Journal of Controlled Release 272 (2018) 159–168

Contents lists available at ScienceDirect

Journal of Controlled Release

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

Review article

Nanotherapeutics in oral and parenteral drug delivery: Key learnings and T

future outlooks as we think small
⁎
Puneet Tyagi, J. Anand Subramony
MedImmune, LLC, USA

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

Keywords: Nanotechnology ushered the field of medicine in to a new era. Miniaturization, increased surface area, and the
Nanotechnology unique physicochemical properties in the nano dimension were explored for new applications. Pharmaceutical
Immunotherapy industry picked up the technology and early success came fast for oral drug delivery through improvement in
Sustained release dissolution properties of the active molecules. Many products were launched using the nanocrystal technology
Nanorobots
on the oral side. Further development of polymeric nanoparticles led to wide spread research of nanocarriers for
EPR
parenteral delivery. While considerable efforts have gone in the last two decades for testing nanoparticles for
Immuno-oncology
Nanocrystals tumor targeting, delivery into tumors has remained challenging and suboptimal. Inadequate in vivo models that
Drug targeting didn't accurately reflect the age and vascularity of human tumors, and inability to reproducibly target ther-
API (active pharmaceutical ingredient) apeutic drugs to the tissue of interest due to intrinsic biodistribution of the particles and hence side effects,
limited the number of studies that advanced to the clinic. Our article addresses the questions commonly asked by
scientific researchers in nanomedicine: “Has nanoparticle technology yielded on its initial promise that scientists
predicted towards improving therapeutic index and avoid toxicity by delivering molecules to target tissues or
was it more of wishful thinking that had several roadblocks?” We answer this question by linking the relevance
of nanoparticles to cancer immunotherapy. The advent of immunotherapy has begun to show the potential
applicability of nanoparticles in a different light, to target the immune system. In this approach, nanoparticles
may positively influence the immune system rather than create the targeted “magic bullet”. Utilizing the in-
trinsic properties of nanoparticles for immune targeting as opposed to targeting the tumor can bring about a
positive difference due to the underlying complex cancer mechanisms that can potentially overlap with the
heterogeneous biodistribution of nanoparticles towards improving the acquired and innate immune responses. In
this review, we have followed the progress of nanotechnology in pharmaceutical applications with key insights
from oral and parenteral drug delivery, and how to modify our thinking to better utilize nanoparticles for
immuno-oncology. In contrast to conventional “local” tumor targeting by nanoparticles, we propose a new
mechanism whereby nanoparticles trigger priming of the T cells towards tumor destruction. The heterogenous
biodistribution of nanoparticles lends itself to stimulating immune cells systemically in a “global” manner and
with the right therapeutic combinations will be able to trigger tumor antigens to continually activate, retain
memory effects and destroy tumor cells.

1. Introduction
Nanotechnology offers new possibilities for creating novel materials
and for the development of unique pharmaceutical dosage forms and
drug delivery systems. Nanoparticle systems are generally classified as
nano-sized active drug particles or nano sized substrates that can en-
capsulate active pharmaceutical ingredients (API) within, complexed or
conjugated, or synthesized in the nano-dimension. According to the
U.S. Food and Drug Administration (FDA), a nanoparticle's dimension is
1–100 nm [1]. Nanotechnologies have been used to improve the
functionality of APIs and excipients. Particularly for oral therapy, na-
noparticle technologies can be used to process APIs by controlling
particle size through bottoms-up crystallization or top-down milling to
create unique nano-sized materials. The substantial improvements in
the dissolution of oral APIs by nanoparticles has led to enhanced ab-
sorption and bioavailability [2]. For parenteral dosage forms, such as
injections containing nanosuspensions, nanoparticles can increase the
volume of distribution and hence alter the pharmacokinetic (PK)
properties of active molecules [3].
Here is what we know and believe nanoparticles can do:

⁎
Corresponding author at: One MedImmune Way, Gaithersburg, MD 20878, USA.
E-mail address: subramonya@medimmune.com (J.A. Subramony).

https://doi.org/10.1016/j.jconrel.2018.01.009
Received 6 December 2017; Received in revised form 11 January 2018; Accepted 11 January 2018
Available online 19 January 2018
0168-3659/ © 2018 Elsevier B.V. All rights reserved.
P. Tyagi, J.A. Subramony
Nanoparticles offer improved dissolution capabilities for the APIs on
the oral side and enable better partition of the APIs in the blood stream
in the case of parenteral due to albumin and other binding mechanisms.
Because many small-molecule drug candidates have potential toxicity
due to high serum concentration (Cmax) upon injection and during
systemic circulation, encapsulating them in a nanoparticle delivery
system allows their release be modulated via controlled release.
Similarly, the rapid clearance of an API on injection can be overcome by
suitable encapsulation and surface decoration of the nanoparticle with
appropriate functional groups for slow release and increased circula-
tion. Nanoparticle-based therapeutics can potentially decrease systemic
toxicity and could improve efficacy by targeting a drug to a tumor site
or by offsetting the drug's release [4]. Active drug targeting using na-
noparticles, another approach for preventing systemic toxicity in APIs,
involves anchoring the nanotherapeutics using a targeting ligand that
can be an antibody fragment, peptide or a small molecule through
surface functionalization. Active targeting relies on the interaction of
targeting ligands with the overexpressed receptors (such as folates and
transferrin) on the tumor cell surface. Active targeting helps with in-
ternalization of the API and its uptake into the tumor. Several pub-
lications have described the use of active targeting and the mechanisms
of uptake [5].
However, data from the descriptive literature presents a different
picture. Even though several years of research have been devoted to the
use of nanoparticles for parenteral applications, the ability to create
reproducible tumor targeting nanoparticle has remained elusive due to
various challenges. A recent review has looked into many of these
challenges in a systematic way [6]. While looking at these difficulties,
we discuss alternate ways of utilizing nanoparticles in immunotherapy
due to their intrinsic features during biodistribution rather than where
we would like them to go. We anticipate that the next wave of na-
notherapeutics in immuno-oncology would require particles that can
tackle and participate in competing events that go on during tumor-
igenesis, such as parallel processing of mechanistic pathways through
different cell types (macrophages, dendritic cells), and heterogeneous
signal transduction occurring away from the tumor in bone marrow and
other places via killer cells for triggering positive immune responses.
In this review, we track the progress of nanotechnology in the last
few decades (Fig. 1) which can be broadly classified into 4 different
waves and describe the proven utility of nanoparticle technologies for
oral products, nanocarriers for parenteral delivery and active targeting,
and the next wave of utility in cancer immunotherapy.
2. Nanocrystals for oral delivery
Oral dosage forms such as tablets and capsules have been the choice
of drug products over the years. Biopharmaceutical Classification
Fig. 1. Evolution of nanotherapeutics. Nanocrystals (Table 1) were utilized for bioa-
vailability enhancement of oral drugs. Liposomes and other nanoparticle systems were
developed as nanocarriers to improve solubility, distribution, and reduce toxicity of drugs
(Table 2). While research continues on active targeting, current focus is more on deli-
vering macromolecules such as RNA and DNA. The future of nanotherapeutics is expected
to be immune targeting by nanoparticle robots, influenced by new learnings in immune
biology.
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Journal of Controlled Release 272 (2018) 159–168
System (BCS) [7] dictates the suitability of active pharmaceutical in-
gredients as candidates for oral dosage form development. Poor drug
solubility of APIs is a major concern that limits the pharmaceutical
development of oral dosage forms; about 40–60% of the early-stage
discovery molecules are poorly soluble [8,9]