions may not efficiently release their cargos.

3.2. Cellular uptake

Author Manuscript
Most of the nucleic acid cargos mentioned, including RNA oligos, mRNA, plasmid DNA,
and immunostimulatory nucleic acids that act in the endosome or cytosol, must be
internalized by target cells in order to function. Because the glycocalyx and lipid bilayer of
cells have a net negative charge on the extracellular surface, cationic nanoparticles, in
addition to their ability to bind nucleic acids, can facilitate cellular binding and uptake via
electrostatic interactions.[47–50] Surface modification with certain moieties, such as cell-
penetrating peptides (CPPs), can be used to further improve overall cellular uptake of
nanoparticles.[51–55] In some cases, the oligonucleotides themselves can lead to cellular
uptake by interaction with scavenger receptors,[56] as in the case of spherical nucleic acids
(SNAs). In SNAs, nucleic acids are bound covalently to the surface of gold nanoparticles,
the three-dimensional architecture of the nucleic acids drives uptake, even in later versions
of SNAs in which the metallic core is dissolved and removed, leaving the nucleic acid shell.
Author Manuscript
[57]
3.3. Endosomal escape and intracellular trafficking

Upon uptake, nanoparticles are generally localized in the endosomal-lysosomal
compartment. Some immunostimulatory nucleic acids, such as agonists of TLR3 (dsRNA),
TLR7/8 (ssRNA), or TLR9 (DNA with unmethylated CpG sequences) act in the endosome,
where their receptors are located, and they can thus act in that compartment.
By contrast, plasmid DNA, mRNA, and RNA oligos avoid degradation in the endosome and
then escape into the cytoplasm in order to be effective. Reversibly protonated cationic
materials, including PEI and PBAEs,[58] can act as a buffer for protons that are pumped into
endosomes, preventing the compartment from becoming overly acidified. This in turn leads
Author Manuscript
to an influx of chloride into the endosome due to the building electrical gradient, followed
by an influx of water due to osmotic pressure, which finally causes the endosome to lyse and
release the nanoparticles into the cytoplasm. This “proton sponge” mechanism is regarded as
one reason for effective endosomal escape by certain cationic nanoparticles.[59] Materials
that can fuse with the endosomal membrane can also facilitate escape,[60] including CPPs,
[33, 52, 53] which also aid in the initial cellular uptake. Other mechanisms have been reported
recently to achieve endosomal escape for combinatorial nucleic acid delivery, including the
inclusion of hydrophobic phenyl groups into dendrimers[61] and the linking of lipid moieties
to a cationic aminoglycoside.[62]
Once out of the endosome, the nucleic acid cargo must be released from the nanoparticle,
which can occur by a decrease in binding between cargo and delivery material and/or
degradation of the material itself.[63, 64] Some common materials like polyesters and PBAEs
Author Manuscript
degrade by hydrolysis, the latter over the course of hours at pH 7 and at physiological
temperature. Interestingly, PBAEs are protonated at endosomal pH and thus less likely to
deprotonate surrounding water molecules to form the stronger −OH nucleophile, and they
are in fact less prone to degradation while inside the slightly acidic endosome than they are
at neutral pH.[65] Thus, they can keep nucleic acid cargo intact while in the endosome and
then release their cargo in the cytoplasm upon escape. Materials like PLGA and PLA have a
wide range of different degradation kinetics, varying from days to weeks or even months,

and their cargo can be released over time as they erode. The intracellular destination of
Author Manuscript
nucleic acids may affect the choice of release mechanism when designing nanoparticles. For
instance, siRNA, miRNA, mRNA, or other cargo that acts in the cytosol would benefit from
a mechanism of fast, triggered cytosolic release. This can be facilitated by bioreducible
moieties in the nanoparticle chemistry, as the cytosol of cells is a relatively reducing
environment compared with the extracellular space.[66] Plasmid DNA, on the other hand,
must enter the nucleus, and the specific barriers to nuclear entry have been reviewed
previously.[67] In brief, diffusion of large biomacromolecules like plasmid DNA through the
cytoplasm to the nucleus is very slow, although this can be improved upon by the attachment
of a nuclear localization signal (NLS) to the plasmid or the nanocarrier,[68] promoting the
binding of the DNA cargo to import proteins that can actively facilitate nuclear transport and
entry.[67] It is therefore unsurprising that plasmid DNA delivery by non-viral nanocarriers
has often been found to be much more efficient in dividing cells,[69–71] and while non-
mitotic mechanisms of nuclear entry have been demonstrated,[70–72] nuclear transport
Author Manuscript
remains a key challenge for non-viral delivery of plasmid DNA. Given the long history of
using uncontrolled cell division as a method of targeting cancer cells while they are in
mitosis,[73] however, this nuclear barrier to plasmid DNA delivery could in fact serve as a
potential avenue to improving the selectivity of an anti-cancer gene therapy.
When combining multiple types of nucleic acids, or co-delivering a drug and nucleic acid,
the desired release kinetics of the cargos must be considered. Sequential delivery may be
desired, for example if a drug is intended to sensitize cells to a nucleic acid therapy or if the
nucleic acid cargo knocks down drug efflux transporters to enhance the potency of a
chemotherapy. In other cases, simultaneous action is preferred, such as for multiple nucleic
acid cargos which act on an oncogenic pathway. To achieve precise release kinetics for each
agent, the components may be incorporated into different compartments within the carrier,
Author Manuscript
where the release of the agent is dependent on the degradation behavior of its encapsulating
material. Layer-by-layer synthesis techniques can be used to deposit different drugs or
nucleic acids to the surface of a carrier in a layered manner, which facilitates sequential
release of the components.[74] Alternatively, nucleic acids may be conjugated to the particle
surface using different chemistries with distinct release kinetics or stimuli-responsive
behavior. To achieve complex release behavior of multiple agents, Badeau et al. have
developed a modular system for logic gated release of various cells from a hydrogel.[75] By
conjugating the cells with different linking moieties in series or in parallel, they achieved
YES/OR/AND outputs from combinations of environmental stimuli, including enzymes and
light. This strategy enables highly complex responsive release kinetics and has clear
applications in combinatorial delivery systems.
Author Manuscript
3.4. In vivo stability of nanoparticles

The physicochemical properties of nanoparticles can affect their fate in vivo after
administration. Nanoparticles are often cleared quickly from circulation by the mononuclear
phagocyte system (MPS),[76, 77] a process that can be prevented or slowed by reducing
interactions between nanoparticles and cells. One common method is to shield the surface of
the nanoparticles by coating them with poly(ethylene glycol) (PEG), or PEGylation.[78]
Grafting of PEG chains to nanoparticles has been reported to increase circulation time of

various types of carriers and therefore allow accumulation at tumor sites,[79–82] as described
Author Manuscript
in detail in the next section.
Because of the many necessary characteristics of nucleic acid delivery nanoparticles, all of
the properties described here should be considered together. For example, while cationic
materials are convenient for encapsulation or complexation of anionic cargo, positively
charged materials can also cause toxicity by disrupting the cell membrane;[83] on the other
hand, decreasing the molecular weight[84] or introducing biodegradable functional groups[85]
into the polymer materials has been shown to increase the safety of the nanomaterial.[86–88]
Thus, a combination of optimized parameters should be used to design an ideal nanoparticle.
4. Tumor Targeting
An overarching challenge in cancer therapeutics, including in nanomedicine, is achieving
Author Manuscript
sufficient concentrations of an anti-cancer agent in the tumor while minimizing off-target

toxicities in healthy tissues. Targeting methods take advantage of features that differentiate
cancer from healthy tissue, including properties of the tumor microenvironment,
overexpressed molecules on the cancer cell surface, and dysregulated gene expression.
Nano-scale delivery vehicles are uniquely capable of passive targeting, active targeting via
ligand functionalization, and controlled or triggered release. Additionally, nucleic acid
cargos can themselves be engineered to take advantage of aberrant gene expression in cancer
cells and achieve cancer specificity through their mechanism of action. Effective targeting is
particularly important for combinatorial nucleic acid delivery. As discussed, certain
combinations of nucleic acids can induce potent apoptotic or immunogenic effects, which
are associated with toxicity in healthy tissues. Further, combining multiple nucleic acids will
proportionally dilute the dose of each. Therefore, tumor targeting must be carefully
Author Manuscript
considered when developing these delivery vehicles to maximize cargo delivery to cancer
cells while avoiding dangerous off-target effects.
4.1. Passive Targeting

Hypervasculature, enhanced vascular permeability, and decreased lymphatic drainage are all
hallmarks of rapidly growing tumors. The abnormal architecture of angiogenic tumor blood
vessels underlies the enhanced permeability and retention (EPR) effect, which describes the
tendency for systemically administered macromolecules within a 10–200 nm size range to
accumulate in solid tumors.[89] Nanoparticle formulations of chemotherapies, such as
Doxil® (for doxorubicin) and Abraxane (for paclitaxel), were developed in response to this
phenomenon and have improved pharmacokinetics, an increased maximum tolerated dose,
less systemic toxicity, and improved therapeutic efficacy compared to their free drug
Author Manuscript
counterparts.[90] Nanoparticle size is a key predictor of passive targeting, because size

affects clearance rate and route, extravasation into tumor tissue, cellular uptake, and
interactions with the immune system. The range for passive targeting by the EPR effect is
around 10–200 nm, although tumor accumulation has been described for slightly smaller or
larger particles. Particle accumulation in the tumor compartment is in competition with
clearance, either by the renal system or the MPS, which is comprised of macrophages
predominately in the liver and spleen. Particles smaller than 10 nm are rapidly cleared by the

renal system and tend to accumulate in the kidney, while larger particles accumulate in the
liver and spleen.[91] Both clearance routes compete with accumulation in tumor tissue, so
Author Manuscript
developing particles with low clearance rate and high circulation time is the primary goal for
passive targeting by EPR. To further elucidate the effect of particle size on EPR and tumor
accumulation, Perrault et al. systematically studied the biodistribution of 10–100 nm PEG-
coated gold nanoparticles (mPEG-GNP) in subcutaneous MDA-MB-435 xenograft tumors.
[92] Their results indicate that, for 40–100 nm particles, longer circulation times directly
correlate with improved tumor accumulation. Smaller particles in the 20 nm range had lower
tumor accumulation than larger particles, but these smaller particles had significantly
enhanced permeation into tumor tissue.[92]
Inspired by the cylindrical or filamentous shapes of many viruses, non-spherical geometries

have been explored for therapeutic drug and nucleic acid delivery. Long cylindrical, rod-like,
disk-shaped, or filamentous particles have been shown to evade phagocytosis by resident
Author Manuscript
macrophages. There is evidence that the contact angle between macrophages and particles
affects phagocytic uptake; contact with a flatter surface, such as a rod or disk, causes
macrophage spreading rather than phagocytosis.[93] Additionally, in dynamic fluid flow,
filamentous particles elongate and align with flow, and hydrodynamic shear forces pull
particles off of macrophages.[94] These effects slow the uptake of filamentous particles by
the MPS and can dramatically increase circulation times. For example, Geng et al.
developed filamentous micelles (filomicelles) comprised of a biodegradable co-polymer that
remain in circulation 1 week after injection, while spherical versions are cleared within 2
days (Figure 2).[95] Increased circulation time is correlated with enhanced therapeutic
efficacy of paclitaxel-loaded filomicelles in a subcutaneous xenograft model.[95] Micelle
length appears to have been be a critical factor: an eight-fold increase in filomicelle length
had the same therapeutic benefit as an eight-fold increase in paclitaxel dose.[95]
Author Manuscript
Along with size and shape, surface properties are a key parameter affecting biodistribution
and cellular uptake. For example, nanoparticles with a strong charge or a hydrophobic
surface attract serum proteins, which adsorb to the surface and form a protein corona.
Adsorption of opsonins, such as IgG or complement factor, tag the protein for clearance by
the immune system. A protein corona can also block targeting ligands that have been
conjugated to the particle surface. Therefore, a neutral hydrophilic charge is typically
desirable for systemically administered nanocarriers. As mentioned above, PEGylation of
particles is often used to reduce non-specific protein adsorption.[96, 97] PEG can be
incorporated into block co-polymers, conjugated to the surface of inorganic particles, or
incorporated into liposomal formulations, making it an attractive option for a variety of
delivery applications. PEGylation reduces uptake by the MPS and dramatically extends
Author Manuscript
circulation time, particularly when particles are coated with high-molecular weight PEG at a
high density.[98, 99] Thus, many nanocarriers for cancer targeting are coated with PEG to
increase blood circulation half-life and enhance passive targeting. However, bioinert
hydrophilic carriers, due to their reduced binding to proteins and cellular components, also
tend to have poor intracellular uptake and endosomal escape, and this related but unintended
consequence is known as the PEG dilemma.[100] Strategies to overcome this dilemma,
including active targeting and stimuli-responsive carriers, will be discussed in subsequent
sections.

While EPR has been a significant discovery in preclinical models, passive targeting is a
Author Manuscript
complex and highly variable process that depends on the size, degree of vascularization, and
location or the tumor.[101, 102] There is also often a high degree of heterogeneity within
tumors, with changes in cell density, interstitial pressure, and extracellular matrix
composition affecting how nanoparticles move through different regions of tumor tissue.[103]
These parameters are not easily recapitulated in vitro, and screening nanoparticles for in
vivo delivery is traditionally low throughput and expensive. Recently, barcoded
nanoparticles have been used to assess systemic nucleic delivery in a high throughput
manner. Dahlman et al. incorporated barcoded oligos into nanoparticles formulated with
different lipid compositions and pooled dozens of distinct formulations into a single
systemic injection.[104] Using Illumina sequencing, this group was able to assess the effects
of nanocarrier formulation on biodistribution and uptake of different nucleic acid
nanoparticles. With this tool, the researchers have identified a successful liposome
formulation for specific mRNA delivery to lung endothelial cells.[105]
Author Manuscript
4.2. Active Targeting

The large surface-to-volume ratio inherent to nanocarriers facilitates their interactions with
biomolecules and cells. While methods described above have been employed to minimize
this, thus extending the circulation time of nanocarriers, such interactions can also be
leveraged as an advantage. Particles may be functionalized with active-targeting molecules
that will specifically interact with the target and enrich particle accumulation in that site.
[106–108] Nanoparticles, including those for combinatorial delivery, have been functionalized
to target various surface macromolecules on cancer cells, including overexpressed or

mutated proteins, altered glycoproteins or glycolipids, and cancer-associated fetal proteins.
An alternative strategy is specifically targeting cell types which are susceptible to the
Author Manuscript
particular nucleic acid combination. For example, targeting drug efflux pumps to deliver a
nucleic acid combination to combat drug resistance, or targeting markers of difficult-to-treat
stem-like tumor cells for combination miRNA delivery. Particles can also target molecules
overexpressed in the tumor microenvironment, such as integrins, which are overexpressed on
tumor vasculature.[109, 110] Optimizing active molecular targeting of nanocarriers requires
careful selection of a conjugation chemistry by which to attach targeting molecules to the
particle surface. Different conjugation strategies can be selected depending on the bulk
particle material, targeting ligand, and desired application. It is possible to harness
hydrophobic or electrostatic interactions to functionalize nanoparticles by nonspecific
adsorption, which enables functionalization without chemical modifications or complex
reactions.[111] However, this requires the use of large amounts of targeting molecule, and
targeting ligands may be displaced by other biological molecules in a physiological
Author Manuscript
environment. Thus, covalent conjugation is commonly used for irreversible attachment of

targeting ligands. Biological molecules such as proteins or peptides contain primary amines,
which react with activated carboxylic acid groups on a nanocarrier to form an amide bond.
[112] Common carboxylic acid-activating compounds include carbodiimidazole (CDI), as
well as carbodiimide compounds, such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide

(EDC), often in combination with N-hydroxysuccinimide (NHS), or
dicyclohexylcarbodiimide (DCC).[113] Alternatively, cysteine residues in proteins or
peptides contain thiol groups that can be reacted with maleimide-containing particles.[114]

Proteins can also be functionalized with biotin for reaction with streptavidin-coated
Author Manuscript
particles. To control ligand orientation on the particle surface, these reactive groups can be
introduced at specific locations on the protein, either during synthesis or after protein
purification.[108, 115] Particles can also be coated with Protein A or Protein G, which bind
the Fc region of antibodies and facilitates properly oriented conjugation. Conjugating
targeting molecules via a flexible linker, such as PEG, allows the conjugated ligand or
antibody to rotate and move freely in space for optimal binding with a target.[116]
Conjugation methods can also dictate ligand density on the particle surface, which has
proven an important parameter for optimization. For example, using various ratios of ligand-
functionalized and unfunctionalized PEG for particle coating can significantly affect uptake
by target cells.[117] While enriching the targeting ligand can lead to multivalent
complexation and enhanced affinity, a saturation effect or even reduced binding has been
reported.[118, 119] This effect has been attributed to multiple factors, including steric
Author Manuscript
hindrance or suboptimal receptor clustering. Nanoparticle size and shape also dictates
surface curvature and contact surface area, which can affect interactions between particles
and target cells.[120] Therefore, although ligand density has been optimized in detail for
particular nanocarriers,[121] optimal parameters vary greatly depending on the size, shape,
and material composition of the particle as well as size, chemistry, and avidity of the
particular targeting ligand.[122, 123] These parameters are summarized in (Figure 3).
4.2.1 Antibodies and Fragments—Antibodies have been extensively explored as

therapeutics because they can be engineered to target almost any antigen with a high degree
of specificity.[124] Monoclonal antibodies have been used in cancer therapy for over 20
years, and patient responses are well-understood, so they are a natural choice for
nanoparticle targeting.[125] While antibody therapies work by blocking or binding a receptor
Author Manuscript
on a cancer cell, nanoparticles harness the specificity of antibodies while incorporating

additional therapeutic modes of action by encapsulating a drug or imaging agent or acting as
a therapeutic itself. For example, trastuzumab is a monoclonal antibody for HER2 that is
used clinically for breast cancer, and conjugating this antibody to gold nanoparticles showed
promise as a photodynamic therapy, a strategy to ablate tumor tissue with a high degree of
precision.[126] Antibodies can also bind to endocytic receptors on target cells and facilitate
cellular uptake, which is necessary for functional nucleic acid delivery.[127, 128] However,
antibodies are bulky and significantly increase the size of conjugated nanoparticles. Safety
concerns have also been raised, even with clinically approved monoclonal antibodies,
regarding their immunogenicity.[129]
With the clinical success of antibody therapies came interest in developing molecules with
Author Manuscript
the same specificity but smaller size. That initiative sparked the invention of next-generation
antibodies, including single chain antibodies, domain antibodies, and nanobodies.[130–132]
Single-chain antibody fragments have been extensively explored for active targeting, since
they maintain the antigen-binding capability, are one fifth the size of full antibodies, and are
considered safer than antibodies.[133] Antibodies and fragments are developed through
display libraries, which preferentially and rapidly select for candidates which bind target
cells and are internalized.[134, 135] Because fragments can be screened for intracellular
uptake, functionalized particles have been explored for nucleic acid delivery. For instance,

liposomes surface-conjugated to a melanoma-targeted antibody fragment showed a

Author Manuscript
significant therapeutic benefit in melanoma lung metastases, while non-targeted control

particles had no significant effect.[136] Because of their small size, antibody fragments
targeting EGFR have been conjugated to very small particles (quantum dots and 10-nm iron
oxide nanoparticles) for high-sensitivity diagnostic imaging.[137]
In particular, antibody targeting can enhance the particular action of nanoparticles for
combinatorial nucleic acid delivery. Certain nucleic acids, such as drug efflux-targeting
RNAi, are designed to act on drug-resistant cells, which can be targeted by surface receptors
overexpressed on the membrane. For example, the drug efflux pump P-glycoprotein has
been targeted using antibody-conjugated nanoparticles to deliver curcumin specifically to
multi drug resistant cervical cancer cells.[138] In this case, active targeting significantly
increased the specific uptake of PLGA nanoparticles and improved the efficacy of the
therapy in vitro.
Author Manuscript
4.2.2. Ligands—Another approach to cancer cell targeting is functionalizing

nanocarriers with ligands for overexpressed receptors, such as epidermal growth factor
receptor (EGFR) and transferrin receptor. Many of these surface receptors are well-
characterized cancer biomarkers, and ligands for these receptors have been identified and
studied, which has led to their extensive development for active targeting. The natural
ligands for overexpressed receptors range from proteins to carbohydrates to small molecules,
such as vitamins.[139] Alternative small molecule ligands may also be developed using
computational modeling and binding experiments, which expands targeting possibilities
beyond the native ligand for a receptor.[140]
Cancer-specific receptors can have a variety of functions, but nucleic acid delivery benefits
Author Manuscript
from active targeting to endocytic receptors, which facilitate cancer-specific uptake and
intracellular delivery. The transferrin receptor is a commonly overexpressed endocytic
receptor on cancer cells and can be targeted by transferrin (Tf) protein-coated particles.[141]
Early preclinical work shows 20-fold higher tumor transfection with Tf-PEI-DNA particles
than with non-targeted PEI-DNA particles, which corresponded with 30-fold lower off-target
transfection in the lungs.[142] Transferrin-functionalized PEG-coated cyclodextrin particle
CALAA-01 was the first RNAi therapeutic to be tested on patients in clinical trials.
Systemically administered CALAA-01 was well-tolerated in phase 1 clinical trials, and there
was dose-dependent nanoparticle accumulation and gene silencing in melanoma tumor
biopsies.[143, 144] In another example, hyaluronic acid (HA) functionalization facilitates
uptake via overexpressed CD44 receptors. HA can serve as a bulk scaffold material for self-
assembled nanoparticles or as a coating to functionalize particles composed of alternate
Author Manuscript
materials, such as mesoporous silica.[145, 146] In mouse studies, pre-treatment blockade with
free hyaluronic acid reduces the concentration of HA-NPs localized in the tumor, indicating
that active HA targeting plays a significant role in tumor accumulation.[147, 148]
Small molecule ligands also offer specificity to overexpressed receptors with minimal
increase in particle size. Folic acid has been extensively studied as a targeting ligand due to
its high affinity of the folate receptor, which is overexpressed in approximately 40% of
cancer types, including breast, lung, ovarian, and colorectal cancers.[149, 150] Folate-

conjugated therapeutics readily bind folate receptors and are internalized into the endosome
Author Manuscript
through receptor-mediated endocytosis, which has enabled many folate-conjugated drugs

and imaging agents to reach clinical trials.[151] Folic acid was one of the earliest molecules
used for nanoparticle targeting, but recent advances have further enhanced its efficacy as a
cancer-targeting ligand.[152] Advances in linker chemistry have enabled efficient recycling of
folate receptors back to the cell membrane after particle internalization, which facilitates
continuous gene delivery to cancer cells.[153] Incorporating PEG into nanoparticle
formulations reduces non-specific endocytosis by healthy cells that do not express folate
receptor, and PEG linkers also enhance folic acid binding with membrane-bound receptors.
[154] Prostate-specific membrane antigen (PSMA) has also been widely investigated as a
receptor target due to its expression in many prostate tumors as well as the neovasculature of
other solid tumors. Small molecule 2-[3-[5-amino-1-carboxypentyl]-ureido] pentanedioic
acid (ACUPA) is a high-affinity small molecule ligand of PSMA which has shown targeting
properties in vivo and in vitro.[155, 156] BIND-14, a PEG-PLGA nanoparticle with ACUPA
Author Manuscript
active targeting for docetaxel developed by BIND Therapeutics, was evaluated in clinical
trials for PSMA-targeted docetaxel delivery to prostate cancer.[123] There was no significant
toxicity, and a 12% overall response rate was observed in phase I clinical trials.[157] ACUPA
has recently been employed for PSMA-targeted delivery of siRNA to prostate cancer cells.
For example, Xu et al. developed a nanocarrier comprised of a pH-responsive polymer blend
coated with PEG-ACUPA for PSMA targeting (Figure 4).[158] This delivery vehicle was
used to deliver siRNA targeting prohibitin 1 (PHB1), which is overexpressed in prostate
cancer.[158] Briefly, nanoparticles extravasate through leaky tumor vasculature and associate
specifically with prostate cancer cells via interactions between ACUPA and PSMA. The pH-
responsive polymer triggers rapid disassembly upon internalization, and the oligoarginine
sharp domains facilitate endosomal escape and efficient siRNA delivery.[158] ACUPA-
targeted NPs significantly suppressed tumor growth over non-targeted NPs after 30 days,
Author Manuscript
showing the benefit of active targeting in this case.[158]
Diseased or cancerous cells often display a different array of surface glycans compared to
healthy cells, offering another targetable feature.[159, 160] Lectins can be used to target drugs
or nanoparticles specifically to cells displaying these abnormal features.[161] One useful
example is targeting asialoglycoprotein receptors on liver cancer cells with galactosamine-
conjugated nanoparticles.[162–164] Lectin conjugation can also facilitate transport across the
blood-brain barrier, which typically serves a major hurdle for delivery to brain tumors. For
example, nanoparticles modified with wheat germ agglutinin enhanced delivery to the brain
two-fold over unmodified nanocarriers.[165]
An alternative to direct ligand conjugation is coating nanocarriers with cancer cell

Author Manuscript
membranes. Recent research describes homotypic cancer cell binding—a phenomenon

where cancer cells preferentially bind to membranes which carry the same surface antigens.
[166] To harness this feature, researchers have coated nanoparticles with modified and
unmodified cancer cell membranes, which provides a stealth coating as well as tumor
homing properties.[167] This straightforward approach enables particle functionalization with
the complete range of tumor ligands in a biomimetic manner and facilitates cancer-specific
accumulation and uptake.

4.2.3 Aptamers—As discussed, many ligand-based active targeting systems are

Author Manuscript
restricted to a biological ligand binding its natural target receptor. Aptamers are single-
stranded DNA or RNA oligos that serve as attractive targeting alternatives. These nucleic
acid molecules form sequence-specific three-dimensional structures and can be engineered
to bind virtually any target, from small molecules and single amino acids to proteins,
carbohydrates, and whole tumor cells. Targeted aptamers are selected from a large library of
random sequences using systematic evolution of ligands by exponential enrichment
(SELEX).[168] In each round of this process, the aptamer library is exposed to the desired
target, unbound sequences are washed away, and bound sequences are selectively eluted and
amplified using PCR. The process is repeated in subsequent selection rounds with more
stringent binding conditions to generate tightly binding aptamers with antibody-like affinity
and specificity.
Aptamers have been extensively explored in the past decade and have yielded novel
Author Manuscript
selection methods for cancer targeting. Cell SELEX selects for aptamers that bind to a
monolayer of cultured cancer cells, rather than purified antigen.[169] This selection method
generates a diverse pool of targeting aptamers that bind to or are internalized by the target
cell population (Figure 5).[169–171] Methods have also been developed for in vivo SELEX,
where aptamers are selected by binding tumors in situ in animal models.[172] This has
resulted in aptamers with minimal off-target binding to healthy tissues that can bind multiple
cell types in heterogeneous tumors.[173] Because aptamers can be generated for a range of
molecules expressed in tumors, this targeting approach circumvents the problem of
resistance, which is common when targeting a single receptor.[174]
Aptamers are attractive for nanoparticle targeting because they have low molecular weight
and can include chemical modifications for particle conjugation.[175] In one example, PSMA
Author Manuscript
aptamer-targeted PLGA-PEG nanoparticles loaded with docetaxel were used to treat animals
with subcutaneous LNCaP prostate tumors, and the survival rate was 100% in treated
animals after 109 days.[176–178] Aptamer-nanoparticle conjugates have also been
investigated for cancer detection and imaging with quantum dots and iron oxide
nanoparticles.[179–181] More recently, peptide nanoparticles functionalized with doxorubicin
and MUC1 aptamer were used for targeted delivery of chemotherapy to cancer cells while
simultaneously monitoring drug release in real time.[182] The anti-VEGF aptamer therapy
pegaptanib was clinically approved in 2004 to treat macular degeneration,[183] and other
aptamers are in clinical development to treat conditions ranging from thrombosis[184, 185] to
leukemia.[186] Aptamer functionalized nanoparticles could be a promising approach for
high-specificity binding to newly emerging cellular targets.
Author Manuscript
4.2.4 Integrin Targeting—The integrin profile of tumors is distinct from that of healthy
tissues, and several integrins are upregulated on tumor endothelial cells and cancer cells.
[187, 188] The α β integrin is significantly upregulated in many cancers and can be targeted
V 3
with a simple RGD peptide motif. RGD-targeted nanoparticles bind selectively to αVβ3
integrins, and this interaction facilitates selective endocytosis into angiogenic endothelial
cells and cancer cells.[189] This approach has been used to efficiently deliver nucleic acids to
tumor vasculature, for example to deliver siRNA against VEGF-R2 and inhibit both
angiogenesis and tumor growth.[190] Because αVβ3 is expressed on both endothelial cells

and cancer cells, RGD facilitates cancer cell targeting in addition to endothelial targeting.
Author Manuscript
This dual targeting is a major advantage of RGD-functionalized nanocarriers. RGD-targeted

chitosan nanoparticles containing siRNA have been used to successfully downregulate drug
efflux transporter P-glycoprotein expression and reverse multidrug resistance in a breast
cancer model.[191] Other integrins can serve as therapeutic targets, including arresten (α1β1),
canstatin (αvβ3 and αvβ5), angiostatin (αvβ3), tumstatin (αvβ3), endostatin (αvβ3, αvβ5, and
α5β1), and endorepellin (α2β1).[192] Nanoparticles have been used to deliver these anti-
angiogenic agents[193] or the genes that encode them.[194]
Despite promising preclinical data for active targeting in nanomedicine, no FDA-approved

nanocarriers have employed active targeting strategies. Active targeting increases the
complexity and potential immunogenicity of a drug delivery system, which makes it more
difficult, time consuming, and expensive to develop. Further complicating their optimization
is the binding site barrier effect, where antibodies or nanoparticles bind target cells with high
Author Manuscript
affinity and cannot penetrate throughout the tumor.[195, 196] BIND-014 was an early targeted
nanocarrier to enter clinical trials, and it benefitted from rigorous and systematic
optimization of particle properties (size, surface properties, drug loading etc.) in preclinical
studies and biodistribution validation in multiple animal models (mouse, rat, and monkey).
[123] This rigor is essential when increasing the complexity of a platform and should be a
model for active targeting in the future.
Target identification remains a major hurdle, and even well-characterized targets are almost
always heterogeneously expressed within tumors. Additionally, many of the receptors
overexpressed on cancer cells, including transferrin and folate receptors, are also expressed
on proliferating healthy cells, so targeting these receptors can lead to off-target side effects
and systemic cytotoxicity.[197] Thus, efforts should be focused on developing companion
Author Manuscript
diagnostic methods to characterize target expression and predict patient response.

Radiolabeled tracers based on the RGD peptide sequence are already in clinical development
for monitoring αVβ3 integrin expression in patients.[198] Such advanced diagnostic tools are
needed to study biomarker distribution within patient populations and assess the feasibility
of actively targeted nanocarriers for personalized medicine.
4.3. Stimulus-Responsive Targeting

As tumors grow and develop, cancer cells exist in a constantly changing environment,
influenced by high cell density and low blood supply. Solid tumors have an abnormally
acidic pH, are subjected to low oxygen, and have a high concentration of certain enzymes.
[199] These properties of the tumor microenvironment are targetable features that can be
exploited for nanoparticle targeting and controlled release. To respond to an environmental

Author Manuscript
stimulus, nanocarriers must include responsive chemistries that change the properties of the
particle when it encounters a trigger. This triggered response can expose binding domains,
dismantle a protective coating, or change particle surface properties to facilitate cancer-
specific uptake.
Zwitterionic polymer nanoparticles are neutrally charged in physiological conditions, which

confers stability, resists serum protein adhesion, and prevents clearance.[199] In the slightly
acidic tumor microenvironment, the zwitterionic polymer becomes protonated and switches

to cationic, facilitating uptake into cancer cells.[200] Cleavable PEG linkers can similarly be
Author Manuscript
used to facilitate tumor cell uptake. Inert PEG coatings are commonly used in drug and gene
delivery to enhance particle stability in circulation and increases the circulation half-life.
However, as mentioned above, PEG coating also tends to decrease cellular uptake of
particles.[100] To address this, researchers have coated particles with cleavable PEG, which
is released in response to a trigger. For example, matrix metalloproteinases (MMPs),
enzymes associated with angiogenesis and tumor growth, have been explored for triggered
PEG de-shielding in a tumor-specific manner.[201] A multifunctional envelope-type nano-
device (MEND) functionalized with MMP-cleavable PEG maintained the prolonged serum
stability characteristic of PEG functionalized particles. Further, carriers conjugated with
cleavable PEG exhibited superior in vitro and in vivo tumor transfection over carriers with
non-cleavable PEG.[202, 203]
Responsive vehicles can also facilitate cytosolic release of nucleic acid cargo, which is
Author Manuscript
particularly important for an efficient therapeutic response from RNA, since these molecules
act in the cytoplasm. Polymer nanoparticles often incorporate disulfide bonds, which
degrade in the reducing environment of the cytoplasm and release their cargo. Using a
bioreducible polymer reduces the toxicity of the nanocarrier while ensuring full protection
of the easily degraded RNA cargo. pH can also trigger release in the acidic endosomal
environment for site-specific cellular delivery. Acidic pH can trigger a shape change or
expansion which disrupts the endosome and releases nucleic acid directly in the cytosol.
Lipid-based liquid crystalline nanoparticles, termed nano-transformers, expand in a pH 5
environment from needle-like structures to nanospheres.[204] This shape transformation was
proposed to have improved endosomal escape by promoting membrane fusion with the
endosome. In another approach, Luo et al used miRNA-catalyzed release to specifically
trigger payload release in the presence of miRNA-21, which is overexpressed in many
Author Manuscript
cancers.[205] Similarly, the DNA “nanosuitcase” developed by Bujold et al. opens

conditionally in the presence of a miRNA or mRNA and releases its therapeutic oligo cargo.
[206] This approach allows triggered intracellular release in response to a genetic biomarker,
limiting its effects to cancer cells. Responsive vehicles for intracellular delivery ensure
efficient and specific cargo release to allow all components in a combinatorial system to act
simultaneously, which is likely important to achieve synergistic effects.
Systemically administered nanocarriers can also be triggered by an external stimulus to

enhance gene delivery at the tumor site. The properties of thermoresponsive polymer
particles change in response to externally applied heat or cold, which can be harnessed for
tumor-specific gene delivery.[207] Poly(N-isopropylacrylamide) (PNIPAM)-based polymer
nanocarriers undergo a hydrophilic to hydrophobic transition when temperature is raised
Author Manuscript
from 37° C to 42°C, which causes the particles to aggregate and enhances endosomal
escape, resulting in 2 orders of magnitude enhanced transfection at hyperthermic sites.[208]
Light-responsive particles have also been used to enhance endosomal escape in a spatially
controlled manner.[209] To harness this for siRNA delivery, a phtotosensitizer was combined
with siRNA and encapsulated using the Lipofectamine™ commercial transfection reagent,
and cells stimulated with light showed 10-fold higher silencing than non-stimulated cells.
[210] Though this strategy has shown efficacy in vitro, photodynamic therapy in vivo is
limited by the penetration depth of visible light through tissue. Magnetic coating or

encapsulation of magnetic material can be used for magnetically guided nucleic acid
delivery to a targeted site.[211, 212] An applied magnetic field is used to concentrate or retain
Author Manuscript
gene delivery particles at the tumor site, which has been used to enhance the delivery of
DNA and siRNA in a process termed magnetofection.[211, 212]
Ultrasound has been extensively used in combination with microbubbles for regio-specific
delivery of anticancer agents. Briefly, a nucleic acid nanocarrier is co-delivered with gas
microbubbles, which are clinically approved for use as a contrast agent.[213] Co-localization
of microbubbles and nanocarriers is essential for successful transfection, so the nanocarriers
must be coupled to the surface of the bubble using covalent conjugation. Then, ultrasound is
applied to the target area, and passage of ultrasound through tissue creates pressure waves,
which cause the microbubbles to undergo cavitation and release energy that opens transient
pores in surrounding cells. The co-delivered nanocarrier enters the cell through these sub-
micron pores, which leads to significantly enhanced transfection at the site where ultrasound
Author Manuscript
was applied. DNA-containing PEI polyplexes conjugated to microbubbles combined with

ultrasound stimulation enabled gene delivery to implanted tumors in a mouse kidney with
40-fold higher expression in tumor tissue than control non-sonicated tissue.[214] In a similar
approach, liposome-bearing microbubbles enhanced the delivery of an anti-fibrotic miRNA
to diseased liver in rats.[215] Microbubble cavitation can also be used to open the blood-brain
barrier and allow systemically administered nanocarriers to reach tumors in the brain.[216]
Because nanocarriers enter through pores in the cell membrane, they bypass endosomal
uptake and are delivered directly to the cytosol.[217] The method is also non-invasive and
provides precise spatial and temporal control over nucleic acid delivery. However, the
precise location of the tumor must be known to effectively apply the ultrasound to the target
site. Image-guided focused ultrasound has been used to localize ultrasound signal more
precisely, particularly for opening the blood brain barrier.[218, 219] Still, this approach is
Author Manuscript
limited to primary tumor sites rather than dispersed metastases.
4.4. Local Administration

When possible, local administration can increase particle concentrations at the target site
while decreasing healthy tissue exposure. Direct intratumoral administration is not an option
in most cases, as accessing the tumor would involve an invasive procedure. Surgical tumor
resection can be used as an opportunity to deliver a therapeutic directly to the site, where any
remaining cancer cells can be treated to prevent recurrence. Implanted drug delivery depots
can deliver a therapeutic at a controlled dose over the course of weeks or months with a
single implantation surgery.[220] Nucleic acid therapeutics have been encapsulated in
hydrogel depots for local delivery and reduced systemic toxicity. Naked DNA can be
incorporated in hydrogels for regenerative medicine and anti-cancer applications, but only
Author Manuscript
low levels of gene transfer have been observed due to the absence of a carrier.[221, 222]
DNA/PEI polyplexes have been successfully encapsulated in hydrogels and show effective
gene transfer in vitro and in choriallantoic membrane assays.[223, 224] siRNA-loaded
micelles have similarly been encapsulated in injectable polyurethane scaffolds for sustained
local gene silencing.[225] The bulk hydrogel can be tuned to achieve the desired release
kinetics, including varying material, molecular weight, crosslinking density, size, and
geometry. Another advantage of hydrogels is the ability to encapsulate multiple separate

agents in a single gel and simultaneously target cancer by multiple modes of action. Locally
Author Manuscript
implanted hydrogel patches have been used to simultaneously deliver chemotherapy, siRNA,
and gold nanoparticles for photothermal therapy.[226] This strategy can also be applied to
deliver nucleic acid cargos with different properties, for example RNA and DNA, at the
same site with particles optimized for each particular cargo. Nanocarrier properties can be
independently tuned for efficient and targeted nucleic acid delivery, but maintaining stability
and bioactivity of encapsulated particles is a significant hurdle to successful transfection.
Certain tumors can be accessed with non-surgical and non-invasive delivery routes, which
reduces the potential for complications and enables frequent repeated particle dosing.
Aerosol delivery provides direct access to lung tissue and can be used as a delivery route for
lung cancer.[227, 228] An inhalable cationic liposome formulation for plasmid DNA is in
clinical trials to treat cystic fibrosis, and a hyperbranched PBAE nanoparticle recently was
used for mRNA delivery to lung epithelium (Figure 6).[229, 230] Patel et al. reported that
Author Manuscript
hDD90–118 polypexes remained stable after aerosolizing with a vibrating mesh nebulizer,
which produced micro-sized droplets ideal for distribution throughout lung tissue.[229] They
were able to transfect 24.6% of lung epithelial cells after a single dose, with transfection
seen in all five loves of the lung.[229] Delivery to other tissues, including the liver, spleen,
and heart, was negligible, and there was no observed local or systemic toxicity.[229] While
this platform has not been employed in lung cancer models, cancer treatment is an obvious
potential application of these new inhalable technologies. The skin is uniquely accessible for
local delivery, so topical applications are being explored for nanoparticle gene delivery to
skin cancer.[231] Nanocarriers can be used to control permeation through the skin, and
transport properties can be controlled independently of the cargo.[232] Chitosan
nanoparticles have been used for antisense oligonucleotide and plasmid DNA delivery to
skin, with reporter gene effects persisting on rat skin for 6–7 days.[233, 234] Free nucleic acid
Author Manuscript
had no measurable transfection, indicating that a drug silvery system is required to

effectively protect and deliver topically applied nucleic acids. Finally, oral delivery of
nanoparticles allows direct access to tumors of the gastrointestinal tract.[235, 236] Chitosan
nanoparticles have also been explored for this purpose, as they are stable upon oral
administration, can encapsulate nucleic acid cargos, facilitate transport across the intestinal
wall, and can be functionalized for active tumor targeting.[237] Delivery to different tissues
requires overcoming different barriers, which can be physical (surfactant, mucous, stratum
corneum), biological (enzymes, resident immune cells, blood brain barrier), and chemical
(harsh pH of the GI tract). Future work in this area must focus on enhancing particle
stability, controlling release kinetics, and improving transport and permeability across these
tissue-specific barriers in orthotopic tumor models.
Author Manuscript
4.5. Cancer-specific nucleic acid therapies

Cancer is fundamentally a condition caused by dysregulated gene expression, and nucleic
acid therapies can treat the genetic basis of the disease by counteracting observed genetic
changes. Advances in high throughput sequencing, microarray technologies, and novel
computational models have resulted in a rapidly growing understanding of the genetic basis
of cancer. We are now beginning to understand how particular genetic mutations and
expression profiles correlate with disease stage, drug resistance, and potential for metastasis.

Therefore, selecting certain nucleic acid cargos can target disease that is more aggressive,
Author Manuscript
drug resistant, or likely to metastasize. Combinatorial strategies greatly increase the number
of possible nucleic acid combinations that may be used to target a heterogeneous tumor
population. Additionally, as illustrated thus far, combinatorial approaches have been
successful in slowing tumor growth in these aggressive and difficult to treat cases.
One common approach is restoring the function of a mutated tumor suppressor with
exogenous nucleic acids. Mutations in tumor suppressors have been associated with
resistance to chemotherapy and radiation.[238, 239] Tumor suppressor function can be
restored by introducing nucleic acids, including DNA and mRNA, that encode for the wild-
type protein.[240] For example, systemic delivery of PTEN mRNA using polymer-lipid
hybrid nanoparticles was shown to slow tumor growth in multiple models of prostate cancer.
[241] Modified mRNA shows enhanced stability compared to plasmid DNA therapies in
systemic circulation with more predictable and desirable protein kinetics. P53 is another
Author Manuscript
tumor suppressor that has been targeted in many clinical and preclinical trials.[242, 243]
Studies have shown that introducing p53 induces apoptosis in many cancer cells, but healthy
cells only experience cell cycle arrest, which suggests inherent cancer-specificity to p53
gene therapy.[244, 245] However, certain healthy cell types, including epithelial and
hemopoietic cells, are sensitive to p53-induced apoptosis.[246, 247] Also, p53 is part of a
complex interconnected web of factors, and the efficacy of these therapies is limited by
mutations in downstream factors and epigenetic changes, as well as heterogeneous p53
expression throughout tumors.[247] While p53 mutations have been explored most
extensively due to their prevalence, other tumor suppressors (e.g., Rb7, PTEN, or mda-7)
have also been targeted.[248]
An alternative approach is silencing of overexpressed oncogenes using RNAi. Many

Author Manuscript
strategies involving siRNA or miRNA have been developed to target oncogenes for cancer
therapy. Ideal targets are upregulated in cancer cells, are vital for cancer progression, and do
not have a rapid turnover rate. Examples of targeted pathways include angiogenesis (VEGF),
proliferation (FAK), survival (Bcl-2, survivin), cell cycle (PLK1, cyclin B1), and resistance
to chemotherapy or radiation (c-myc).[249] Knocking down these pathways can have a potent
anti-cancer effect but can also affect the viability of healthy cells and tissues, leading to
systemic toxicity. Brummelkamp et al. approached this problem by developing siRNA that
specifically targets the oncogenic K-RASV12 allele without any effect on wild-type K-RAS
expression, which is required for normal cell survival.[250] They showed that knocking down
K-RASV12 with a viral vector completely prevented CAPAN-1 pancreatic cancer cell growth
in vitro as well as in a subcutaneous tumor model. This approach is promising for
specifically targeting cancers with mutations or chromosomal translocations that produce
Author Manuscript
mRNA transcripts distinct from those expressed in healthy cells.
Cancer cells are also more vulnerable to cell death through certain proapoptotic pathways,
including TNF,[251] Fas,[252] and Bcl.[253] Tumor necrosis factor (TNF)-related apoptosis-
inducing ligand (TRAIL) induces apoptosis with a strong cancer selectivity, due to
overexpression of TRAIL-binding death receptors on cancer cells.[254] Because recombinant
TRAIL proteins have poor pharmacokinetics and a short circulation half-life, nucleic acid
therapies are a promising approach to achieve sustained TRAIL expression. Co-delivery

with small molecule sensitizers, including clinically approved chemotherapies, has been
Author Manuscript
shown to reverse TRAIL-resistance and improve antitumor efficacy in a synergistic manner.

A dendrimer nanocarrier co-encapsulating doxorubicin and a plasmid DNA expressing
human TRAIL induced synergistic growth inhibition in U87 glioma cells.[255] This
treatment administered intravenously induced observable apoptosis in an orthotopic murine
glioma model, and the co-delivery vehicle extended median survival to 57 days, compared to
34 days with doxorubicin alone. Another strategy for TRAIL therapy is to transfect or
transduce tumor-homing stem cells ex vivo to secrete TRAIL in the vicinity of the tumor.
[256] This strategy combines regio-selective delivery of the stem cells with the cancer-
specific TRAIL therapy and shows significant survival benefit in an aggressive brainstem
glioma model.[257] A secretable TRAIL construct has also been developed to enhance the
bystander effect to neighboring cancer cells and further potentiate the antitumor effect.[258]
Gene expression in cancer cells is modulated by differential expression of transcription

Author Manuscript
factors, which can be exploited to restrict the expression of therapeutic genes to tumor cells.
By placing transgenes under the control of certain promoters, it is possible to achieve tissue-
specific, cell-specific, or exogenously stimulated expression.[259] For example, hTR and
hTERT promoter drive telomerase activity, which is a common feature of most cancers and
can activate genes in a cancer-targeted manner.[260] Tumor-specific promoters ideally have
strong expression in cancer cells and little to no expression in healthy cells. For example,
alpha fetoprotein (AFP) is transcriptionally silent in adult liver but is expressed in 70–80%
of hepatocellular carcinoma cases.[261] When potent pro-apoptotic genes are placed under
the control of the AFP-promoter, cell death is restricted to AFP-producing HCC cells, and
no acute systemic toxicity is observed.[262] Transcriptionally targeted BikDD DNA delivered
by DOPC-cholesterol liposomes prolonged survival in multiple xenograft and syngeneic
orthotopic murine HCC models (Figure 7).[262] Histological staining indicated that
Author Manuscript
treatment-induced apoptosis was restricted to liver tumor cells, and cell death was not
observed in healthy liver.[262] Many other tumor-specific promoters have been identified for
particular cancer types, each with a different prevalence, expression profile, promoter
strength, and tumor target. Another approach is to use promoters that respond to the tumor
microenvironment. Hypoxia response elements (HREs) can be used to drive gene expression
in hypoxic tumor environments, where cells are typically resistant to chemotherapy and
radiation. Glucose-responsive promoters, such as hexokinase 2 and GRP78, respond to low
glucose and high catabolism in tumors.[263–265] Finally, inducible promoters have been
developed to respond to exogenous stimulation. Examples of stimuli include radiation,
hyperthermia, and small molecule drugs.[266–268] These promoters do not depend on the
expression profile of the tumor, so the strength and duration of activation can be controlled.
Ultimately, transcriptional targeting offers an opportunity to control expression at the
Author Manuscript
cellular level, which can dramatically reduce off-target toxicity.
Combinatorial delivery can also be used to counteract phenotypic changes in a particular

subset of cancer cells, such as drug resistant or stem-like cells. A combination of miRNAs
can be used to target multiple pathways involved in the stem-like phenotype of brain tumor
initiating cells. This minor population of glioblastoma cells has been associated with tumor
reoccurrence and drug resistance. Using pooled miRNAs to revert the stem-like traits in
these cells has proven successful in inhibiting growth, neurosphere formation, and shrinking

orthotopic xenografts in mouse glioblastoma models.[269, 270] Therefore, particular

Author Manuscript
combinations of nucleic acids can target certain phenotypes associated with aggressive or
reoccurring disease. Pooled siRNAs have also been used as a treatment strategy to overcome
multiple drug resistance. In one example, two siRNAs (anti-Pgp and anti-Bcl-2) were
combined with epirubicin in a calcium phosphate inorganic nanoparticle as a treatment for
drug-resistant liver cancer cells and tumors.[271] The study concluded that the combination
therapy was effective due to simultaneous targeting of two drug resistance mechanisms:
pump (anti-Pgp) and non-pump (anti-Bcl-2).[271] Therefore, pooled nucleic acid
combination therapies can act synergistically and address tumor heterogeneity by acting on
multiple pathways with spatial and temporal synchronization.
Nucleic acid therapeutics are distinct in that Watson-Crick base pairing allows for sequence-
dependent manipulation of three-dimensional structure. Changing the architecture of the
nucleic acid can confer favorable properties or additional specificity. For example, Conde et
Author Manuscript
al. developed an RNA triple helix comprised of two different tumor suppressor miRNAs and
one antagomir which inhibits a pro-cancer oncomiR.[271] Combining these molecules into a
triple helical structure imparts high structural stability and facilitates co-delivery.[271]
Similarly, Li et al. developed tetrahedral RNA structures with precise control over their
geometry.[272] The 2’ -F modified RNA tetrahedrons were both thermodynamically and
enzymatically more stable compared with DNA and non-modified RNA.[272] The
tetrahedrons were also functionalized to include siRNAs and RNA aptamers, which
facilitated siRNA delivery in vitro and in vivo with aptamer-specific tumor targeting.[272]
The enhanced stability and targeting properties of RNA structures facilitate carrier-free
nucleic acid delivery, which reduces the design complexity and the number of potentially
toxic or immunogenic components
Author Manuscript
While many combinations of nucleic acid and drug cargos have been shown to have additive
or synergistic effects, new data regarding the pathways involved in tumor growth,
progression, and metastasis should be considered. As our understanding of cancer
progression evolves, combination treatments must be continuously optimized to identify
potent combinations of anticancer agents. These combinations should act broadly on many
types of cancers, particularly in drug-resistant, aggressive, and metastatic disease.
4.6 Multifunctional Targeted Nanocarriers

The use of a cancer-specific cargo reduces the burden of developing a perfectly targeted
nanocarrier, since off-target delivery will have minimal effect in normal cells. Further,
combinatorial nucleic acid therapies can be selected to address tumor heterogeneity or to
target a particular cancer cell subtype. The targeting methods described here work at
Author Manuscript
different levels: local delivery to the tissue of interest, responsive uptake in the tumor
microenvironment, active targeting to certain cell types, and genetic targeting to particular
expression profiles. Thus, these orthogonal targeting mechanisms can be combined into
highly targeted multifunctional nanocarriers. The multifunctional envelope-type nano-device
(MEND) developed by Hatakeyama et al. integrates multiple strategies for successful
cancer-specific nucleic acid delivery.[203] MEND is a nanocarrier comprised of nucleic acid
condensed by a cationic polymer coated with a lipid envelope, which has been

functionalized with various combinations of responsive and targeting features. For example,
Author Manuscript
one iteration of the platform combines MMP-cleavable PEG and pH-sensitive fusogenic
peptides to overcome the PEG dilemma and specifically enhance cellular uptake of siRNA
in solid tumors.[273] Combining transcriptional targeting with particle targeting is another
promising dual-targeting strategy, demonstrated by Cocco et al.[274] They developed a
PLGA-PBAE blend nanoparticle functionalized with tumor targeting c-CPE peptides and
used these particles to deliver a diphtheria toxin subunit A (DT-A) gene under the control of
the cancer-specific p16 promoter. The dual-targeted particles efficiently transfected primary
patient cells and significantly slowed tumor growth in chemotherapy-resistant ovarian cancer
models.
Multifunctional particles also have the potential to achieve stepwise release of therapeutic
agents. For example, core-shell particles can release siRNA molecules from an outer
polymer layer in a glutathione-responsive manner followed by slow release of a
Author Manuscript
chemotherapy to overcome multi-drug resistance.[275] While multifunctional targeted

particles show promise in preclinical studies, the lack of targeted particles in clinical trials
for nucleic acid delivery is a testament to the complexity of implementing active targeting in
a translational and scalable way. Developing targeted nanocarriers requires additional
testing, time, and expense, and the resulting product is often expensive to manufacture.
Despite these hurdles, several next-generation actively targeted nanomedicines for nucleic
acid delivery have entered clinical trials, as summarized in Table 2. As more potent nucleic
acid therapies are developed, particularly immunotherapies, a high degree of specificity will
be essential to avoid dangerous off-target effects. Thus, these targeting strategies may serve
as enabling technologies to bring nucleic therapies to patients.
5. Nanomaterials for combinatorial nucleic acid delivery

Author Manuscript
Nucleic acids confer many advantages as therapeutic agents, one of which is their ability to
be co-delivered for combinatorial therapy. Viruses are an obvious vehicle for gene delivery,
as they have evolved to be highly efficient at nucleic acid transfer.[276] Presently, the
majority of gene therapy clinical trials have relied on viral vectors, and the only FDA-
approved gene therapy uses a viral vector.[277] However, while new generations of viral
vectors are continually being developed,[278] viruses can have several limitations as delivery
vehicles, including safety concerns related to their immunogenicity,[279] risks of
mutagenesis,[280] and limited cargo capacity.[281] The last of these, in particular, is an
important concern when considering combinatorial delivery of multiple nucleic acid
sequences or types. In this section, we focus on non-viral delivery vectors engineered to
prevent the problems generally seen with viruses, and their uses for combinatorial nucleic
Author Manuscript
acid delivery will be described. Examples of some prominent types of materials used for this
purpose are illustrated in Figure 8.
5.1. Lipid-based nanoparticles

Lipid-based materials have been studied extensively for delivery of nucleic acids. Easily
manufactured, lipid-based nanoparticles can carry many types of nucleic acid within a
hydrophilic liposome core, including in combination,[282] and they have been of high

interest in particular for the delivery of oligonucleotides like siRNA.[283, 284] Cationic lipids,
Author Manuscript
which can associate with anionic nucleic acids and negatively charged target cell surfaces,
can be synthesized using combinatorial chemistry to form a wide range of lipids or lipidoids
with varying properties,[285–287] allowing researchers to formulate different nanoparticles in
a facile way. Lipid nanoparticles can facilitate cellular uptake by interaction with lipids in
the cell membrane, which also aids in endosomal escape,[288–291] or by interacting with
endogenous lipoproteins that are taken up via binding to specific cell receptors.[292–294]
However, lipid nanoparticles carry certain drawbacks. The lipid-lipid interactions that help
these nanoparticles to enter cells and escape the endosome may also result in high toxicity,
[295] and some lipids have been found to be inherently immunogenic on their own, even
without the addition of nucleic acid cargo.[296] Thus, careful study of the chemical
properties of the lipids used is critical for the use of this type of nanocarrier. Lipid
nanoparticles are also prone to interactions with components of the blood that may cause
Author Manuscript
aggregation or destabilization. PEGylation can been used to prevent clearance of lipid

nanoparticles,[297] and SNALPs, stable nucleic acid lipid particles, were developed to have
both internally stabilizing components as well as a PEG shield.[298] Though challenges
remain, lipid-based nanoparticles are among the most highly studied nanocarriers for nucleic
acids, both pre-clinically and in clinical trials,[299] especially for delivery of
oligonucleotides. Alnylam Pharmaceuticals reported a phase I trial in 2013 that used a lipid
nanoparticle carrying two siRNA sequences to target vascular endothelial growth factor
(VEGF) and kinesin spindle protein (KSP),[300] with multiple siRNAs being loaded into the
same particles. VEGF is upregulated in many solid cancers due to the tumor cells’ need to
rapidly increase angiogenesis to provide nutrients to the growing tumor; KSP is involved in
mitosis and is overexpressed in quickly dividing cells. In cancer patients with tumors in the
liver, the authors reported toxicities comparable to or lower than those seen from traditional
Author Manuscript
chemotherapy and demonstrated anti-tumor activity after administration of their formulation.

The biodistribution of these nanoparticles was largely in the spleen and liver, allowing them
to passively target hepatic tumors.[300] The authors noted that their therapeutic was
detectable in extrahepatic tumors as well and that these tumors were also controlled by the
treatment; though an explanation is not provided, it may be that the size of the nanoparticles
allowed them to accumulate in these other tumors via the EPR effect. It should be noted that
an increase in cytokine levels was noted after treatment,[300] suggesting that, while siRNAs
can be modified to elicit minimal immune responses, some immunostimulatory effects may
still occur using this and similar systems.
The NOV340 liposomal delivery system, or SMARTICLES®, developed by Mirna

Therapeutics Inc. and Marina Biotech, addresses some of the drawbacks of lipid-based
Author Manuscript
nanoparticles.[301] The system uses ionizable lipids that are negatively charged or neutral at
neutral pH or higher, allowing the nanoparticles to avoid interaction with cell membranes or
tissues that might prevent their free circulation. At lower pH, such as that found at tumor
sites, NOV340 becomes cationic, and this triggered change facilitates its interaction with
and uptake by cancer cells. A phase I clinical trial using the NOV340 system to deliver a
miR-34a mimic to patients with advanced malignancies suggested some anti-tumor activity,
though severe adverse immune responses were also observed in some patients.[302] A pre-
clinical study using the NOV340 liposomal system to deliver a combination of miRNA

mimics (miR-34a and let-7b), showed that the combination of both sequences was more
Author Manuscript
effective than either on its own in a mouse model of non-small cell lung cancer (Figure 9).
[303] Thus, while, once again, careful work must be undertaken to better understand the
safety profile of lipid-based systems in a clinical setting, combinatorial delivery of nucleic

acids still holds promise for therapeutic effect.
In another illustrative example, Chen et al. used a lipid-based nanoparticle formulation for
the co-delivery of miR-34a, an miRNA commonly downregulated in human cancers that can
cause apoptosis via multiple mechanisms upon administration to cancer cells, and three
siRNA sequences against c-Myc, MDM2, and VEGF, all genes commonly upregulated in
solid tumors.[136] Their formulation consisted of hyaluronic acid, miRNA, and siRNA pre-
complexed with cationic molecule protamine and then encapsulated into cationic, PEGylated
lipids for enhanced stability. The final nanoparticle was surface-modified with the tumor-
targeting small chain antibody fragment (scFv) GC4, specific for melanoma. Exploiting the
Author Manuscript
combined effects of multiple RNA cargos, the encapsulation capability of a hydrophilic

polymer and cationic agent, the enhanced circulation time conferred by PEG, and the
targeting capacity of the scFv, they were able to show not only accumulation of the
nanoparticles at the tumor site in a B16-F10 murine melanoma model but also additive
inhibition of metastatic tumor growth after delivery of miRNA and siRNAs in combination.
[136]
As well as being multiplexed with each other, RNA oligos can also be delivered in
combination with traditional chemotherapy drugs. Given a known mechanism of drug
resistance by cancer cells, researchers can use siRNA to knock down a gene that promotes
resistance and simultaneously deliver a chemotherapeutic. Saad et al. used a cationic
liposomal formulation to co-deliver siRNAs against MRP1, an efflux pump that removes
Author Manuscript
drugs from cells, and BCL2, which confers resistance by preventing apoptosis, along with
the common anticancer agent doxorubicin.[304] While treatment of lung cancer cells with
doxorubicin led to upregulation of both of these resistance-causing genes, co-delivery with
either the two siRNA sequences decreased resistance and led to significantly greater
susceptibility of the cells to doxorubicin-mediated killing.[304]
As the above examples demonstrate, lipid-based nanoparticles have been, and continue to
be, studied extensively for the delivery of nucleic acids, especially siRNA and miRNA. This
may be due in part to the greater challenge of binding short oligonucleotides via
complexation compared to larger nucleic acids with greater multivalency, as discussed in
Section 3; liposomal formulations do not rely on high-avidity electrostatic interactions with
nucleic acids in order to encapsulate them in their hydrophilic core and may therefore by
Author Manuscript
favorable for delivery of smaller cargo. This type of formulation is also easily adaptable to
the co-encapsulation of multiple cargoes for combinatorial delivery. However, despite the
long history of their use in research and in clinical development, lipid-based carriers must
still contend with problems of excessive toxicity and potentially lethal side effects described
above. Intrinsic immunogenicity as well as enhancement of the immunogenicity of RNA
cargo has been of particular concern in the cases described above, including examples that
reached the stage of clinical trials.[300, 302]

5.2. Cationic polymeric nanoparticles

Author Manuscript
As previously mentioned, PLL-based NPs are poorly able to escape the endosome, unlike
newer cationic polymers,[305, 306] which contain reversibly protonatable amines that can aid
in escape to the cytosol. PEI and its branched or functionalized derivatives, on the other
hand, has been a basis for many types of polymer-based intracellular delivery systems in
large part because of its reversibly protonated state: PEI, with a mixture of primary,
secondary, and sometimes tertiary amines, can exist in a positively charged state that allows
tight binding of nucleic acids, while other amines can still buffer additional protons once
within the endosome, leading to escape.
An important hurdle to the use of PEI alone as a delivery agent is its toxicity, as the cationic
properties that aid in cargo complexation and cellular internalization can also disrupt the cell
membrane and lead to adverse effects. High-molecular weight (MW) PEI is effective in gene
Author Manuscript
transfer but tends to be toxic, while low-MW PEI is less toxic but can also be less effective.
[307, 308] To address this challenge, researchers have developed PEI-based polymers that
have high MW but can degrade into smaller segments.[307] This can be done by linking
multiple low-molecular-weight PEI oligomers with ester linkages, which degrade by
hydrolysis; reducible disulfide bridges; or other degradable functional groups.[308]
Condensation of nucleic acids into nanoparticles by PEI and similar polymers is driven
largely by electrostatic interactions, and some of the properties of such particles can be
tuned by changing the ratio of cationic polymer to anionic genetic material. This type of
flexibility allows researchers to easily mix multiple different nucleic acids together to co-
complex with PEI and form nanoparticles for combination therapy. Chen et al. used
branched PEI, which allows the use of relatively low-MW PEI while retaining higher
efficacy due to the increased amine density of the branching structure, to co-deliver siRNAs
Author Manuscript
against VEGF receptor 2 (VEGFR2) and epidermal growth factor receptor (EGFR).[309]
Both VEGFR2 and EGFR are upregulated in many solid tumors and participate in
angiogenesis and tumor growth, respectively. They were able to achieve knockdown of both
genes in non-small cell lung cancer cells in vitro and in vivo. Interestingly, knockdown of
VEGFR2 alone was more effective in tumor control than knockdown of both genes together;
however, the high dosage of VEGFR2 siRNA needed caused adverse side effects, which
were mitigated by the combination of both genes at a lower dose. Similarly, the combination
of this dual-gene knockdown with a low dose of traditional chemotherapeutic agent cisplatin
led to a strong therapeutic effect while minimizing the toxic effects normally seen with
higher doses of the drug, illustrating the ability of combination therapy to achieve similar
results to a single-agent therapy while reducing toxicity.[309] Although the commercially
available branched PEI polymer was not chemically modified in any way for this study, gene
Author Manuscript
knockdown in vitro was demonstrated in the absence of serum, and a therapeutic effect was
seen in vivo after local intratumoral injection.[309] As mentioned, unmodified PEI-based
polyplexes can be destabilized by interaction with serum proteins and cells after systemic
administration, but the methods used by the authors allowed them to sidestep these hurdles,
a potentially viable therapeutic strategy in the case of solid tumors that are accessible for
local administration of nanoparticles.

Another class of cationic polymers, PBAEs, shares some of the advantages of PEI while also
Author Manuscript
being less toxic due to the presence of degradable ester linkages and has been widely studied
for gene delivery.[310] As with lipid-based nanoparticles, combinatorial chemistry has been
used to study PBAEs for DNA or RNA delivery,[311–313] generating PBAEs with a variety of
different physical and chemical properties. The ease of manufacture of these polymeric
formulations makes them a versatile group of materials for gene delivery compatible with
large plasmid DNA, RNA oligomers, and smaller CDNs. Aside from their hydrolytically
degradable ester linkages, PBAEs can be synthesized with disulfide bridges that target
nucleic acid cargo to the cytoplasm.[270, 314, 315]
While PBAEs, like other cationic polymers that form electrostatic complexes, bind to
oligonucleotides less efficiently than to larger plasmids due to reduced avidity, the relatively
low toxicity of PBAEs permits the use of higher amounts of the polymer or branched
polymer structures with greater amine density to compensate for lower binding affinity.
Author Manuscript
Using these strategies, these biomaterials have been shown to be effective for delivery of
short sequences like siRNA,[312, 314–317] miRNA,[270] and CDNs.[43] A recent report by
Lopez-Bertoni et al. used disulfide-containing PBAEs to co-deliver miR-148a and miR-296–
5p, both miRNAs that inhibit the stem cell-like phenotype of human glioblastoma (GBM)
cells in an effort to prevent tumor growth in a mouse model.[270] Not only did the PBAE-
delivered miRNAs affect the expression of stem cell markers and formation of tumorigenic
neurospheres in vitro, but they also inhibited tumor growth in an orthotopic human GBM
mouse model. Importantly, the two miRNA mimics in combination were more effective than
either of the sequences alone, when measured by tumor size over time, and 67% of the
animals treated with the miRNA combination were long-term survivors (>133 days post-
tumor implantation, with 67% of these survivors having no detectable tumor), compared to a
median survival of <70 days in the control group,[270] demonstrating the power of
Author Manuscript
nanoparticle-based nucleic acid therapies that target multiple biochemical pathways (Figure
10).[270]
Another method of indirectly delivering RNA oligos is to deliver plasmid DNA that encodes
short hairpin RNA (shRNA) that will be processed into siRNA once in the cytoplasm. Yin et
al. used a disulfide-containing PBAE to co-deliver shRNAs encoding siRNA against survivin
and Mdr-1, both important for tumor cell survival and drug resistance.[318] They showed in
vitro that PBAE-mediated transfection of drug-resistant human breast cancer cells with
either of these shRNAs facilitated increased susceptibility of the cells to doxorubicin
treatment. In a mouse model, the combination of both shRNAs with doxorubicin was
synergistic and led to slower tumor growth than any of the three components on its own.[318]
Author Manuscript
Related to PBAEs are poly(amido amine)s (PAMAMs), which contain the more stable amide
linkages in place of degradable ester linkages but can be synthesized to contain disulfide
bridges that release cargo in the cytoplasm.[319, 320] Branching PAMAM structures, or
dendrimers, can be synthesized in a highly controlled manner to have high amine density for
complexation with nucleic acids.[321] Like other cationic polymers, PAMAM dendrimers
condense anionic genetic material into nanoscale particles, and the properties thereof are
dependent in part on the balance of positive and negative charges in the polyplex.
Maksimenko et al. used anionic oligomers, including DNA olionucleotides and anionic

dextran sulfate, as a binding component in their PAMAM dendrimer nanoparticles, and, by

Author Manuscript
this method, they were able to improve the efficacy of transfection and decrease the required
dose of the therapeutic gene.[322] In this case, the co-delivered oligonucleotide was not
intended to have a direct biological target but rather served to alter the physicochemical
properties of the nanoparticle formulation. This method of stabilizing PAMAM dendrimer
formulations with an oligonucleotide was also used by Vincent et al. to form nanoparticles
for the co-delivery of two genes, angiostatin and TIMP-2, in the form of DNA plasmids.[323]
Both of these genes were intended to inhibit tumor angiogenesis, with the former
specifically inhibiting endothelial cell proliferation and the latter regulating the activity of
matrix metalloproteinases (MMPs) involved in tissue remodeling and new vessel formation.
Using their PAMAM dendrimers complexed with a DNA oligonucleotide and carrying two
other DNA plasmids encoding functional genes, they showed that the oligonucleotide
enhanced transfection efficacy and that co-delivery of both anti-angiogenic genes was more
effective at slowing tumor growth in vivo than delivery of either gene separately.[323]
Author Manuscript
An advantage of synthetic polymers such as the ones described here is the ease with which
they can be tuned or chemically modified to exhibit desirable properties and to bear
functional groups of interest, including chemical functionalities as well as biological groups
like targeting moieties. For instance, PBAE or PAMAM nanoparticles can be PEGylated to
improve biodistribution after administration.[324, 325] The chemical versatility of polymers is
a major advantage for increasing functionality or modifying nanoparticle properties, like
toxicity. However, unlike liposomal nanocarriers, cationic polymer-based polyplexes do not
strictly encapsulate nucleic acids but rather bind them electrostatically, and they therefore
bind less strongly to cargos with lower MW (i.e., lower avidity). It is interesting to note that,
while both lipid- and polymer-based nanoparticles have been used to deliver many different
types of nucleic acids, many of the polymer-based systems tend to be used for larger plasmid
Author Manuscript
DNA molecules rather than siRNA or miRNA, in contrast to lipid-based systems, and/or use
various strategies to improve binding, including the addition of non-coding oligomers as
binding help[322] and the use of excess cationic polymer to enhance binding stability,
[270, 312, 314, 315] the latter of which in turn requires a polymer with low toxicity to allow the
increased amount of material to be safely added to the nanoparticle system.
Polymers can also be combined with other classes of materials to take advantage of a wider
range of favorable characteristics. Dahlman et al. used lipid-polymer hybrids to achieve in
vivo siRNA delivery with specific biodistribution.[326] The authors conjugated a lipid to a
low-MW polyamine, and the resulting novel material was formulated with PEGylated lipids
and RNA oligos to form lipid-polymer hybrid nanoparticles. Importantly, these hybrids were
found to target endothelial cells, rather than the liver,[326] which is the most common target
Author Manuscript
of in vivo siRNA delivery due to the high accumulation of lipid nanoparticles in the liver. In
a follow-up study, taking advantage of the ability of a single nanoparticle to carry multiple
oligos, the authors delivered an miR-34a mimic, whose expression has been shown to limit
tumor growth due to regulation of genes involved in cell cycle progression and apoptosis,
and an siRNA against the oncogene Kras in a murine model of lung cancer.[327] This
formulation’s material properties allowed it to accumulate in the lungs, and delivery of both
miR-34a and siKras simultaneously was more effective at controlling tumor progression
than either sequence alone. Additionally, the combination nucleic acid delivery also

enhanced the effect of paclitaxel, resulting in significantly longer survival times (Figure 11),
[327]
Author Manuscript
further demonstrating the benefits of combining multiple nucleic acids as well as other
traditional therapeutic cargos. Designs such as this combine the advantages of multiple
systems in an effort to optimize nucleic acid delivery. In this case, the lipid component could
interact with cells as well as improving the stability of the nanoparticles; the polymer could
be easily modified and used as the basis for reaction with the other materials; and an
additional lipid component brought with it PEG functionalization for better in vivo stability,
serving as an example of combinatorial use of delivery material as well as cargo.
5.3. Other types of polymeric nanoparticles

One of the most commonly studied types of polymers for gene and drug delivery is
polyesters,[328] including poly(lactic acid) (PLA), poly(glycolic acid) (PGA), and the co-
polymer poly(lactic-co-glycolic acid) (PLGA). As PLGA and related polymers are
Author Manuscript
components of FDA-approved devices, they are generally considered to be safe, a major

advantage over other biomaterials that have significant toxicity.[329] However, encapsulation
efficiency of nucleic acids in PLGA-based nanoparticles is generally poor compared to the
loading efficiency in liposomes or cationic polymers and normally requires additional
emulsifying excipients, and nucleic acids have been found to be unstable after encapsulation.
[330] To improve association with nucleic acids, cellular uptake, and endosomal escape, a
common strategy is to use blends or co-polymers of PLGA with cationic materials, such as
PEI,[331, 332] PLL,[333, 334] chitosan,[335] or spermidine,[336] thereby combining the assets of
cargo-binding polycations with the biocompatible matrix within which nucleic acids can be
encapsulated. For instance, Wang et al. synthesized a co-polymer consisting of a
hydrophobic portion based on cholesterol [N-(2-bromoethyl)carbamoyl cholesterol] and a
hydrophilic, cationic main chain [poly(N-methyldietheneamine sebacate)] and used the
Author Manuscript
resulting amphiphilic polymer to form core-shell nanoparticles co-encapsulating the

chemotherapeutic paclitaxel and either DNA plasmids or siRNA oligos.[337] The authors
showed in a 4T1 murine breast cancer model that co-delivery of a DNA plasmid encoding
IL-12, an anti-tumor cytokine, could enhance the effects of paclitaxel and slow the growth of
the 4T1 tumor. The same nanoparticle system could also be used to co-deliver paclitaxel and
siRNA against BCL2 in vitro, knocking down an anti-apoptotic of tumor cells while
delivering a cytotoxic agent and demonstrating the ability of the same nanoparticle system to
delivery multiple types of nucleic acid due to similarities in their electrostatic properties.[337]
In addition to synthetic polymers, natural polymers like cyclodextrins (CDs) have been used
for gene delivery, including in clinical trial.[144] As cyclic oligosaccharides, CDs naturally
form an amphiphilic structure that allows them to encapsulate cargo while shielding the
cargo from potentially destabilizing interactions with other biomolecules.[338] The
Author Manuscript
encapsulation capacity, the availability of multiple functional groups for chemical

modification, and the common use of CDs as excipients and absorption enhancers make CDs
a versatile platform for gene delivery. Many efforts to use CDs to deliver nucleic acids use a
combination of CDs and other components like PEI[339] or lipids[340] to enhance gene
delivery or targeting ligands[341] to prevent off-target effects on healthy tissue.

5.4. Inorganic nanoparticles

Author Manuscript
Calcium phosphate (CaP) was used in early efforts to achieve DNA delivery by co-
precipitating DNA with CaP to form nanoparticles.[342, 343] Once internalized into cells, CaP
has been shown to dissolve at the low endosomal pH, which increases the osmotic pressure
of the endosomal compartment and leads to cytoplasmic release of nucleic acid cargo.[344]
While CaP crystal growth can be challenging to control, CaP nanoparticles have been used
in combination with organic biomaterials to improve gene delivery[344–347] to aid in
overcoming delivery hurdles like uptake, stability, or endosomal escape. An interesting
example is that of lipid-coated CaP nanoparticles (LCPs). In LCPs, nucleic acid is loaded
into the CaP core, which is then stabilized with a surrounding lipid bilayer. Yang et al. used
LCPs to encapsulate a pool of siRNAs, then coated the core with PEGylated lipids and the
tumor-targeting ligand anisamide, which has been used in a number of cancer-targeted
nanomedicines.[348] The authors co-delivered siRNAs against HDM2, a suppressor of p53
Author Manuscript
activity; the oncogene c-myc; and the pro-angiogenic factor VEGF. The PEGylated lipid
coating and the targeting ligand together promoted accumulation of the LCPs in non-small-
cell lung cancer after injection in vivo. While the authors do not show the anti-tumor effects
of each siRNA alone, if any, the pool of three siRNAs in the lipid-modified CaP
nanoparticles was able to significantly slow tumor growth and promote cancer cell death.
[348]
Mesoporous silica nanoparticles (MSNs) have also been used to facilitate gene and drug
delivery due to the biocompatibility of silica and the tunability of the porous architecture of
these solid particles.[349] Highly porous MSNs can be loaded with therapeutics like nucleic
acids, as well as functionalized for various chemical properties. Taratula et al. developed a
multimodal MSN-based delivery system, in which thiol surface-functionalization was used
to bind thiol-terminated siRNA to the nanoparticles; surface thiols were also used for
Author Manuscript
PEGylation of the particles; and the porous structure was used to load traditional
chemotherapy drugs like cisplatin and doxorubicin.[350] Using inhalation to deliver their
loaded and functionalized MSNs directly to lung cancer cells, the authors report that
simultaneous knockdown of BCL2 and MRP1, both genes that are important for different
mechanisms of drug resistance, led to increased cell death and greater susceptibility to
doxorubicin and/or cisplatin.[350] This strategy takes advantage of the various surfaces and
compartments available for loading and functionalization on particles like MSNs, which
allows different cargoes and materials to be utilized for simultaneous delivery (Figure 12).
Among inorganic materials, gold nanoparticles (AuNPs) are among the most well studied
today. Because their surface can be easily chemically functionalized,[351–353] there are
multiple ways in which they can be used to bind nucleic acids. They can be surface-coated
Author Manuscript
layer-by-layer (LbL) with alternating layers of anionic nucleic acids and polycations like
PEI[354, 355] or PAMAMs and PBAEs.[74] In one study by Bishop et al., AuNPs were
functionalized with anionic 11-mercaptoundecanoic acid (11-MUA), allowing them to be
coated with cationic PAMAM or PBAE.[74] These positively charged NPs could then be
coated with layers of siRNA or DNA, alternating with layers of cationic polymer. The
cationic polymers facilitated cellular uptake and endosomal escape, and reducible disulfide
bond-containing PAMAMs were used to cause release of siRNA in the cytoplasm following

hydrolytic degradation of PBAEs and release of DNA.[74] AuNPs bring additional

advantages, such as their ability to act as sensors due to their optical properties[356] and to
Author Manuscript
deal cause damage to cancer cells via their photothermal properties.[357, 358] Spherical
nucleic acids (SNAs) have become prominent in nucleic acid delivery research and are
composed of AuNP cores coated with a shell of nucleic acids, using DNA in the earlier
studies and later extending to other types of cargo like siRNA.[39] As mentioned briefly
above, the properties of SNAs depend heavily on their three-dimensional structure, which
affects their interaction with other biomolecules as well as their ability to be taken up by
target cells. Later versions of SNAs used a coating of silica between the core and the nucleic
acid shell, allowing the AuNP core to be dissolved entirely, leaving a hollow, spherical shell
of nucleic acid while minimizing the inorganic delivery vehicle itself.[57]
Like gold, other inorganic nanoparticles can also facilitate simultaneous imaging and
therapeutic delivery. Magnetic nanoparticles are easily and scalably manufactured, and they
Author Manuscript
can be detected by MRI[359] for tracking or diagnostic purposes as well as moved toward
target cells using a magnetic field.[360] Quantum dots (QDs) are an interesting option as a
delivery vehicle with optical properties, as they are designed to be bright, photostable
imaging agents and are normally of small size amenable to in vivo trafficking and cellular
uptake.[361, 362] As with other inorganic nanoparticles mentioned here, many of these are
generally used in combination with other gene delivery materials, such as coatings of PEI,
[363] PEG,[364] and other polymers[365, 366] to allow binding and other capabilities required
for gene delivery. For example, Dong et al. reported graphene quantum dots (GQDs)
conjugated with PEG-PLA for stability, and miRNAs and antisense oligodeoxynucleotides
(ASODNs), which have a similar function but greater chemical stability than antisense RNA
oligos, were loaded onto the functionalized GQDs via surface absorption.[367] The extremely
high surface area of GQDs allowed the co-loading and -delivery of an miRNA inhibitor to
Author Manuscript
suppress miR-21, often upregulated in solid tumors, and an ASODN targeting survivin
(Figure 13). The GQDs demonstrated stable optical properties after functionalization,
allowing them to be imaged via fluorescence, and they were able to transfect HeLa cells in
vitro, with the combination of both inhibitors having a stronger effect on cells than either of
them separately.[367] While inorganic nanoparticles have promise and can provide interesting
properties to nanocarriers, they have not proceeded to the clinic to the same extent as organic
nanoparticles, though strong research on them is ongoing.
5.5. DNA-based nanostructures

The base complementarity of DNA allows researchers to form self-assembled structures at
the nanoscale[323] with the potential for a high degree of precision and complexity, as
reviewed in more detail elsewhere.[368] Oligonucleotides can be self-assembled into simple
Author Manuscript
geometric shapes, branching junction structures, and higher-order periodic structures by

selecting the nucleotide sequence to control conformation and binding among strands.[369]
While these nanostructures can be well controlled, high purity and precise stoichiometry are
required to prevent errors. More complex structures, termed “DNA origami,” can be formed
from long single-stranded DNA stabilized with short oligonucleotide sequences. This type of
design allows more flexibility in the required stoichiometry as well as greater complexity of
the final structure. Importantly, unmodified as well as functionalized DNA nanostructures

have been shown to be able to be internalized by cells, normally a challenge for naked DNA,
and do not cause the type of cytotoxicity often seen with cationic materials.[368] Nucleic
Author Manuscript
acids can also be easily loaded into DNA-based nanostructures through traditional chemical
functionalization or Watson-Crick base complementarity, either as the therapeutic material
or as a targeting moiety, as in the case of aptamers. This class of nanomaterials, therefore,
can use DNA itself as a structural component as well as facilitating the delivery of
biologically active nucleic acids. Lee et al. used self-assembled DNA tetrahedrons to deliver
siRNA to HeLa cells.[370] Because tight control of oligonucleotide stoichiometry affords this
method a high degree of precision in the geometric shapes formed, they were able to
fabricate monodisperse, “molecularly identical” nanoparticles using base hybridization. Pre-
conjugation of folic acid to the siRNA strands also allowed the researchers to achieve gene
knockdown specifically in cancer cells overexpressing the folate receptor.[370]
The precision and detailed bottom-up planning needed for base hybridization-mediated
Author Manuscript
DNA self-assembly can itself be a hurdle to efficient nanocarrier design, in addition to

requiring a large amount of high-purity DNA sequences to use as building blocks. Rolling
circle replication (RCR) allows researchers to generate large quantities of long DNA strands
via an enzymatic process, and these DNA strands form nanoscale liquid crystals without
requiring careful selection of complementary base sequences. This DNA self-assembly
method results in “nanoflowers” (NFs) that can be used as a carrier for other therapeutics or
even be used as the therapeutic molecule itself, with greater flexibility in the DNA
sequences that can be used. Jin et al. fabricated NFs using DNA containing built-in
functionalities, including DNAzymes targeting early growth response 1 (EGR-1) and
survivin, both involved in the development and progression of breast cancer, and an aptamer
binding cancer cells that overexpress nucleolin.[371] By incorporating magnesium
pyrophosphate during the self-assembly, they formed NFs that were stable at neutral pH but
Author Manuscript
collapsed at acidic conditions, such as that found in the endosomal component, due to the
dissolution of magnesium pyrophosphate. This provided a source of Mg2+ ions to be used as
co-factors for the DNAzymes, which subsequently resulted in knockdown of survivin and
EGR-1 expression by the cells. Importantly, decreasing the expression of both genes led to
better tumor control in vivo than decreased expression of either gene alone. Although in vivo
NF administrations were intratumoral rather than systemic, their in vitro studies suggested
that their aptamer targeting ligand could potentially lead to specific uptake of NFs by only
the cancer cell type of interest.[371]
As with other types of nanoparticles, NFs can be combined with other types of materials to
take advantage of their different characteristics. For instance, Zhu et al. designed
intertwining DNA-RNA nanocapsules (iDR-NCs) to deliver DNA and RNA together for
Author Manuscript
anti-tumor immunotherapy.[372] They designed DNA with a CpG sequence to stimulate

TLR9 and amplified this using RCR; an shRNA sequence targeting Stat3, which has
immunosuppressive functions in the presence of tumors, was encoded in DNA and also
amplified by rolling circle transcription (RCT) in the same pot to. The resulting DNA and
RNA sequences were self-assembled into “microflowers” (MFs), structures on the order of
1–2 μm in diameter, kept fairly large in part because of mutually repelling negative charges.
The MFs were then condensed into nanocapsules using a PPT-g-PEG, a cationic polypeptide
grafted with PEG chains. Finally, the authors complexed the iDR-NCs with a tumor

neoantigen, Adpkg. The resulting particle, consisting of (1) CpG-rich DNA, (2) Stat3-
Author Manuscript
targeting shRNA, (3) a cationic polymer, and (4) a tumor antigen was able affect multiple
aspects of the tumor immune environment simultaneously and lead to a durable, specific T
cell-mediated anti-tumor response (Figure 14).[372]
5.6. Genome-editing delivery systems

A growing niche in intersection of gene therapy and combinatorial delivery is the field of
genome editing. Genome editing technologies [373] are special cases that illustrate the
importance of nanoparticles that can reliably co-deliver multiple nucleic acid cargos. A
number of genome-editing tools exist, including zinc-finger nucleases (ZFNs)[374] and
transcription activator-like effector nucleases (TALENs),[375], both of which consist of the
enzyme Fok1 that cleaves DNA at a specific site determined by the DNA-binding domains
flanking the endonuclease, and this cleavage event and subsequent repair results in
Author Manuscript
modification of the original sequence. Because the site specificity relies on the binding
affinity of the protein domains for a particular DNA sequence, designing new ZFNs and
TALENs to target different sequences requires potentially extensive protein engineering.
While these nucleases can be encoded in nucleic acids like DNA or mRNA and delivered as
such, we focus here on the CRISPR-Cas [clustered regularly interspaced short palindromic
repeat/CRISPR-associated protein (Cas)] platform, which is most commonly accomplished
by co-delivery of two separate nucleic acids. In the CRISPR-Cas system, which has been
reviewed in detail elsewhere,[373] the DNA sequence specificity is determined by a single
guide RNA sequence (sgRNA) rather than by the binding affinity of a protein, simplifying
the process of designing systems with new specificities, and a nuclease, commonly Cas9,
serves to cut the genomic DNA at the specific site. Traditionally, viral vectors were used to
deliver (1) DNA or mRNA encoding the Cas9 nuclease along with (2) DNA or mRNA
Author Manuscript
encoding the sgRNA,[376, 377] but researchers have since developed non-viral methods of co-
delivering Cas9- and sgRNA-expressing nucleic acids or sgRNA itself in order to improve
the translational potential of this technology.[378]
The delivery of plasmid DNA and mRNA faces challenges similar to those described
previously—the former must be trafficked into the nucleus while the latter acts in the
cytosol; mRNA also avoids the risk of insertion into the nucleus but may be less stable—but
must also contend with the need for both components, Cas9 and sgRNA, to complex with
one another before entering the nucleus themselves to edit the genomic DNA. As a result,
many groups have developed modified versions of the types of nanoparticles described
above to co-deliver the larger Cas9-expressing nucleic acid and the smaller sgRNA. Due to
differences in expression kinetics and persistence of the two components, some studies have
Author Manuscript
also found that mRNA encoding Cas9 should ideally be delivered before sgRNA to optimize
efficiency.[379] In this study, Miller et al. used zwitterionic amino lipids (ZALs) to co-deliver
sgRNA and mRNA encoding Cas9, taking advantage of the ability of lipids with high
positive charge to bind to smaller nucleic acids (sgRNA) and the ability of zwitterionic lipids
to bind to longer mRNA, resulting in co-encapsulated cargo. Though they noted the
preference for sequential delivery of mRNA and sgRNA, concerns about delivery kinetics
and persistence must be balanced against the necessity for both nucleic acids to reach the

same cell, which is most easily facilitated by delivering both components within the same
Author Manuscript
nanoparticle, and the authors demonstrated successful in vivo editing by increasing the ratio
of Cas9 mRNA to sgRNA within the same ZAL particles.[379]
An example of polymer-mediated genome editing was recently reported by Rui et al.[380] In

order to co-encapsulate Cas9 plasmid DNA, which is large and must enter the nucleus, and
sgRNA, which is much smaller and must complex with the Cas9 protein in the cytosol, the
authors used modified PBAEs termed reducible branched ester-amine quadpolymers
(rBEAQs). The hyperbranched architecture of rBEAQs increased the amine density of the
polymer, permitting tighter electrostatic binding, and the presence of reducible disulfide
bridges in the polymer backbone permits targeted cytosolic release of smaller nucleic acids
like sgRNA.[380] These PBAE-derivatives were able to achieve approximately 40% gene
knockout in vitro by co-delivery of the two cargos.
Author Manuscript
For successful genome editing using CRISPR/Cas technology, nanoparticles must be

designed for successful co-encapsulation and co-delivery of both components, generally
achieved using nucleic acid cargos. The differences in the sites of action and the physical
and chemical properties of the two components requires that the particles be designed for
optimal delivery of both types of cargo. Because this technology is intended to cause
permanent genetic change, unlike many of the previously discussed nucleic acid delivery
methods that result in only transient retention of the nucleic acid cargo, any non-specific or
off-target effects present an important risk. One strategy for addressing this is to design
plasmids with promoters allow expression of either Cas or the sgRNA only in certain
circumstances, such as in the presence of tetracycline.[381, 382] While this field is still
growing, genome editing with nanocarriers has already proven to be a powerful approach
and highlights the benefits of being able to co-deliver different nucleic acids.[383]
Author Manuscript
6. Conclusions and Perspectives

Nucleic acids have the ability to specifically target single genes, affording them high
precision, and multiple different nucleic acid cargos can be delivered in combination,
providing a strategy for overcoming drug resistance mechanisms in heterogeneous cancer
cell populations. The physical and chemical properties of nucleic acids, however, requires
that they be carefully complexed or encapsulated in a vehicle that will facilitate safe and
effective intracellular delivery to target cells. Various aspects of the nucleic acid sequence,
the activity of the nucleic acid or its product, the physicochemical properties of the
nanoparticle, or administration method can also determine the tissue or cell type affected.
While there are many different types of nucleic acids, their broadly similar physical and
Author Manuscript
chemical characteristics allows them to be co-loaded or co-complexed into a vehicle for

combinatorial delivery, although specific delivery hurdles must still be addressed for
different types of nucleic acids. A wide range of different biomaterials can be used to
complex or encapsulate genetic material, with cationic lipids and polymers being the most
prominent in the field. While a large proportion of the early work in the field of gene
delivery focused on development of materials or nanoparticles that could facilitate effective
transfection, either in vitro or in vivo, as our understanding of transfection agents improves,

as well as our understanding of the biological mechanisms underlying cancer, an increasing

Author Manuscript
number of reports have aimed to achieve enhanced therapeutic effects through combinatorial
delivery of genes that target multiple distinct biochemical pathways.
Apart from the challenges of designing the optimal nucleic acids for the desired biological
effects and the challenges of engineering the chemical vehicle to overcome delivery
limitations in a controlled laboratory setting, for a new translational genetic medicine to be
successful, there are also important manufacturing and scale-up considerations. For
example, while many bottom-up self-assembly processes allow polymers, lipids, and other
biomolecules to bind to and encapsulate nucleic acids into nanostructures, these processes
can often be variable and heterogeneous, generating a polydisperse population of
nanostructures.[384, 385] This heterogeneity can be exacerbated by scaling up from small
scale bulk mixing conditions found in a laboratory to larger size volumes, especially as
mixing environments vary with different types of processing equipment. To help alleviate
Author Manuscript
these concerns, increasing attention in the nanomedicine field has turned to continuous
manufacture processes such as microfluidic mixing and extrusion techniques that can be
more easily scaled in a homogenous and robust manner.[386–388] Similarly, to minimize
variability in patient outcomes, homogeneity in nanomedicine properties is critical and may
necessitate improvements to both separation and characterization methods to ensure
precision. Finally, as these nanostructured materials encapsulate fragile nucleic acids and are
often wholly composed of degradable materials, the shelf-life and durability of the materials
is also an important concern for clinical translation. Excipient selection, storage conditions,
and often freeze-drying procedures (lyophilization) must be optimized and can make the
difference between a suitable nanomaterial for genetic medicine in the clinic and one that
unsuitable due to aggregation, degradation, or altered biophysical properties.[389, 390]
Author Manuscript
Thus, for successful nanoparticle-mediated combinatorial nucleic acid delivery to cancer,

there are important challenges that span the biological, chemical, and engineering
disciplines. Advanced nanomaterials are capable of overcoming these obstacles and progress
in this field is accelerating as new approaches enter the clinic. The continuing development
of nanomaterials for targeted nucleic acid delivery hold enormous promise for the treatment
of cancer.
Acknowledgements
The authors thank the NIH for support (R01CA228133 and R01EB022148).
Biographies
Author Manuscript
Hannah J. Vaughan received her B.S. in Biomedical Engineering from Duke University
and came to the Johns Hopkins University School of Medicine in 2016 as a PhD candidate.

Her research under the mentorship of Dr. Jordan Green is focused on targeted non-viral
Author Manuscript
nucleic acid delivery for cancer therapy and diagnostics.
Jordan J. Green is a Professor of Biomedical Engineering, Chemical & Biomolecular

Engineering, Materials Science & Engineering, Oncology, Neurosurgery, and
Ophthalmology at the Johns Hopkins University School of Medicine. He received his Ph.D.
in Biological Engineering from the Massachusetts Institute of Technology in 2007. Prof.
Green’s main research interests are in creating biomaterials and nanobiotechnology to
Author Manuscript
engineer cells and developing advanced therapeutics.
Stephany Y. Tzeng earned her PhD in Biomedical Engineering at the Johns Hopkins
University School of Medicine in 2014 and joined their research faculty in 2017. Her
research focuses on nanomedicine for cancer treatment, with particular focus on gene
therapy and immunotherapy.
Author Manuscript
References
[1]. Barbalat R, Ewald SE, Mouchess ML, Barton GM, Annual review of immunology 2011, 29, 185.
[2]. Lechardeur D, Verkman AS, Lukacs GL, Adv Drug Deliver Rev 2005, 57, 755.
[3]. Kauffman KJ, Webber MJ, Anderson DG, Journal of controlled release: official journal of the
Controlled Release Society 2016, 240, 227. [PubMed: 26718856]
[4]. Youn H, Chung JK, Expert opinion on biological therapy 2015, 15, 1337. [PubMed: 26125492]
[5]. Fire A, Xu SQ, Montgomery MK, Kostas SA, Driver SE, Mello CC, Nature 1998, 391, 806.
[PubMed: 9486653]
[6]. Kuwabara PE, Coulson A, Parasitology Today 2000, 16, 347. [PubMed: 10900483]
[7]. Wu W, Sun M, Zou GM, Chen J, International Journal of Cancer 2007, 120, 953. [PubMed:
17163415]
[8]. Yadav S, van Vlerken LE, Little SR, Amiji MM, Cancer Chemotherapy and Pharmacology 2009,
63, 711. [PubMed: 18618115]
Author Manuscript
[9]. Wilson RC, Doudna JA, Annual review of biophysics 2013, 42, 217.
[10]. Hannon GJ, Nature 2002, 418, 244. [PubMed: 12110901]
[11]. Stark GR, Kerr IM, Williams BRG, Silverman RH, Schreiber RD, Annual Review of
Biochemistry 1998, 67, 227.
[12]. Zambon RA, Vakharia VN, Wu LP, Cellular microbiology 2006, 8, 880. [PubMed: 16611236]
[13]. Li Y, Lu J, Han Y, Fan X, Ding SW, Science 2013, 342, 231. [PubMed: 24115437]
[14]. Scheiermann J, Klinman DM, Vaccine 2014, 32, 6377. [PubMed: 24975812]
[15]. Diebold SS, Kaisho T, Hemmi H, Akira S, Reis e Sousa C, Science 2004, 303, 1529. [PubMed:
14976261]

[16]. Lund JM, Alexopoulou L, Sato A, Karow M, Adams NC, Gale NW, Iwasaki A, Flavell RA,
Proceedings of the National Academy of Sciences of the United States of America 2004, 101,
Author Manuscript
5598. [PubMed: 15034168]

[17]. Alexopoulou L, Holt AC, Medzhitov R, Flavell RA, Nature 2001, 413, 732. [PubMed: 11607032]
[18]. O’Neill LA, Golenbock D, Bowie AG, Nature reviews. Immunology 2013, 13, 453.
[19]. Krasteva PV, Sondermann H, Nat Chem Biol 2017, 13, 350. [PubMed: 28328921]
[20]. Judge AD, Sood V, Shaw JR, Fang D, McClintock K, MacLachlan I, Nature biotechnology 2005,
23, 457.
[21]. Dass CR, Choong PF, Khachigian LM, Molecular cancer therapeutics 2008, 7, 243. [PubMed:
18281510]
[22]. Rybinski B, Yun K, Oncotarget 2016, 7, 72322. [PubMed: 27608848]
[23]. Iyer AK, Singh A, Ganta S, Amiji MM, Adv Drug Deliv Rev 2013, 65, 1784. [PubMed:
23880506]
[24]. Xu X, Ho W, Zhang X, Bertrand N, Farokhzad O, Trends in molecular medicine 2015, 21, 223.
[PubMed: 25656384]
Author Manuscript
[25]. Hu CM, Zhang L, Biochemical pharmacology 2012, 83, 1104. [PubMed: 22285912]
[26]. Dewey RA, Morrissey G, Cowsill CM, Stone D, Bolognani F, Dodd NJF, Southgate TD,
Klatzmann D, Lassmann H, Castro MG, Lowenstein PR, Nature Medicine 1999, 5, 1256.
[27]. Verma IM, Mol Ther 2000, 2, 415. [PubMed: 11082313]
[28]. Cornetta K, Morgan RA, Anderson WF, Hum Gene Ther 1991, 2, 5. [PubMed: 1863639]
[29]. Check E, Nature 2003, 421, 678.
[30]. Check E, Nature 2005, 433, 561.
[31]. Putnam D, Nat. Mater 2006, 5, 439. [PubMed: 16738681]
[32]. Wu GY, Wu CH, Journal of Biological Chemistry 1987, 262, 4429. [PubMed: 3558345]
[33]. Wagner E, Plank C, Zatloukal K, Cotten M, Birnstiel ML, P Natl Acad Sci USA 1992, 89, 7934.
[34]. Ball RL, Hajj KA, Vizelman J, Bajaj P, Whitehead KA, Nano letters 2018, 18, 3814. [PubMed:
29694050]
[35]. Vadiei K, Lopez-Berestein G, Perez-Soler R, Luke DR, International Journal of Pharmaceutics
1989, 57, 133.
Author Manuscript
[36]. Scherphof GL, Dijkstra J, Spanjer HH, Derksen JT, Roerdink FH, Ann N Y Acad Sci. 1985, 446,
368. [PubMed: 2409883]
[37]. Alving CR, Steck EA, Chapman WL Jr., Waits VB, Hendricks LD, Swartz GM Jr., Hanson WL,
P Natl Acad Sci USA 1978, 75, 2959.
[38]. Harguindey A, Domaille DW, Fairbanks BD, Wagner J, Bowman CN, Cha JN, Advanced
materials 2017, 29.
[39]. Cutler JI, Auyeung E, Mirkin CA, Journal of the American Chemical Society 2012, 134, 1376.
[PubMed: 22229439]
[40]. Ding Y, Jiang Z, Saha K, Kim CS, Kim ST, Landis RF, Rotello VM, Mol Ther 2014, 22, 1075.
[PubMed: 24599278]
[41]. Bolcato-Bellemin AL, Bonnet ME, Creusatt G, Erbacher P, Behr JP, Proceedings of the National
Academy of Sciences of the United States of America 2007, 104, 16050. [PubMed: 17913877]
[42]. Li SD, Chen YC, Hackett MJ, Huang L, Mol Ther 2008, 16, 163. [PubMed: 17923843]
[43]. Wilson DR, Sen R, Sunshine JC, Pardoll DM, Green JJ, Kim YJ, Nanomedicine: nanotechnology,
Author Manuscript
biology, and medicine 2018, 14, 237.

[44]. Brus C, Petersen H, Aigner A, Czubayko F, Kissel T, European journal of pharmaceutics and
biopharmaceutics: official journal of Arbeitsgemeinschaft fur Pharmazeutische Verfahrenstechnik
e.V 2004, 57, 427. [PubMed: 15093589]
[45]. Zou W, Liu C, Chen Z, Zhang N, Int J Pharm 2009, 370, 187. [PubMed: 19073241]
[46]. Leong KW, Mao HQ, Truong-Le VL, Roy K, Walsh SM, August JT, Journal of controlled
release: official journal of the Controlled Release Society 1998, 53, 183. [PubMed: 9741926]
[47]. Hafez I, Maurer N, Cullis P, Gene Therapy 2001, 8, 1188. [PubMed: 11509950]
[48]. Xu Y, Szoka FC Jr, Biochemistry 1996, 35, 5616. [PubMed: 8639519]

[49]. Zelphati O, Szoka FC Jr, P Natl Acad Sci USA 1996, 93, 11493.
[50]. Verma A, Stellacci F, Small 2010, 6, 12. [PubMed: 19844908]
Author Manuscript
[51]. Heitz F, Morris MC, Divita G, British journal of pharmacology 2009, 157, 195. [PubMed:
19309362]
[52]. Niidome T, Ohmori N, Ichinose A, Wada A, Mihara H, Hirayama T, Aoyagi H, Journal of
Biological Chemistry 1997, 272, 15307. [PubMed: 9182558]
[53]. Plank C, Oberhauser B, Mechtler K, Koch C, Wagner E, Journal of Biological Chemistry 1994,
269, 12918. [PubMed: 8175709]
[54]. Frankel AD, Pabo CO, Cell 1988, 55, 1189. [PubMed: 2849510]
[55]. Green M, Loewenstein PM, Cell 1988, 55, 1179. [PubMed: 2849509]
[56]. Choi CH, Hao L, Narayan SP, Auyeung E, Mirkin CA, Proceedings of the National Academy of
Sciences of the United States of America 2013, 110, 7625. [PubMed: 23613589]
[57]. Young KL, Scott AW, Hao L, Mirkin SE, Liu G, Mirkin CA, Nano letters 2012, 12, 3867.
[PubMed: 22725653]
[58]. Boussif O, Lezoualch F, Zanta MA, Mergny MD, Scherman D, Demeneix B, Behr JP,
Author Manuscript
7297. [PubMed: 7638184]
[59]. Nel AE, Madler L, Velegol D, Xia T, Hoek EMV, Somasundaran P, Klaessig F, Castranova V,
Thompson M, Nat Mater 2009, 8, 543. [PubMed: 19525947]
[60]. Meyer M, Philipp A, Oskuee R, Schmidt C, Wagner E, Journal of the American Chemical
Society 2008, 130, 3272. [PubMed: 18288843]
[61]. Chang H, Zhang J, Wang H, Lv J, Cheng Y, Biomacromolecules 2017, 18, 2371. [PubMed:
28686016]
[62]. Habrant D, Peuziat P, Colombani T, Dallet L, Gehin J, Goudeau E, Evrard B, Lambert O,
Haudebourg T, Pitard B, Journal of medicinal chemistry 2016, 59, 3046. [PubMed: 26943260]
[63]. Gary DJ, Puri N, Won YY, Journal of Controlled Release 2007, 121, 64. [PubMed: 17588702]
[64]. Luo D, Saltzman WM, Nature biotechnology 2000, 18, 33.
[65]. Lynn DM, Langer R, Journal of the American Chemical Society 2000, 122, 10761.
[66]. Son S, Namgung R, Kim J, Singha K, Kim WJ, Accounts Chem Res 2012, 45, 1100.
Author Manuscript
[67]. Lechardeur D, Lukacs GL, Human gene therapy 2006, 17, 882. [PubMed: 16972756]
[68]. Xu Y, Liang W, Qiu Y, Cespi M, Palmieri GF, Mason AJ, Lam JK, Molecular pharmaceutics
2016, 13, 3141. [PubMed: 27458925]
[69]. Kirchenbuechler I, Kirchenbuechler D, Elbaum M, Experimental cell research 2016, 345, 1.
[PubMed: 25556666]
[70]. Grosse S, Thevenot G, Monsigny M, Fajac I, J Gene Med 2006, 8, 845. [PubMed: 16685744]
[71]. Ross NL, Sullivan MO, Biotechnology and bioengineering 2016, 113, 2686. [PubMed:
27241022]
[72]. Matz RL, Erickson B, Vaidyanathan S, Kukowska-Latallo JF, Baker JR Jr., Orr BG, Banaszak
Holl MM, Molecular pharmaceutics 2013, 10, 1306. [PubMed: 23458572]
[73]. Chan KS, Koh CG, Li HY, Cell death & disease 2012, 3, e411. [PubMed: 23076219]
[74]. Bishop CJ, Tzeng SY, Green JJ, Acta biomaterialia 2015, 11, 393. [PubMed: 25246314]
[75]. Badeau BA, Comerford MP, Arakawa CK, Shadish JA, DeForest CA, Nature chemistry 2018, 10,
251.
Author Manuscript
[76]. Wightman L, Kircheis R, Rossler V, Carotta S, Ruzicka R, Kursa M, Wagner E, J Gene Med
2001, 3, 362. [PubMed: 11529666]
[77]. Ogris M, Steinlein P, Kursa M, Mechtler K, Kircheis R, Wagner E, Gene Therapy 1998, 5, 1425.
[PubMed: 9930349]
[78]. Ogris M, Brunner S, Schuller S, Kircheis R, Wagner E, Gene Therapy 1999, 6, 595. [PubMed:
10476219]
[79]. Suk JS, Suh J, Choy K, Lai SK, Fu J, Hanes J, Biomaterials 2006, 27, 5143. [PubMed:
16769110]

[80]. Hatakeyama H, Akita H, Kogure K, Oishi M, Nagasaki Y, Kihira Y, Ueno M, Kobayashi H,

Kikuchi H, Harashima H, Gene Therapy 2007, 14, 68. [PubMed: 16915290]
Author Manuscript
[81]. Kawano T, Yamagata M, Takahashi H, Niidome Y, Yamada S, Katayama Y, Niidome T, Journal

of Controlled Release 2006, 111, 382. [PubMed: 16487614]
[82]. Wang W, Balk M, Deng Z, Wischke C, Gossen M, Behl M, Ma N, Lendlein A, Journal of
controlled release: official journal of the Controlled Release Society 2016, 242, 71. [PubMed:
27498020]
[83]. Spagnou S, Miller AD, Keller M, Biochemistry 2004, 43, 13348. [PubMed: 15491141]
[84]. Hill IR, Garnett MC, Bignotti F, Davis SS, Biochimica et Biophysica Acta 1999, 1427, 161.
[PubMed: 10216233]
[85]. Lim YB, Han SO, Kong HU, Lee Y, Park JS, Jeong B, Kim SW, Pharmaceut Res 2000, 17, 811.
[86]. Forrest ML, Koerber JT, Pack DW, Bioconjugate Chem 2003, 14, 934.
[87]. Sutton D, Kim SJ, Shuai XT, Leskov K, Marques JT, Williams BRG, Boothman DA, Gao JM, Int
J Nanomed 2006, 1, 155.
[88]. Grayson ACR, Doody AM, Putnam D, Pharmaceut Res 2006, 23, 1868.
Author Manuscript
[89]. Matsumura Y, Maeda H, Cancer Res 1986, 46, 6387. [PubMed: 2946403]
[90]. O’Brien ME, Wigler N, Inbar M, Rosso R, Grischke E, Santoro A, Catane R, Kieback DG,
Tomczak P, Ackland SP, Orlandi F, Mellars L, Alland L, Tendler C, Group CBCS, Annals of
oncology: official journal of the European Society for Medical Oncology 2004, 15, 440.
[PubMed: 14998846]
[91]. Steichen SD, Caldorera-Moore M, Peppas NA, Eur J Pharm Sci 2013, 48, 416. [PubMed:
23262059]
[92]. Perrault SD, Walkey C, Jennings T, Fischer HC, Chan WC, Nano letters 2009, 9, 1909. [PubMed:
19344179]
[93]. Champion JA, Mitragotri S, Proceedings of the National Academy of Sciences of the United
States of America 2006, 103, 4930. [PubMed: 16549762]
[94]. Truong NP, Whittaker MR, Mak CW, Davis TP, Expert opinion on drug delivery 2015, 12, 129.
[PubMed: 25138827]
[95]. Geng Y, Dalhaimer P, Cai S, Tsai R, Tewari M, Minko T, Discher DE, Nature nanotechnology
2007, 2, 249.
Author Manuscript
[96]. Peracchia MT, Harnisch S, Pinto-Alphandary H, Gulik A, Dedieu JC, Desmaele D, d’Angelo J,
Muller RH, Couvreur P, Biomaterials 1999, 20, 1269. [PubMed: 10403044]
[97]. Gref R, Luck M, Quellec P, Marchand M, Dellacherie E, Harnisch S, Blunk T, Muller RH,
Colloids and surfaces. B, Biointerfaces 2000, 18, 301. [PubMed: 10915952]
[98]. Bazile D, Prud’homme C, Bassoullet MT, Marlard M, Spenlehauer G, Veillard M, Journal of
pharmaceutical sciences 1995, 84, 493. [PubMed: 7629743]
[99]. Gref R, Minamitake Y, Peracchia MT, Trubetskoy V, Torchilin V, Langer R, Science 1994, 263,
1600. [PubMed: 8128245]
[100]. Hatakeyama H, Akita H, Harashima H, Biological & pharmaceutical bulletin 2013, 36, 892.
[PubMed: 23727912]
[101]. Prabhakar U, Maeda H, Jain RK, Sevick-Muraca EM, Zamboni W, Farokhzad OC, Barry ST,
Gabizon A, Grodzinski P, Blakey DC, Cancer Res 2013, 73, 2412. [PubMed: 23423979]
[102]. Maeda H, Adv Drug Deliv Rev 2015, 91, 3. [PubMed: 25579058]
[103]. Jain RK, Stylianopoulos T, Nature reviews. Clinical oncology 2010, 7, 653.
Author Manuscript
[104]. Dahlman JE, Kauffman KJ, Xing Y, Shaw TE, Mir FF, Dlott CC, Langer R, Anderson DG,
Wang ET, Proceedings of the National Academy of Sciences of the United States of America
2017, 114, 2060. [PubMed: 28167778]
[105]. Sago CD, Lokugamage MP, Paunovska K, Vanover DA, Monaco CM, Shah NN, Gamboa
Castro M, Anderson SE, Rudoltz TG, Lando GN, Munnilal Tiwari P, Kirschman JL, Willett N,
Jang YC, Santangelo PJ, Bryksin AV, Dahlman JE, Proceedings of the National Academy of
Sciences of the United States of America 2018, 115, E9944. [PubMed: 30275336]
[106]. Bazak R, Houri M, El Achy S, Kamel S, Refaat T, Journal of cancer research and clinical
oncology 2015, 141, 769. [PubMed: 25005786]

[107]. Zhong Y, Meng F, Deng C, Zhong Z, Biomacromolecules 2014, 15, 1955. [PubMed: 24798476]
[108]. Byrne JD, Betancourt T, Brannon-Peppas L, Adv Drug Deliv Rev 2008, 60, 1615. [PubMed:
Author Manuscript
18840489]
[109]. Weis SM, Cheresh DA, Cold Spring Harbor perspectives in medicine 2011, 1, a006478.
[PubMed: 22229119]
[110]. Desgrosellier JS, Cheresh DA, Nature reviews. Cancer 2010, 10, 9. [PubMed: 20029421]
[111]. Harris TJ, Green JJ, Fung PW, Langer R, Anderson DG, Bhatia SN, Biomaterials 2010, 31, 998.
[PubMed: 19850333]
[112]. Noga DE, Petrie TA, Kumar A, Weck M, Garcia AJ, Collard DM, Biomacromolecules 2008, 9,
2056. [PubMed: 18576683]
[113]. Nicolas J, Mura S, Brambilla D, Mackiewicz N, Couvreur P, Chemical Society reviews 2013,
42, 1147. [PubMed: 23238558]
[114]. Li J, Feng L, Fan L, Zha Y, Guo L, Zhang Q, Chen J, Pang Z, Wang Y, Jiang X, Yang VC, Wen
L, Biomaterials 2011, 32, 4943. [PubMed: 21470674]
[115]. Kumar S, Aaron J, Sokolov K, Nature protocols 2008, 3, 314. [PubMed: 18274533]
Author Manuscript
[116]. Kawano K, Maitani Y, Journal of drug delivery 2011, 2011, 160967. [PubMed: 21490746]
[117]. Nasongkla N, Shuai X, Ai H, Weinberg BD, Pink J, Boothman DA, Gao J, Angewandte Chemie
2004, 43, 6323. [PubMed: 15558662]
[118]. Elias DR, Poloukhtine A, Popik V, Tsourkas A, Nanomedicine: nanotechnology, biology, and
medicine 2013, 9, 194.
[119]. Haun JB, Hammer DA, Langmuir: the ACS journal of surfaces and colloids 2008, 24, 8821.
[PubMed: 18630976]
[120]. Chithrani BD, Ghazani AA, Chan WC, Nano letters 2006, 6, 662. [PubMed: 16608261]
[121]. Choi CH, Alabi CA, Webster P, Davis ME, Proceedings of the National Academy of Sciences of
the United States of America 2010, 107, 1235. [PubMed: 20080552]
[122]. Nel AE, Madler L, Velegol D, Xia T, Hoek EM, Somasundaran P, Klaessig F, Castranova V,
Thompson M, Nat Mater 2009, 8, 543. [PubMed: 19525947]
[123]. Hrkach J, Von Hoff D, Mukkaram Ali M, Andrianova E, Auer J, Campbell T, De Witt D, Figa
M, Figueiredo M, Horhota A, Low S, McDonnell K, Peeke E, Retnarajan B, Sabnis A, Schnipper
E, Song JJ, Song YH, Summa J, Tompsett D, Troiano G, Van Geen Hoven T, Wright J, LoRusso
Author Manuscript
P, Kantoff PW, Bander NH, Sweeney C, Farokhzad OC, Langer R, Zale S, Science translational
medicine 2012, 4, 128ra39.
[124]. Reichert JM, Current pharmaceutical biotechnology 2008, 9, 423. [PubMed: 19075682]
[125]. Scott AM, Wolchok JD, Old LJ, Nature reviews. Cancer 2012, 12, 278. [PubMed: 22437872]
[126]. Stuchinskaya T, Moreno M, Cook MJ, Edwards DR, Russell DA, Photochemical &
photobiological sciences: Official journal of the European Photochemistry Association and the
European Society for Photobiology 2011, 10, 822.
[127]. Kirpotin DB, Drummond DC, Shao Y, Shalaby MR, Hong K, Nielsen UB, Marks JD, Benz CC,
Park JW, Cancer Res 2006, 66, 6732. [PubMed: 16818648]
[128]. Mamot C, Drummond DC, Noble CO, Kallab V, Guo Z, Hong K, Kirpotin DB, Park JW, Cancer
Res 2005, 65, 11631. [PubMed: 16357174]
[129]. Hansel TT, Kropshofer H, Singer T, Mitchell JA, George AJ, Nature reviews. Drug discovery
2010, 9, 325. [PubMed: 20305665]
[130]. Kijanka M, Dorresteijn B, Oliveira S, van Bergen en Henegouwen PM, Nanomedicine (Lond)
Author Manuscript
2015, 10, 161. [PubMed: 25597775]

[131]. Muyldermans S, Annu Rev Biochem 2013, 82, 775. [PubMed: 23495938]
[132]. Lipovsek D, Protein engineering, design & selection: PEDS 2011, 24, 3.
[133]. Holliger P, Hudson PJ, Nature biotechnology 2005, 23, 1126.
[134]. Winter G, Griffiths AD, Hawkins RE, Hoogenboom HR, Annual review of immunology 1994,
12, 433.
[135]. Kretzschmar T, von Ruden T, Current opinion in biotechnology 2002, 13, 598. [PubMed:
12482520]

[136]. Chen Y, Zhu X, Zhang X, Liu B, Huang L, Mol Ther 2010, 18, 1650. [PubMed: 20606648]
[137]. Yang L, Mao H, Wang YA, Cao Z, Peng X, Wang X, Duan H, Ni C, Yuan Q, Adams G, Smith
Author Manuscript
MQ, Wood WC, Gao X, Nie S, Small 2009, 5, 235. [PubMed: 19089838]
[138]. Punfa W, Yodkeeree S, Pitchakarn P, Ampasavate C, Limtrakul P, Acta pharmacologica Sinica
2012, 33, 823. [PubMed: 22580738]
[139]. Saha RN, Vasanthakumar S, Bende G, Snehalatha M, Molecular membrane biology 2010, 27,
215. [PubMed: 20939772]
[140]. Hornick JR, Xu J, Vangveravong S, Tu Z, Mitchem JB, Spitzer D, Goedegebuure P, Mach RH,
Hawkins WG, Molecular cancer 2010, 9, 298. [PubMed: 21092190]
[141]. Bartlett DW, Su H, Hildebrandt IJ, Weber WA, Davis ME, Proceedings of the National
[142]. Kircheis R, Wightman L, Schreiber A, Robitza B, Rossler V, Kursa M, Wagner E, Gene Ther
2001, 8, 28. [PubMed: 11402299]
[143]. Davis ME, Zuckerman JE, Choi CH, Seligson D, Tolcher A, Alabi CA, Yen Y, Heidel JD, Ribas
A, Nature 2010, 464, 1067. [PubMed: 20305636]
[144]. Zuckerman JE, Gritli I, Tolcher A, Heidel JD, Lim D, Morgan R, Chmielowski B, Ribas A,
Author Manuscript
Davis ME, Yen Y, Proceedings of the National Academy of Sciences of the United States of
America 2014, 111, 11449. [PubMed: 25049380]
[145]. Ganesh S, Iyer AK, Weiler J, Morrissey DV, Amiji MM, Molecular therapy. Nucleic acids 2013,
2, e110. [PubMed: 23900224]
[146]. Yu M, Jambhrunkar S, Thorn P, Chen J, Gu W, Yu C, Nanoscale 2013, 5, 178. [PubMed:
23076766]
[147]. Ganesh S, Iyer AK, Morrissey DV, Amiji MM, Biomaterials 2013, 34, 3489. [PubMed:
23410679]
[148]. Choi KY, Chung H, Min KH, Yoon HY, Kim K, Park JH, Kwon IC, Jeong SY, Biomaterials
2010, 31, 106. [PubMed: 19783037]
[149]. Low PS, Kularatne SA, Current opinion in chemical biology 2009, 13, 256. [PubMed:
19419901]
[150]. Zwicke GL, Mansoori GA, Jeffery CJ, Nano reviews 2012, 3.
[151]. Hilgenbrink AR, Low PS, Journal of pharmaceutical sciences 2005, 94, 2135. [PubMed:
Author Manuscript
16136558]
[152]. Stella B, Arpicco S, Peracchia MT, Desmaele D, Hoebeke J, Renoir M, D’Angelo J, Cattel L,
Couvreur P, Journal of pharmaceutical sciences 2000, 89, 1452. [PubMed: 11015690]
[153]. Zhao F, Yin H, Zhang Z, Li J, Biomacromolecules 2013, 14, 476. [PubMed: 23323627]
[154]. Benns JM, Mahato RI, Kim SW, Journal of controlled release: official journal of the Controlled
Release Society 2002, 79, 255. [PubMed: 11853936]
[155]. Maresca KP, Hillier SM, Femia FJ, Keith D, Barone C, Joyal JL, Zimmerman CN, Kozikowski
AP, Barrett JA, Eckelman WC, Babich JW, Journal of medicinal chemistry 2009, 52, 347.
[PubMed: 19111054]
[156]. Hillier SM, Maresca KP, Femia FJ, Marquis JC, Foss CA, Nguyen N, Zimmerman CN, Barrett
JA, Eckelman WC, Pomper MG, Joyal JL, Babich JW, Cancer Res 2009, 69, 6932. [PubMed:
19706750]
[157]. Von Hoff DD, Mita MM, Ramanathan RK, Weiss GJ, Mita AC, LoRusso PM, Burris HA 3rd,
Hart LL, Low SC, Parsons DM, Zale SE, Summa JM, Youssoufian H, Sachdev JC, Clinical
Author Manuscript
cancer research: an official journal of the American Association for Cancer Research 2016, 22,
3157. [PubMed: 26847057]
[158]. Xu X, Wu J, Liu Y, Saw PE, Tao W, Yu M, Zope H, Si M, Victorious A, Rasmussen J, Ayyash
D, Farokhzad OC, Shi J, ACS nano 2017, 11, 2618. [PubMed: 28240870]
[159]. Adamczyk B, Tharmalingam T, Rudd PM, Biochim Biophys Acta 2012, 1820, 1347. [PubMed:
22178561]
[160]. Dube DH, Bertozzi CR, Nature reviews. Drug discovery 2005, 4, 477. [PubMed: 15931257]
[161]. Bies C, Lehr CM, Woodley JF, Adv Drug Deliv Rev 2004, 56, 425. [PubMed: 14969751]

[162]. Zhu D, Tao W, Zhang H, Liu G, Wang T, Zhang L, Zeng X, Mei L, Acta biomaterialia 2016, 30,
144. [PubMed: 26602819]
Author Manuscript
[163]. Liang HF, Chen SC, Chen MC, Lee PW, Chen CT, Sung HW, Bioconjug Chem 2006, 17, 291.
[PubMed: 16536458]
[164]. Seymour LW, Ferry DR, Anderson D, Hesslewood S, Julyan PJ, Poyner R, Doran J, Young AM,
Burtles S, Kerr DJ, I. I. I. C. T. c. Cancer Research Campaign Phase, Journal of clinical
oncology: official journal of the American Society of Clinical Oncology 2002, 20, 1668.
[PubMed: 11896118]
[165]. Gao X, Tao W, Lu W, Zhang Q, Zhang Y, Jiang X, Fu S, Biomaterials 2006, 27, 3482. [PubMed:
16510178]
[166]. Fang RH, Hu CM, Luk BT, Gao W, Copp JA, Tai Y, O’Connor DE, Zhang L, Nano letters 2014,
14, 2181. [PubMed: 24673373]
[167]. Sun H, Su J, Meng Q, Yin Q, Chen L, Gu W, Zhang P, Zhang Z, Yu H, Wang S, Li Y, Advanced
materials 2016, 28, 9581. [PubMed: 27628433]
[168]. Tuerk C, Gold L, Science 1990, 249, 505. [PubMed: 2200121]
[169]. Daniels DA, Chen H, Hicke BJ, Swiderek KM, Gold L, Proceedings of the National Academy
Author Manuscript
of Sciences of the United States of America 2003, 100, 15416. [PubMed: 14676325]
[170]. Xiao Z, Shangguan D, Cao Z, Fang X, Tan W, Chemistry 2008, 14, 1769. [PubMed: 18092308]
[171]. Xiao Z, Levy-Nissenbaum E, Alexis F, Luptak A, Teply BA, Chan JM, Shi J, Digga E, Cheng J,
Langer R, Farokhzad OC, ACS nano 2012, 6, 696. [PubMed: 22214176]
[172]. Mi J, Liu Y, Rabbani ZN, Yang Z, Urban JH, Sullenger BA, Clary BM, Nat Chem Biol 2010, 6,
22. [PubMed: 19946274]
[173]. Liu H, Mai J, Shen J, Wolfram J, Li Z, Zhang G, Xu R, Li Y, Mu C, Zu Y, Li X, Lokesh GL,
Thiviyanathan V, Volk DE, Gorenstein DG, Ferrari M, Hu Z, Shen H, Theranostics 2018, 8, 31.
[PubMed: 29290791]
[174]. Kaur J, Tikoo K, Oncogene 2015, 34, 5216. [PubMed: 25639877]
[175]. Levy-Nissenbaum E, Radovic-Moreno AF, Wang AZ, Langer R, Farokhzad OC, Trends in
biotechnology 2008, 26, 442. [PubMed: 18571753]
[176]. Dhar S, Gu FX, Langer R, Farokhzad OC, Lippard SJ, Proceedings of the National Academy of
Author Manuscript
[177]. Farokhzad OC, Cheng J, Teply BA, Sherifi I, Jon S, Kantoff PW, Richie JP, Langer R,
6315. [PubMed: 16606824]
[178]. Farokhzad OC, Jon S, Khademhosseini A, Tran TN, Lavan DA, Langer R, Cancer Res 2004, 64,
7668. [PubMed: 15520166]
[179]. Liu G, Mao X, Phillips JA, Xu H, Tan W, Zeng L, Analytical chemistry 2009, 81, 10013.
[PubMed: 19904989]
[180]. Wang AZ, Bagalkot V, Vasilliou CC, Gu F, Alexis F, Zhang L, Shaikh M, Yuet K, Cima MJ,
Langer R, Kantoff PW, Bander NH, Jon S, Farokhzad OC, ChemMedChem 2008, 3, 1311.
[PubMed: 18613203]
[181]. Bagalkot V, Zhang L, Levy-Nissenbaum E, Jon S, Kantoff PW, Langer R, Farokhzad OC, Nano
letters 2007, 7, 3065. [PubMed: 17854227]
[182]. Fan Z, Sun L, Huang Y, Wang Y, Zhang M, Nature nanotechnology 2016, 11, 388.
[183]. Gragoudas ES, Adamis AP, Cunningham ET Jr., Feinsod M, Guyer DR, V. I. S. i. O. N. C. T.
Author Manuscript
Group, The New England journal of medicine 2004, 351, 2805. [PubMed: 15625332]
[184]. Povsic TJ, Cohen MG, Mehran R, Buller CE, Bode C, Cornel JH, Kasprzak JD, Montalescot G,
Joseph D, Wargin WA, Rusconi CP, Zelenkofske SL, Becker RC, Alexander JH, American heart
journal 2011, 161, 261. [PubMed: 21315207]
[185]. Lincoff AM, Mehran R, Povsic TJ, Zelenkofske SL, Huang Z, Armstrong PW, Steg PG, Bode
C, Cohen MG, Buller C, Laanmets P, Valgimigli M, Marandi T, Fridrich V, Cantor WJ, Merkely
B, Lopez-Sendon J, Cornel JH, Kasprzak JD, Aschermann M, Guetta V, Morais J, Sinnaeve PR,
Huber K, Stables R, Sellers MA, Borgman M, Glenn L, Levinson AI, Lopes RD, Hasselblad V,
Becker RC, Alexander JH, R.-P. Investigators, Lancet 2016, 387, 349. [PubMed: 26547100]

[186]. Rosenberg JE, Bambury RM, Van Allen EM, Drabkin HA, Lara PN Jr., Harzstark AL, Wagle N,
Figlin RA, Smith GW, Garraway LA, Choueiri T, Erlandsson F, Laber DA, Investigational new
Author Manuscript
drugs 2014, 32, 178. [PubMed: 24242861]

[187]. Guo W, Giancotti FG, Nature reviews. Molecular cell biology 2004, 5, 816. [PubMed:
15459662]
[188]. Mbeunkui F, Johann DJ Jr., Cancer Chemother Pharmacol 2009, 63, 571. [PubMed: 19083000]
[189]. Danhier F, Le Breton A, Preat V, Molecular pharmaceutics 2012, 9, 2961. [PubMed: 22967287]
[190]. Schiffelers RM, Ansari A, Xu J, Zhou Q, Tang Q, Storm G, Molema G, Lu PY, Scaria PV,
Woodle MC, Nucleic acids research 2004, 32, e149. [PubMed: 15520458]
[191]. Jiang J, Yang SJ, Wang JC, Yang LJ, Xu ZZ, Yang T, Liu XY, Zhang Q, European journal of
pharmaceutics and biopharmaceutics: official journal of Arbeitsgemeinschaft fur
Pharmazeutische Verfahrenstechnik e.V 2010, 76, 170. [PubMed: 20600887]
[192]. Kim J, Mirando AC, Popel AS, Green JJ, Adv Drug Deliv Rev 2017, 119, 20. [PubMed:
27913120]
[193]. Li W, Zhao X, Du B, Li X, Liu S, Yang XY, Ding H, Yang W, Pan F, Wu X, Qin L, Pan Y,
Scientific reports 2016, 6, 30619. [PubMed: 27470938]
Author Manuscript
[194]. Zheng Y, Chen H, Zeng X, Liu Z, Xiao X, Zhu Y, Gu D, Mei L, Nanoscale research letters
2013, 8, 161. [PubMed: 23570619]
[195]. Juweid M, Neumann R, Paik C, Perez-Bacete MJ, Sato J, van Osdol W, Weinstein JN, Cancer
Res 1992, 52, 5144. [PubMed: 1327501]
[196]. Allen TM, Nature reviews. Cancer 2002, 2, 750. [PubMed: 12360278]
[197]. Cheng Z, Al Zaki A, Hui JZ, Muzykantov VR, Tsourkas A, Science 2012, 338, 903. [PubMed:
23161990]
[198]. Gaertner FC, Kessler H, Wester HJ, Schwaiger M, Beer AJ, European journal of nuclear
medicine and molecular imaging 2012, 39 Suppl 1, S126. [PubMed: 22388629]
[199]. Whiteside TL, Oncogene 2008, 27, 5904. [PubMed: 18836471]
[200]. Yuan YY, Mao CQ, Du XJ, Du JZ, Wang F, Wang J, Advanced materials 2012, 24, 5476.
[PubMed: 22886872]
[201]. Coussens LM, Fingleton B, Matrisian LM, Science 2002, 295, 2387. [PubMed: 11923519]
[202]. Hatakeyama H, Akita H, Kogure K, Oishi M, Nagasaki Y, Kihira Y, Ueno M, Kobayashi H,
Author Manuscript
Kikuchi H, Harashima H, Gene therapy 2007, 14, 68. [PubMed: 16915290]

[203]. Hatakeyama H, Akita H, Harashima H, Adv Drug Deliv Rev 2011, 63, 152. [PubMed:
20840859]
[204]. He S, Fan W, Wu N, Zhu J, Miao Y, Miao X, Li F, Zhang X, Gan Y, Nano letters 2018, 18,
2411. [PubMed: 29561622]
[205]. Luo X, Li Z, Wang G, He X, Shen X, Sun Q, Wang L, Yue R, Ma N, ACS applied materials &
interfaces 2017, 9, 33624. [PubMed: 28915002]
[206]. Bujold KE, Hsu JCC, Sleiman HF, Journal of the American Chemical Society 2016, 138, 14030.
[PubMed: 27700075]
[207]. Du F, Wang Y, Zhang R, Li Z, Soft Matter 2010, 835.
[208]. Zintchenko A, Ogris M, Wagner E, Bioconjug Chem 2006, 17, 766. [PubMed: 16704216]
[209]. Oliveira S, Hogset A, Storm G, Schiffelers RM, Current pharmaceutical design 2008, 14, 3686.
[PubMed: 19075744]
Author Manuscript
[210]. Oliveira S, Fretz MM, Hogset A, Storm G, Schiffelers RM, Biochim Biophys Acta 2007, 1768,
1211. [PubMed: 17343820]
[211]. Mykhaylyk O, Zelphati O, Rosenecker J, Plank C, Current opinion in molecular therapeutics
2008, 10, 493. [PubMed: 18830925]
[212]. Schillinger U, Brill T, Rudolph C, Huth S, Gersting S, Krötz F, Hirschberger J, Bergemann C,
Plank C, Journal of Magnetism and Magnetic Materials 2005, 293, 501.
[213]. Hernot S, Klibanov AL, Adv Drug Deliv Rev 2008, 60, 1153. [PubMed: 18486268]

[214]. Sirsi SR, Hernandez SL, Zielinski L, Blomback H, Koubaa A, Synder M, Homma S, Kandel JJ,
Yamashiro DJ, Borden MA, Journal of controlled release: official journal of the Controlled
Author Manuscript

[215]. Yang D, Gao YH, Tan KB, Zuo ZX, Yang WX, Hua X, Li PJ, Zhang Y, Wang G, Gene Ther
2013, 20, 1140. [PubMed: 23966015]
[216]. McDannold N, Vykhodtseva N, Hynynen K, Physics in medicine and biology 2006, 51, 793.
[PubMed: 16467579]
[217]. Mura S, Nicolas J, Couvreur P, Nat Mater 2013, 12, 991. [PubMed: 24150417]
[218]. Fan CH, Ting CY, Lin HJ, Wang CH, Liu HL, Yen TC, Yeh CK, Biomaterials 2013, 34, 3706.
[PubMed: 23433776]
[219]. Kinoshita M, McDannold N, Jolesz FA, Hynynen K, Proceedings of the National Academy of
[220]. Wolinsky JB, Colson YL, Grinstaff MW, Journal of controlled release: official journal of the
[221]. Megeed Z, Haider M, Li D, O’Malley BW Jr., Cappello J, Ghandehari H, Journal of controlled
release: official journal of the Controlled Release Society 2004, 94, 433. [PubMed: 14744493]
Author Manuscript
[222]. Jang JH, Rives CB, Shea LD, Mol Ther 2005, 12, 475. [PubMed: 15950542]
[223]. Lei Y, Huang S, Sharif-Kashani P, Chen Y, Kavehpour P, Segura T, Biomaterials 2010, 31, 9106.
[PubMed: 20822811]
[224]. Lei Y, Rahim M, Ng Q, Segura T, Journal of controlled release: official journal of the Controlled
[225]. Nelson CE, Gupta MK, Adolph EJ, Shannon JM, Guelcher SA, Duvall CL, Biomaterials 2012,
33, 1154. [PubMed: 22061489]
[226]. Conde J, Oliva N, Zhang Y, Artzi N, Nat Mater 2016, 15, 1128. [PubMed: 27454043]
[227]. Sung JC, Pulliam BL, Edwards DA, Trends in biotechnology 2007, 25, 563. [PubMed:
17997181]
[228]. Koshkina NV, Knight V, Gilbert BE, Golunski E, Roberts L, Waldrep JC, Cancer Chemother
Pharmacol 2001, 47, 451. [PubMed: 11391862]
[229]. Patel AK, Kaczmarek JC, Bose S, Kauffman KJ, Mir F, Heartlein MW, DeRosa F, Langer R,
Anderson DG, Advanced materials 2019, e1805116. [PubMed: 30609147]
Author Manuscript
[230]. Alton E, Armstrong DK, Ashby D, Bayfield KJ, Bilton D, Bloomfield EV, Boyd AC, Brand J,
Buchan R, Calcedo R, Carvelli P, Chan M, Cheng SH, Collie DDS, Cunningham S, Davidson
HE, Davies G, Davies JC, Davies LA, Dewar MH, Doherty A, Donovan J, Dwyer NS, Elgmati
HI, Featherstone RF, Gavino J, Gea-Sorli S, Geddes DM, Gibson JSR, Gill DR, Greening AP,
Griesenbach U, Hansell DM, Harman K, Higgins TE, Hodges SL, Hyde SC, Hyndman L, Innes
JA, Jacob J, Jones N, Keogh BF, Limberis MP, Lloyd-Evans P, Maclean AW, Manvell MC,
McCormick D, McGovern M, McLachlan G, Meng C, Montero MA, Milligan H, Moyce LJ,
Murray GD, Nicholson AG, Osadolor T, Parra-Leiton J, Porteous DJ, Pringle IA, Punch EK,
Pytel KM, Quittner AL, Rivellini G, Saunders CJ, Scheule RK, Sheard S, Simmonds NJ, Smith
K, Smith SN, Soussi N, Soussi S, Spearing EJ, Stevenson BJ, Sumner-Jones SG, Turkkila M,
Ureta RP, Waller MD, Wasowicz MY, Wilson JM, Wolstenholme-Hogg P, U. K. C. F. G. T.
Consortium, The Lancet. Respiratory medicine 2015, 3, 684. [PubMed: 26149841]
[231]. Zhang Z, Tsai PC, Ramezanli T, Michniak-Kohn BB, Wiley interdisciplinary reviews.
Nanomedicine and nanobiotechnology 2013, 5, 205. [PubMed: 23386536]
[232]. Guterres SS, Alves MP, Pohlmann AR, Drug target insights 2007, 2, 147. [PubMed: 21901071]
Author Manuscript
[233]. Ozbas-Turan S, Akbuga J, Drug delivery 2011, 18, 215. [PubMed: 21226549]
[234]. Ozbas-Turan S, Akbuga J, Sezer AD, Oligonucleotides 2010, 20, 147. [PubMed: 20180684]
[235]. Yang SJ, Lin FH, Tsai KC, Wei MF, Tsai HM, Wong JM, Shieh MJ, Bioconjug Chem 2010, 21,
679. [PubMed: 20222677]
[236]. Jain A, Jain SK, Ganesh N, Barve J, Beg AM, Nanomedicine: nanotechnology, biology, and
medicine 2010, 6, 179.
[237]. Bowman K, Leong KW, Int J Nanomedicine 2006, 1, 117. [PubMed: 17722528]

[238]. Tsai CM, Chang KT, Wu LH, Chen JY, Gazdar AF, Mitsudomi T, Chen MH, Perng RP, Cancer
Res 1996, 56, 206. [PubMed: 8548764]
Author Manuscript
[239]. Buttitta F, Marchetti A, Gadducci A, Pellegrini S, Morganti M, Carnicelli V, Cosio S, Gagetti O,

Genazzani AR, Bevilacqua G, British journal of cancer 1997, 75, 230. [PubMed: 9010031]
[240]. Wang K, Huang Q, Qiu F, Sui M, Current medicinal chemistry 2015, 22, 4118. [PubMed:
26423086]
[241]. Islam MA, Xu Y, Tao W, Ubellacker JM, Lim M, Aum D, Lee GY, Zhou K, Zope H, Yu M, Cao
W, Oswald JT, Dinarvand M, Mahmoudi M, Langer R, Kantoff PW, Farokhzad OC, Zetter BR,
Shi J, Nature Biomedical Engineering 2018, 2, 850.
[242]. Zhang SW, Xiao SW, Liu CQ, Sun Y, Su X, Li DM, Xu G, Cai Y, Zhu GY, Xu B, Lu YY,
Zhonghua yi xue za zhi 2003, 83, 2023. [PubMed: 14703408]
[243]. Lang FF, Bruner JM, Fuller GN, Aldape K, Prados MD, Chang S, Berger MS, McDermott MW,
Kunwar SM, Junck LR, Chandler W, Zwiebel JA, Kaplan RS, Yung WK, Journal of clinical
oncology: official journal of the American Society of Clinical Oncology 2003, 21, 2508.
[PubMed: 12839017]
[244]. Vousden KH, Cancer cell 2002, 2, 351. [PubMed: 12450789]
Author Manuscript
[245]. Seoane J, Le HV, Massague J, Nature 2002, 419, 729. [PubMed: 12384701]
[246]. Komarova EA, Gudkov AV, Biochemistry. Biokhimiia 2000, 65, 41. [PubMed: 10702639]
[247]. Zeimet AG, Marth C, The Lancet. Oncology 2003, 4, 415. [PubMed: 12850192]
[248]. Gottesman MM, Cancer gene therapy 2003, 10, 501. [PubMed: 12833130]
[249]. Resnier P, Montier T, Mathieu V, Benoit JP, Passirani C, Biomaterials 2013, 34, 6429. [PubMed:
23727262]
[250]. Brummelkamp TR, Bernards R, Agami R, Cancer cell 2002, 2, 243. [PubMed: 12242156]
[251]. Wu Y, Zhou BP, British journal of cancer 2010, 102, 639. [PubMed: 20087353]
[252]. O’Brien DI, Nally K, Kelly RG, O’Connor TM, Shanahan F, O’Connell J, Expert opinion on
therapeutic targets 2005, 9, 1031. [PubMed: 16185156]
[253]. Adams JM, Cory S, Oncogene 2007, 26, 1324. [PubMed: 17322918]
[254]. Stuckey DW, Shah K, Trends in molecular medicine 2013, 19, 685. [PubMed: 24076237]
[255]. Liu S, Guo Y, Huang R, Li J, Huang S, Kuang Y, Han L, Jiang C, Biomaterials 2012, 33, 4907.
Author Manuscript
[PubMed: 22484049]
[256]. Jiang X, Fitch S, Wang C, Wilson C, Li J, Grant GA, Yang F, Proceedings of the National
[257]. Choi SA, Hwang SK, Wang KC, Cho BK, Phi JH, Lee JY, Jung HW, Lee DH, Kim SK, Neuro-
oncology 2011, 13, 61. [PubMed: 21062796]
[258]. Kim CY, Jeong M, Mushiake H, Kim BM, Kim WB, Ko JP, Kim MH, Kim M, Kim TH,
Robbins PD, Billiar TR, Seol DW, Gene Ther 2006, 13, 330. [PubMed: 16195699]
[259]. Robson T, Hirst DG, Journal of biomedicine & biotechnology 2003, 2003, 110. [PubMed:
12721516]
[260]. De Cian A, Lacroix L, Douarre C, Temime-Smaali N, Trentesaux C, Riou JF, Mergny JL,
Biochimie 2008, 90, 131. [PubMed: 17822826]
[261]. Hallenbeck PL, Chang YN, Hay C, Golightly D, Stewart D, Lin J, Phipps S, Chiang YL, Human
gene therapy 1999, 10, 1721. [PubMed: 10428217]
[262]. Li LY, Dai HY, Yeh FL, Kan SF, Lang J, Hsu JL, Jeng LB, Chen YH, Sher YP, Lin WC, Hung
Author Manuscript
MC, Oncogene 2011, 30, 1773. [PubMed: 21151169]

[263]. Little E, Ramakrishnan M, Roy B, Gazit G, Lee AS, Critical reviews in eukaryotic gene
expression 1994, 4, 1. [PubMed: 7987045]
[264]. Lee AS, Trends in biochemical sciences 2001, 26, 504. [PubMed: 11504627]
[265]. Katabi MM, Chan HL, Karp SE, Batist G, Human gene therapy 1999, 10, 155. [PubMed:
10022541]
[266]. Kawashita Y, Ohtsuru A, Kaneda Y, Nagayama Y, Kawazoe Y, Eguchi S, Kuroda H, Fujioka H,
Ito M, Kanematsu T, Yamashita S, Human gene therapy 1999, 10, 1509. [PubMed: 10395376]

[267]. Braiden V, Ohtsuru A, Kawashita Y, Miki F, Sawada T, Ito M, Cao Y, Kaneda Y, Koji T,
Yamashita S, Human gene therapy 2000, 11, 2453. [PubMed: 11119417]
Author Manuscript
[268]. Orth P, Schnappinger D, Hillen W, Saenger W, Hinrichs W, Nature structural biology 2000, 7,
215. [PubMed: 10700280]
[269]. Esposito CL, Nuzzo S, Kumar SA, Rienzo A, Lawrence CL, Pallini R, Shaw L, Alder JE, Ricci-
Vitiani L, Catuogno S, de Franciscis V, Journal of controlled release: official journal of the
[270]. Lopez-Bertoni H, Kozielski KL, Rui Y, Lal B, Vaughan H, Wilson DR, Mihelson N, Eberhart
CG, Laterra J, Green JJ, Nano letters 2018, 18, 4086. [PubMed: 29927251]
[271]. Chen W, Liu X, Xiao Y, Tang R, Small 2015, 11, 1775. [PubMed: 25641804]
[272]. Li H, Zhang K, Pi F, Guo S, Shlyakhtenko L, Chiu W, Shu D, Guo P, Advanced materials 2016,
28, 7501. [PubMed: 27322097]
[273]. Hatakeyama H, Ito E, Akita H, Oishi M, Nagasaki Y, Futaki S, Harashima H, Journal of
controlled release: official journal of the Controlled Release Society 2009, 139, 127. [PubMed:
19540888]
[274]. Cocco E, Deng Y, Shapiro EM, Bortolomai I, Lopez S, Lin K, Bellone S, Cui J, Menderes G,
Author Manuscript
Black JD, Schwab CL, Bonazzoli E, Yang F, Predolini F, Zammataro L, Altwerger G, de Haydu
C, Clark M, Alvarenga J, Ratner E, Azodi M, Silasi DA, Schwartz PE, Litkouhi B, Saltzman
WM, Santin AD, Molecular cancer therapeutics 2017, 16, 323. [PubMed: 27956521]
[275]. Sun L, Wang D, Chen Y, Wang L, Huang P, Li Y, Liu Z, Yao H, Shi J, Biomaterials 2017, 133,
219. [PubMed: 28441616]
[276]. Kay MA, Nature reviews. Genetics 2011, 12, 316.
[277]. Russell S, Bennett J, Wellman JA, Chung DC, Yu ZF, Tillman A, Wittes J, Pappas J, Elci O,
McCague S, Cross D, Marshall KA, Walshire J, Kehoe TL, Reichert H, Davis M, Raffini L,
George LA, Hudson FP, Dingfield L, Zhu X, Haller JA, Sohn EH, Mahajan VB, Pfeifer W,
Weckmann M, Johnson C, Gewaily D, Drack A, Stone E, Wachtel K, Simonelli F, Leroy BP,
Wright JF, High KA, Maguire AM, Lancet 2017, 390, 849. [PubMed: 28712537]
[278]. Naldini L, Nature 2015, 526, 351. [PubMed: 26469046]
[279]. Bessis N, GarciaCozar FJ, Boissier MC, Gene Ther 2004, 11 Suppl 1, S10. [PubMed:
15454952]
Author Manuscript
[280]. Baum C, Kustikova O, Modlich U, Li Z, Fehse B, Human gene therapy 2006, 17, 253.
[PubMed: 16544975]
[281]. Thomas CE, Ehrhardt A, Kay MA, Nature reviews. Genetics 2003, 4, 346.
[282]. Zhao Y, Huang L, Advances in genetics 2014, 88, 13. [PubMed: 25409602]
[283]. Leung AK, Tam YY, Cullis PR, Advances in genetics 2014, 88, 71. [PubMed: 25409604]
[284]. Kozielski KL, Tzeng SY, Green JJ, Wiley interdisciplinary reviews. Nanomedicine and
nanobiotechnology 2013, 5, 449. [PubMed: 23821336]
[285]. Akinc A, Zumbuehl A, Goldberg M, Leshchiner ES, Busini V, Hossain N, Bacallado SA,
Nguyen DN, Fuller J, Alvarez R, Borodovsky A, Borland T, Constien R, de Fougerolles A,
Dorkin JR, Jayaprakash KN, Jayaraman M, John M, Koteliansky V, Manoharan M, Nechev L,
Qin J, Racie T, Raitcheva D, Rajeev KG, Sah DWY, Soutschek J, Toudjarska I, Vornlocher HP,
Zimmermann TS, Langer R, Anderson DG, Nature biotechnology 2008, 26, 561.
[286]. Chen D, Love KT, Chen Y, Eltoukhy AA, Kastrup C, Sahay G, Jeon A, Dong Y, Whitehead KA,
Anderson DG, Journal of the American Chemical Society 2012, 134, 6948. [PubMed: 22475086]
Author Manuscript
[287]. Whitehead KA, Dorkin JR, Vegas AJ, Chang PH, Veiseh O, Matthews J, Fenton OS, Zhang Y,
Olejnik KT, Yesilyurt V, Chen D, Barros S, Klebanov B, Novobrantseva T, Langer R, Anderson
DG, Nature communications 2014, 5, 4277.
[288]. Zelphati O, Szoka FC Jr., Proceedings of the National Academy of Sciences of the United States
of America 1996, 93, 11493. [PubMed: 8876163]
[289]. Schroeder A, Levins CG, Cortez C, Langer R, Anderson DG, Journal of internal medicine 2010,
267, 9. [PubMed: 20059641]
[290]. Litzinger DC, Huang L, Biochimica et Biophysica Acta 1992, 1113, 201. [PubMed: 1510997]
[291]. Hafez IM, Cullis PR, Adv Drug Deliver Rev 2001, 47, 139.

[292]. Akinc A, Querbes W, De S, Qin J, Frank-Kamenetsky M, Jayaprakash KN, Jayaraman M,

Rajeev KG, Cantley WL, Dorkin JR, Butler JS, Qin L, Racie T, Sprague A, Fava E, Zeigerer A,
Author Manuscript
Hope MJ, Zerial M, Sah DW, Fitzgerald K, Tracy MA, Manoharan M, Koteliansky V,
Fougerolles A, Maier MA, Mol Ther 2010, 18, 1357. [PubMed: 20461061]
[293]. Lu JJ, Langer R, Chen JZ, Molecular pharmaceutics 2009, 6, 763. [PubMed: 19292453]
[294]. Umeda M, Nojima S, Inoue K, J Biochem-Tokyo 1985, 97, 1301. [PubMed: 2993266]
[295]. Xue HY, Liu S, Wong HL, Nanomedicine (Lond) 2014, 9, 295. [PubMed: 24552562]
[296]. Vangasseri DP, Cui Z, Chen W, Hokey DA, Falo LD Jr., Huang L, Molecular membrane biology
2006, 23, 385. [PubMed: 17060156]
[297]. Huang L, Liu Y, Annual review of biomedical engineering 2011, 13, 507.
[298]. Lin Q, Chen J, Zhang Z, Zheng G, Nanomedicine (Lond) 2014, 9, 105. [PubMed: 24354813]
[299]. Ozcan G, Ozpolat B, Coleman RL, Sood AK, Lopez-Berestein G, Adv Drug Deliv Rev 2015,
87, 108. [PubMed: 25666164]
[300]. Tabernero J, Shapiro GI, LoRusso PM, Cervantes A, Schwartz GK, Weiss GJ, Paz-Ares L, Cho
DC, Infante JR, Alsina M, Gounder MM, Falzone R, Harrop J, White AC, Toudjarska I, Bumcrot
D, Meyers RE, Hinkle G, Svrzikapa N, Hutabarat RM, Clausen VA, Cehelsky J, Nochur SV,
Author Manuscript
Gamba-Vitalo C, Vaishnaw AK, Sah DW, Gollob JA, Burris HA 3rd, Cancer discovery 2013, 3,
406. [PubMed: 23358650]
[301]. Bader AG, Frontiers in genetics 2012, 3, 120. [PubMed: 22783274]
[302]. Beg MS, Brenner AJ, Sachdev J, Borad M, Kang YK, Stoudemire J, Smith S, Bader AG, Kim S,
Hong DS, Investigational new drugs 2017, 35, 180. [PubMed: 27917453]
[303]. Kasinski AL, Kelnar K, Stahlhut C, Orellana E, Zhao J, Shimer E, Dysart S, Chen X, Bader AG,
Slack FJ, Oncogene 2015, 34, 3547. [PubMed: 25174400]
[304]. Saad M, Garbuzenko OB, Minko T, Nanomedicine (Lond) 2008, 3, 761. [PubMed: 19025451]
[305]. Curiel DT, Agarwal S, Wagner E, Cotten M, Proceedings of the National Academy of Sciences
of the United States of America 1991, 88, 8850. [PubMed: 1681545]
[306]. Midoux P, Monsigny M, Bioconjugate Chem 1999, 10, 406.
[307]. Jere D, Jiang HL, Arote R, Kim YK, Choi YJ, Cho MH, Akaike T, Cho CS, Expert opinion on
drug delivery 2009, 6, 827. [PubMed: 19558333]
[308]. Breunig M, Lungwitz U, Liebl R, Fontanari C, Klar J, Kurtz A, Blunk T, Goepferich A, J Gene
Author Manuscript
Med 2005, 7, 1287. [PubMed: 15906395]

[309]. Chen S, Liu X, Gong W, Yang H, Luo D, Zuo X, Li W, Wu P, Liu L, Xu Q, Ji A, Oncology
reports 2013, 29, 260. [PubMed: 23117577]
[310]. Akinc A, Anderson DG, Lynn DM, Langer R, Bioconjug Chem 2003, 14, 979. [PubMed:
13129402]
[311]. Tzeng SY, Yang PH, Grayson WL, Green JJ, Int J Nanomed 2012, 6, 3309.
[312]. Tzeng SY, Green JJ, Advanced Healthcare Materials 2013, 2, 467.
[313]. Green JJ, Langer R, Anderson DG, Accounts Chem Res 2008, 41, 749.
[314]. Kozielski KL, Tzeng SY, Green JJ, Chemical Communications 2013.
[315]. Tzeng SY, Hung BP, Grayson WL, Green JJ, Biomaterials 2012, 33, 8142. [PubMed: 22871421]
[316]. Liu Y, Chen J, Tang Y, Li S, Dou Y, Zheng J, Molecular pharmaceutics 2018, 15, 4558.
[PubMed: 30103607]
[317]. Dosta P, Ramos V, Borrós S, Molecular Systems Design & Engineering 2018, 3, 677.
Author Manuscript
[318]. Yin Q, Shen J, Chen L, Zhang Z, Gu W, Li Y, Biomaterials 2012, 33, 6495. [PubMed:
22704597]
[319]. Jeong JH, Christensen LV, Yockman JW, Zhong ZY, Engbersen JFJ, Kim WJ, Feijen J, Kim SW,
Biomaterials 2007, 28, 1912. [PubMed: 17218006]
[320]. Christensen LV, Chang CW, Kim WJ, Kim SW, Zhong Z, Lin C, Engbersen JF, Feijen J,
Bioconjug Chem 2006, 17, 1233. [PubMed: 16984133]
[321]. Nanjwade BK, Bechra HM, Derkar GK, Manvi FV, Nanjwade VK, Eur J Pharm Sci 2009, 38,
185. [PubMed: 19646528]

[322]. Maksimenko AV, Mandrouguine V, Gottikh MB, Bertrand JR, Majoral JP, Malvy C, J Gene Med
2003, 5, 61. [PubMed: 12516052]
Author Manuscript
[323]. Vincent L, Varet J, Pille JY, Bompais H, Opolon P, Maksimenko A, Malvy C, Mirshahi M, Lu
H, Vannier JP, Soria C, Li H, Int J Cancer 2003, 105, 419. [PubMed: 12704680]
[324]. Vader P, van der Aa LJ, Engbersen JFJ, Storm G, Schiffelers RM, Pharmaceut Res 2012, 29,
352.
[325]. Kim J, Kang Y, Tzeng SY, Green JJ, Acta biomaterialia 2016, 41, 293. [PubMed: 27262740]
[326]. Dahlman JE, Barnes C, Khan O, Thiriot A, Jhunjunwala S, Shaw TE, Xing Y, Sager HB, Sahay
G, Speciner L, Bader A, Bogorad RL, Yin H, Racie T, Dong Y, Jiang S, Seedorf D, Dave A,
Sandu KS, Webber MJ, Novobrantseva T, Ruda VM, Lytton-Jean AKR, Levins CG, Kalish B,
Mudge DK, Perez M, Abezgauz L, Dutta P, Smith L, Charisse K, Kieran MW, Fitzgerald K,
Nahrendorf M, Danino D, Tuder RM, von Andrian UH, Akinc A, Schroeder A, Panigrahy D,
Kotelianski V, Langer R, Anderson DG, Nature nanotechnology 2014, 9, 648.
[327]. Xue W, Dahlman JE, Tammela T, Khan OF, Sood S, Dave A, Cai W, Chirino LM, Yang GR,
Bronson R, Crowley DG, Sahay G, Schroeder A, Langer R, Anderson DG, Jacks T, Proceedings
of the National Academy of Sciences of the United States of America 2014, 111, E3553.
Author Manuscript
[PubMed: 25114235]
[328]. Jain RA, Biomaterials 2000, 21, 2475. [PubMed: 11055295]
[329]. Anderson JM, Shive MS, Adv Drug Deliver Rev 1997, 28, 5.
[330]. Walter E, Moelling K, Pavlovic J, Merkle HP, Journal of controlled release: official journal of
the Controlled Release Society 1999, 61, 361. [PubMed: 10477808]
[331]. Chumakova OV, Liopo AV, Andreev VG, Cicenaite I, Evers BM, Chakrabarty S, Pappas TC,
Esenaliev RO, Cancer letters 2008, 261, 215. [PubMed: 18164806]
[332]. Bivas-Benita M, Romeijn S, Junginger HE, Borchard G, European journal of pharmaceutics and
biopharmaceutics: official journal of Arbeitsgemeinschaft fur Pharmazeutische Verfahrenstechnik
e.V 2004, 58, 1. [PubMed: 15207531]
[333]. Capan Y, Woo BH, Gebrekidan S, Ahmed S, DeLuca PP, Journal of controlled release: official
journal of the Controlled Release Society 1999, 60, 279. [PubMed: 10425333]
[334]. Zhou J, Patel TR, Fu M, Bertram JP, Saltzman WM, Biomaterials 2012, 33, 583. [PubMed:
22014944]
Author Manuscript
[335]. Yuan X, Shah BA, Kotadia NK, Li J, Gu H, Wu Z, Pharm Res 2010, 27, 1285. [PubMed:
20309616]
[336]. Woodrow KA, Cu Y, Booth CJ, Saucier-Sawyer JK, Wood MJ, Saltzman WM, Nat Mater 2009,
8, 526. [PubMed: 19404239]
[337]. Wang Y, Gao S, Ye WH, Yoon HS, Yang YY, Nat Mater 2006, 5, 791. [PubMed: 16998471]
[338]. Ortiz Mellet C, Garcia Fernandez JM, Benito JM, Chemical Society reviews 2011, 40, 1586.
[PubMed: 21042619]
[339]. Pun SH, Bellocq NC, Liu A, Jensen G, Machemer T, Quijano E, Schluep T, Wen S, Engler H,
Heidel J, Davis ME, Bioconjug Chem 2004, 15, 831. [PubMed: 15264871]
[340]. Li HY, Seville PC, Williamson IJ, Birchall JC, J Gene Med 2005, 7, 1035. [PubMed: 15756712]
[341]. Davis ME, Molecular pharmaceutics 2009, 6, 659. [PubMed: 19267452]
[342]. Chen C, Okayama H, Molecular and Cellular Biology 1987, 7, 2745. [PubMed: 3670292]
[343]. Jordan M, Schallhorn A, Wurm FM, Nucleic acids research 1996, 24, 596. [PubMed: 8604299]
[344]. Li J, Chen YC, Tseng YC, Mozumdar S, Huang L, Journal of controlled release: official journal
Author Manuscript
of the Controlled Release Society 2010, 142, 416. [PubMed: 19919845]

[345]. Kakizawa Y, Kataoka K, Langmuir: the ACS journal of surfaces and colloids 2002, 18, 4539.
[346]. Kakizawa Y, Furukawa S, Ishii A, Kataoka K, Journal of Controlled Release 2006, 111, 368.
[PubMed: 16504335]
[347]. Pittella F, Zhang M, Lee Y, Kim HJ, Tockary T, Osada K, Ishii T, Miyata K, Nishiyama N,
Kataoka K, Biomaterials 2011, 32, 3106. [PubMed: 21272932]
[348]. Yang Y, Hu Y, Wang Y, Li J, Liu F, Huang L, Molecular pharmaceutics 2012, 9, 2280.
[PubMed: 22686936]

[349]. Slowing II, Vivero-Escoto JL, Wu CW, Lin VS, Adv Drug Deliv Rev 2008, 60, 1278. [PubMed:
18514969]
Author Manuscript
[350]. Taratula O, Garbuzenko OB, Chen AM, Minko T, Journal of drug targeting 2011, 19, 900.
[PubMed: 21981718]
[351]. Ghosh PS, Kim CK, Han G, Forbes NS, Rotello VM, ACS nano 2008.
[352]. Love JC, Estroff LA, Kriebel JK, Nuzzo RG, Whitesides GM, Chemical Reviews-Columbus
2005, 105, 1103.
[353]. Mirkin CA, Letsinger RL, Mucic RC, Storhoff JJ, Nature 1996.
[354]. Decher G, Hong JD, “Buildup of ultrathin multilayer films by a self-assembly process, 1
consecutive adsorption of anionic and cationic bipolar amphiphiles on charged surfaces”,
presented at Makromolekulare Chemie. Macromolecular Symposia, 1991.
[355]. Lee Y, Lee SH, Kim JS, Maruyama A, Chen X, Park TG, Journal of controlled release: official
journal of the Controlled Release Society 2011, 155, 3. [PubMed: 20869409]
[356]. Pissuwan D, Niidome T, Cortie MB, Journal of controlled release: official journal of the
[357]. Huang X, Jain PK, El-Sayed IH, El-Sayed MA, Lasers in medical science 2008, 23, 217.
Author Manuscript
[PubMed: 17674122]
[358]. Loo C, Lowery A, Halas N, West J, Drezek R, Nano letters 2005, 5, 709. [PubMed: 15826113]
[359]. Perez JM, O’Loughin T, Simeone FJ, Weissleder R, Josephson L, Journal of the American
Chemical Society 2002, 124, 2856. [PubMed: 11902860]
[360]. Fouriki A, Dobson J, Nanomedicine (Lond) 2014, 9, 989. [PubMed: 23901783]
[361]. Derfus AM, Chen AA, Min DH, Ruoslahti E, Bhatia SN, Bioconjug Chem 2007, 18, 1391.
[PubMed: 17630789]
[362]. Matea CT, Mocan T, Tabaran F, Pop T, Mosteanu O, Puia C, Iancu C, Mocan L, Int J
Nanomedicine 2017, 12, 5421. [PubMed: 28814860]
[363]. Liu G, Xie J, Zhang F, Wang Z, Luo K, Zhu L, Quan Q, Niu G, Lee S, Ai H, Chen X, Small
2011, 7, 2742. [PubMed: 21861295]
[364]. Yoon TJ, Kim JS, Kim BG, Yu KN, Cho MH, Lee JK, Angewandte Chemie 2005, 44, 1068.
[PubMed: 15635729]
[365]. Kievit FM, Veiseh O, Bhattarai N, Fang C, Gunn JW, Lee D, Ellenbogen RG, Olson JM, Zhang
Author Manuscript
M, Advanced functional materials 2009, 19, 2244. [PubMed: 20160995]

[366]. Duan H, Nie S, Journal of the American Chemical Society 2007, 129, 3333. [PubMed:
17319667]
[367]. Dong H, Dai W, Ju H, Lu H, Wang S, Xu L, Zhou SF, Zhang Y, Zhang X, ACS applied
materials & interfaces 2015, 7, 11015. [PubMed: 25942410]
[368]. Li J, Fan C, Pei H, Shi J, Huang Q, Advanced materials 2013, 25, 4386. [PubMed: 23765613]
[369]. Doye JP, Ouldridge TE, Louis AA, Romano F, Sulc P, Matek C, Snodin BE, Rovigatti L,
Schreck JS, Harrison RM, Smith WP, Physical chemistry chemical physics: PCCP 2013, 15,
20395. [PubMed: 24121860]
[370]. Lee H, Lytton-Jean AK, Chen Y, Love KT, Park AI, Karagiannis ED, Sehgal A, Querbes W,
Zurenko CS, Jayaraman M, Peng CG, Charisse K, Borodovsky A, Manoharan M, Donahoe JS,
Truelove J, Nahrendorf M, Langer R, Anderson DG, Nature nanotechnology 2012, 7, 389.
[371]. Jin Y, Li Z, Liu H, Chen S, Wang F, Wang L, Li N, Ge K, Yang X, Liang X-J, Zhang J, Npg
Asia Materials 2017, 9, e365.
Author Manuscript
[372]. Zhu G, Mei L, Vishwasrao HD, Jacobson O, Wang Z, Liu Y, Yung BC, Fu X, Jin A, Niu G,
Wang Q, Zhang F, Shroff H, Chen X, Nature communications 2017, 8, 1482.
[373]. Yin H, Kauffman KJ, Anderson DG, Nature reviews. Drug discovery 2017, 16, 387. [PubMed:
28337020]
[374]. Urnov FD, Rebar EJ, Holmes MC, Zhang HS, Gregory PD, Nature reviews. Genetics 2010, 11,
636.
[375]. Joung JK, Sander JD, Nature reviews. Molecular cell biology 2013, 14, 49. [PubMed:
23169466]

[376]. Senis E, Fatouros C, Grosse S, Wiedtke E, Niopek D, Mueller AK, Borner K, Grimm D,
Biotechnology journal 2014, 9, 1402. [PubMed: 25186301]
Author Manuscript
[377]. Wang D, Mou H, Li S, Li Y, Hough S, Tran K, Li J, Yin H, Anderson DG, Sontheimer EJ, Weng
Z, Gao G, Xue W, Human gene therapy 2015, 26, 432. [PubMed: 26086867]
[378]. Rui Y, Wilson DR, Green JJ, Trends in biotechnology 2019, 37, 281. [PubMed: 30278987]
[379]. Miller JB, Zhang S, Kos P, Xiong H, Zhou K, Perelman SS, Zhu H, Siegwart DJ, Angewandte
Chemie 2017, 56, 1059. [PubMed: 27981708]
[380]. Rui Y, Wilson D, Sanders K, Green JJ, ACS applied materials & interfaces 2019.
[381]. Dow LE, Fisher J, O’Rourke KP, Muley A, Kastenhuber ER, Livshits G, Tschaharganeh DF,
Socci ND, Lowe SW, Nature biotechnology 2015, 33, 390.
[382]. de Solis CA, Ho A, Holehonnur R, Ploski JE, Frontiers in molecular neuroscience 2016, 9, 70.
[PubMed: 27587996]
[383]. He X-Y, Liu B-Y, Peng Y, Zhuo R-X, Cheng S-X, ACS applied materials & interfaces 2019, 11,
226. [PubMed: 30540162]
[384]. Agmo Hernandez V, Karlsson G, Edwards K, Langmuir: the ACS journal of surfaces and
colloids 2011, 27, 4873. [PubMed: 21391645]
Author Manuscript
[385]. Burgos-Mármol JJ, Patti A, Polymer 2017, 113, 92.

[386]. Lim JM, Swami A, Gilson LM, Chopra S, Choi S, Wu J, Langer R, Karnik R, Farokhzad OC,
ACS nano 2014, 8, 6056. [PubMed: 24824296]
[387]. He Z, Santos JL, Tian H, Huang H, Hu Y, Liu L, Leong KW, Chen Y, Mao HQ, Biomaterials
2017, 130, 28. [PubMed: 28359018]
[388]. Wilson DR, Mosenia A, Suprenant MP, Upadhya R, Routkevitch D, Meyer RA, Quinones-
Hinojosa A, Green JJ, Journal of biomedical materials research. Part A 2017, 105, 1813.
[PubMed: 28177587]
[389]. Schulze J, Kuhn S, Hendrikx S, Schulz-Siegmund M, Polte T, Aigner A, Small 2018, 14,
e1701810. [PubMed: 29430833]
[390]. Rietscher R, Thum C, Lehr CM, Schneider M, Pharm Res 2015, 32, 1859. [PubMed: 25547536]
Author Manuscript
Author Manuscript

Author Manuscript
Author Manuscript
Author Manuscript
Figure 1. Challenges of nucleic acid delivery to tumors.

Effective and specific delivery of nucleic acids to tumors requires encapsulation or
condensation of the cargo into nanoparticles. Nanoparticles must then remain stable in
circulation, evading clearance and avoiding aggregation with other particles, and then leave
the circulation to accumulate at the tumor site. Once there, particles must enter cells, and
various intracellular barriers must be overcome depending on the type of nucleic acid cargo
being delivered.
Author Manuscript

Author Manuscript
Author Manuscript
Author Manuscript
Figure 2. Shape effects of spherical vs filamentous micelles.

Filomicelles are self-assembled from diblock co-polymers (a) with nano-scale diameter and
micro-scale length. The filomicelles extend in flow (b) and evade phagocytosis while
spherical micelles in flow are internalized. When the micelles are injected systemically in
mice, they persist in circulation for days, and longer micelles have a longer circulation half-
life than shorter micelles. Filomicelles are efficiently internalized (d) by lung epithelial cells
in static culture. Reproduced from Geng et al., “Shape effects of filaments versus spherical
particles in flow and drug delivery,” Nature Nanotechnology 2:249–255, 2007,[95] with
permission from Springer Nature.
Author Manuscript

Author Manuscript
Author Manuscript
Author Manuscript
Figure 3. Optimization parameters for cancer-specific nanocarriers.

Physical and chemical properties of delivery vehicles affect tumor accumulation, particle
internalization and cargo delivery, and ultimately the therapeutic outcome. Classes of
targeting moieties and their sizes are also summarized.
Author Manuscript

Author Manuscript
Author Manuscript
Author Manuscript
Figure 4.
A prostate cancer targeted multifunctional envelope-like nano device (MEND) (A)
nanocarrier is synesized by siRNA self-assembly with two block co-polymers: sharp
oligoarginine functionalized pH responsive Meo-PEG-b-P(DPA-co-GMA-Rn) and PSMA
Author Manuscript
targeted ACUPA-PEG-b-PDPA. Schematic shows targteted intracellular siRNA delivery

after IV administration of MENDs. This strategy enables efficient gene silencing and
significantly slows LNCaP tumor growth (B) compared with control and non-targeted NPs.
Representative images of tumor bearing mice on day 18 (C) and photographs of harvested
LNCaP tumors afetr 30 days (D). Reprinted with permission from Xu, Xiaoding, et al.,
“Multifunctional envelope-type siRNA delivery nanoparticle platform for prostate cancer
therapy,” ACS Nano 11(3): 2618–2627.[158] Copyright (2017) American Chemical Society.

Author Manuscript
Author Manuscript
Author Manuscript
Figure 5.
Schematic (A) illustrating the selection process for prostate cancer-specific internalizing
RNA aptamers. Nanoparticles coated with prostate cancer-specific internalizing aptamers are
specifically taken up in target PC3 cells (B) to a higher degree than in non-target HeLa cells.
Bare particles without aptamer are taken up at low levels in both target and non-target cells,
so aptamer conjugation is necessary for target-specific uptake. Uptake is distributed
Author Manuscript
throughout the cytosol of targeted calls (C). When particles are loaded with Docetaxel (D),
the aptamer conjugated particles (Dtxl-NP-Apt) are significantly more potent non-targeted
particles (Dtxl-NP) at killing target cells. Reprinted with permission from Xiao, Zeyu, et al.,
“Engineering of targeted nanoparticles for cancer therapy using internalizing aptamers
isolated by cell-uptake selection.” ACS Nano 6(1): 696–704.[171] Copyright (2012)
American Chemical Society.

Author Manuscript
Author Manuscript
Author Manuscript
Figure 6.
A vibrating mesh nebulizer (A) was used to prepare luciferase mRNA delivery vectors for
Author Manuscript
aerosol administration. Nano-scale polyplexes were encapsulated in micron-sized droplets

and administered to a whole-body chamber. Hyperbranched PBAE hDD90–118 polyplexes
enabled high levels of luciferase delivery in the lungs (B) after 24 hours, and local delivery
by inhallation resulted in highly specific delivery to lung tissue and negligible off-target
luciferase (c) measured by bioluminescence. Particles maintained a similar size and
morphology before and after nebulization, characterized by electron microscopy (D).
Particles also have a narrow size distribution before and after nebulization (E). Reprinted
with permission from Patel, Asha Kumari, et al., “Inhaled Nanoformulated mRNA

Polyplexes for Protein Production in Lung Epithelium,” Advanced Materials

(2019):e1805116,[229] with permission from John Wiley and Sons.
Author Manuscript
Author Manuscript
Author Manuscript
Author Manuscript

Author Manuscript
Author Manuscript
Author Manuscript
Figure 7.
DOTAP-cholesterol liposomes loaded with transcriptionally targeted eAFP-VISA-BikDD or
non-targeted CMV-BikDD were I.V. injected in orthotopic ML-1 tumor-bearing mice. Both
particles significantly reduced tumor burden (a) and representative photos are shown from 1
week after the last treatment. Mouse survival (b) was significant on treatment groups, and
the transcriptionally targeted DNA therapy extended survival significantly compared with
Author Manuscript
the non-targeted DNA. Tissue samples from mice in (a) were fixed and stained for apoptosis
using a TUNEL assay (c). The percentage of apoptotic cells were quantified in random
fields from both tumor and healthy liver. While the targeted and non-trageted therapies
induced similar numbers of apoptotic cells in the tumor, the transcriptionally targeted DNA
induced less apoptosis in the healthy liver tissue. Reprinted with permission from Li, L. Y.,
et al., “Targeted hepatocellular carcinoma proapoptotic BikDD gene therapy,” Oncogene
30(15):1773, 2011,[262] with permission from Springer Nature.

Author Manuscript
Author Manuscript
Figure 8. Types of nanomaterials used for nucleic acid delivery.

Broad classes of materials and nanostructures used as nucleic acid delivery vehicles are
Author Manuscript
summarized, including lipid-based nanoparticles (A), cationic polymer-based nanoparticles

(B), nanoparticles based on other polymer types (C), inorganic nanoparticles (D), and
nanostructures that use DNA itself as a structural component (E). Part E adapted from Doye
et al., “Coarse-graining DNA for simulations of DNA nanotechnology,” Physical Chemistry
Chemical Physics 15(47):20381–20772, 2013,[369] with permission from the Royal Society
of Chemistry.
Author Manuscript

Author Manuscript
Author Manuscript
Author Manuscript
Figure 9.
A liposomal formulation (NOV340) facilitated co-delivery of miR-34a and let-7b. After
multiple injections (a), tumor lesions in the left lobe of lungs in animals treated with one or
both miRNAs were fewer and smaller in number as seen by hematoxylin and eosin staining
(b) and quantified in (c). Tumor sizes were lower in animals treated with both miRNAs
compared to each individual miRNA (d), and tumor proliferation was lowe after treatment
Author Manuscript
(e-f). Survival was also extended by treatment with miR-34a. Figure reprinted by permission
from Springer Nature: Kasinski et al., “A combinatorial microRNA therapeutics approach to
suppressing non-small cell lung cancer,” Oncogene 34(27):3547–3555, 2015.[303]

Author Manuscript
Author Manuscript
Author Manuscript
Figure 10.
A cationic and bioreducible PBAE was used to form nanoparticles with each of two
miRNAs (miR-148a and miR-296–5p) or a combination of both in order to prevent the
growth and tumorigenicity of stem-like GBM cells (A). After intratumoral injection of
nanoparticles, the combination of both miRNAs was more effective than either individual
sequence in reducing tumor size (B) and causing necrosis of tumor tissue (C). Combination
miRNA delivery also significantly extended survival (D). Figure adapted with permission
from Lopez-Bertoni et al., “Bioreducible Polymeric Nanoparticles Containing Multiplexed
Author Manuscript
Cancer Stem Cell Regulating miRNAs Inhibit Glioblastoma Growth and Prolong Survival,”
Nano Letters 18(7):4086–4094.[270] Copyright 2018 American Chemical Society.

Author Manuscript
Author Manuscript
Author Manuscript
Figure 11.
Lipid-polymer hybrid nanoparticles were used to co-deliver an miRNA (miR-34a) and
siRNA (siKras) to lung tumor. The combination of both decreased the number of cancer
cells in vitro (A) and significantly slowed tumor growth in vivo (B). The RNA combination
also increased the number of CC3+ apoptotic cells in the tumor (C), and combining miRNA
and siRNA with cisplatin further improved animal survival over any of the component
Author Manuscript
treatments alone (D), indicating that combining these modalities can have an additive effect
on tumors. Figure reproduced from Xue et al., “Small RNA combination therapy for lung
cancer,” Proceedings of the National Academy of Sciences of the United States of America,
111(34):E3553-E3561.[327] Copyright 2014 National Academy of Sciences.

Author Manuscript
Author Manuscript
Figure 12.
Author Manuscript
Mesoporous silica nanoparticles allow the co-delivery of multiple cargos and

functionalization with a range of materials. In this example, MSNs were functionalized with
thiol-reactive groups. The pores were loaded with chemotherapeutics doxorubicin or
cisplatin, and the thiol-reactive groups were used to load two siRNA sequences and a tumor-
targeting peptide sequence to the surface. Figure reprinted with permission of Taylor &
Francis, Ltd, from Taratula et al., “Innovative strategy for treatment of lung cancer: targeted
nanotechnology-based inhalation co-delivery of anticancer drugs and siRNA,” Journal of
Drug Targeting 19(10):900–914, 2011.[350]
Author Manuscript

Author Manuscript
Author Manuscript
Figure 13.
The high surface area of graphene quantum dots allowed them to be functionalized with two
probes to inhibit miR-21 and survivin as well as polymers PLA and PEG. The resulting
GQDs were biocompatible in addition to retaining favorable optical properties. Figure
reprinted with permission from Dong et al., “Multifunctional Poly(L-lactide)–Polyethylene
Glycol-Grafted Graphene Quantum Dots for Intracellular MicroRNA Imaging and
Combined Specific-Gene-Targeting Agents Delivery for Improved Therapeutics,” ACS
Applied Materials and Interfaces, 7(20):11015–11023.[367] Copyright 2015, American
Chemical Society.
Author Manuscript
Author Manuscript

Author Manuscript
Author Manuscript
Author Manuscript
Figure 14.
Intertwining DNA-RNA nanocapsules (iDR-NCs) were fabricated into a multimodal
delivery vehicle. A CpG-rich DNA sequence and Stat3 shRNA sequence were amplified by
rolling circle replication or transcription, respectively, forming microflowers (MFs) (A). The
MFs were shrunk, or condensed, into iDR-NCs using PEG grafted to a cationic,
hydrophobic polypeptide (PPT-g-PEG), which was also used to load tumor antigens into the
NCs (B). These iDR-NCs could then be delivered to APCs in lymph nodes as a vaccine to
promote gene knockdown, immunostimulation, and a tumor-specific response (C). Figure is
Author Manuscript
reproduced with permission from Zhu et al., “Intertwining DNA-RNA nanocapsules loaded
with tumor neoantigens as synergistic nanovaccines for cancer immunotherapy,” Nature
Communications 8:1482 (2017).[372]

Table 1.
Examples of types of nucleic acid cargo, their properties, and delivery challenges.
Author Manuscript
Nucleic Acid Cargo Intended Action Site of Action Chemical Properties Delivery Challenges
Plasmid DNA (pDNA) Gene Transcription to • Double-stranded • Large size
overexpression mRNA in
nucleus • Circular • Endosomal escape
• Varying size • Entry into nucleus
(103-105 bp)
• Off-target immune
stimulation (CpG
sequences)
Messenger RNA Gene Translation to • Single-stranded • Endosomal escape

(mRNA) overexpression protein in
cytoplasm • Linear • Release into cytoplasm
• 102-104 bases • High susceptibility to
RNAses (ssRNA in
endosomes)
Author Manuscript
Small interfering RNA Gene knockdown RNAi pathway • Double-stranded • Endosomal escape
(siRNA) (affects specific in cytoplasm
gene) • Linear • Release into cytoplasm
• approx. 20 bp • Susceptibility to
RNAses
stimulation (dsRNA)
Micro RNA (miRNA) Gene regulation RNAi pathway • Double-stranded • Endosomal escape
(affects pool of in cytoplasm
genes) • Linear • Release into cytoplasm
• approx. 20 bp • Susceptibility to
RNAses
stimulation (dsRNA)
• Non-specific gene
effects
Author Manuscript
CpG Immune Endosome • Single-stranded • Off-target/excessive

oligodeoxynucleotides stimulation immune stimulation
(ODNs) • Linear
• approx. 10–30
bases
Cyclic dinucleotides Immune Cytoplasm • Heterocyclic • Endosomal escape

(CDNs) stimulation
• 2 nucleotides • Off-target/excessive
immune stimulation
Author Manuscript

Table 2.
Nucleic acid delivery vehicles that have been investigated in clinical trials. SD = Stable Disease, PR = Partial
Author Manuscript
Response by RECIST criteria
Name Nanocarrier Cargo Targeting Response Phase Identifier

ALN-VSP02 Liposome 1:1 anti-KF11 and Passive SD in 6/15 and PR in 1 Completed NCT01262235
anti-VEGF 1/15 patients at high NCT01158079
dose
Atu27 Liposome Anti-PKN3 siRNA Passive SD in 14/34 patients 2, Completed NCT00938574
CALAA-01 Cyclodextrin NP Anti-RRM2 siRNA Transferrin SD in 1/24 patients 1, Terminated NCT00689065
EPHARNA Liposome Anti-EPHA2 siRNA Passive 1, Recruiting NCT01591356
MK-4621–002 JetPEI™ MK-4621 RNA Intratumoral SD in 4/15 patients 2, Recruiting NCT03065023
targeting RIG-1 Injection NCT03739138
NU-0129 SNA Gold NP Anti-Bcl2L12 siRNA Passive 1, Recruiting NCT03020017
PNT2258 Liposome DNA oligo blocking Passive SD in 4/15, PR in 2, Completed NCT01733238
BCL2 2/15, and CR in 3/15
Author Manuscript
patients
SGT-53 Liposome p53 DNA plasmid Anti-transferrin SD in 7/11 patients 2, Recruiting NCT02354547
receptor scFv NCT02340156
NCT02340117
NCT03554707
TargomiRs EDV™nanocells miRNA mimic Anti-EGFR SD in 4/6 and PR in 1, Completed NCT02369198
antibody 1/6 patients
TherGAP JetPEI™ Plasmid DNA Local delivery SD in 6/22 patients 2, Recruiting NCT01274455
(DCK::UMK fusion by endoscopic NCT02806687
gene) ultrasound
TKM-080301 Liposome Anti-PLK1 siRNA Passive SD in 3/6 and PR in 1/2, Completed NCT01262235
1/6 patients NCT02191878
NCT01437007
Author Manuscript
Author Manuscript

Article - Cancer-Targeting Nanoparticles For Combinatorial Nucleic Acid Delivery

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Article - Cancer-Targeting Nanoparticles For Combinatorial Nucleic Acid Delivery

Uploaded by

Copyright:

Available Formats

HHS Public Access

Published in final edited form as:

Cancer-Targeting Nanoparticles for Combinatorial Nucleic Acid

combinations of nucleic acids will be reviewed.

Adv Mater. Author manuscript; available in PMC 2021 April 01.

2. Nucleic acids as cargo

DNA for overexpression of a gene is normally delivered as a double-stranded (ds) circular

Translation of mRNA can be interrupted by RNAi, leading to decreased expression of a

Adv Mater. Author manuscript; available in PMC 2021 April 01.

intracellular locations, and/or entry into the nucleus.

Adv Mater. Author manuscript; available in PMC 2021 April 01.

Non-viral, synthetic nanoparticles, including polymeric or lipid-based nanoparticles and

3. Nanoparticles as nucleic acid delivery vehicles

3.1. Nucleic acid binding or encapsulation

Adv Mater. Author manuscript; available in PMC 2021 April 01.

poly(L-lysine) (PLL), polyethylenimine (PEI), polyamidoamine (PAMAM), and poly(beta-

Adv Mater. Author manuscript; available in PMC 2021 April 01.

3.2. Cellular uptake

3.3. Endosomal escape and intracellular trafficking

Adv Mater. Author manuscript; available in PMC 2021 April 01.

3.4. In vivo stability of nanoparticles

Adv Mater. Author manuscript; available in PMC 2021 April 01.

in detail in the next section.

sufficient concentrations of an anti-cancer agent in the tumor while minimizing off-target

4.1. Passive Targeting

counterparts.[90] Nanoparticle size is a key predictor of passive targeting, because size

Adv Mater. Author manuscript; available in PMC 2021 April 01.

Inspired by the cylindrical or filamentous shapes of many viruses, non-spherical geometries

Adv Mater. Author manuscript; available in PMC 2021 April 01.

4.2. Active Targeting

to target various surface macromolecules on cancer cells, including overexpressed or

environment. Thus, covalent conjugation is commonly used for irreversible attachment of

well as carbodiimide compounds, such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide

Adv Mater. Author manuscript; available in PMC 2021 April 01.

4.2.1 Antibodies and Fragments—Antibodies have been extensively explored as

on a cancer cell, nanoparticles harness the specificity of antibodies while incorporating

Adv Mater. Author manuscript; available in PMC 2021 April 01.

liposomes surface-conjugated to a melanoma-targeted antibody fragment showed a

significant therapeutic benefit in melanoma lung metastases, while non-targeted control

4.2.2. Ligands—Another approach to cancer cell targeting is functionalizing

Adv Mater. Author manuscript; available in PMC 2021 April 01.

through receptor-mediated endocytosis, which has enabled many folate-conjugated drugs

showing the benefit of active targeting in this case.[158]

An alternative to direct ligand conjugation is coating nanocarriers with cancer cell

membranes. Recent research describes homotypic cancer cell binding—a phenomenon

Adv Mater. Author manuscript; available in PMC 2021 April 01.

4.2.3 Aptamers—As discussed, many ligand-based active targeting systems are

Adv Mater. Author manuscript; available in PMC 2021 April 01.

This dual targeting is a major advantage of RGD-functionalized nanocarriers. RGD-targeted

Despite promising preclinical data for active targeting in nanomedicine, no FDA-approved

model for active targeting in the future.

diagnostic methods to characterize target expression and predict patient response.

4.3. Stimulus-Responsive Targeting

exploited for nanoparticle targeting and controlled release. To respond to an environmental

Zwitterionic polymer nanoparticles are neutrally charged in physiological conditions, which

Adv Mater. Author manuscript; available in PMC 2021 April 01.

cancers.[205] Similarly, the DNA “nanosuitcase” developed by Bujold et al. opens

Systemically administered nanocarriers can also be triggered by an external stimulus to

Adv Mater. Author manuscript; available in PMC 2021 April 01.

was applied. DNA-containing PEI polyplexes conjugated to microbubbles combined with

limited to primary tumor sites rather than dispersed metastases.

4.4. Local Administration

Adv Mater. Author manuscript; available in PMC 2021 April 01.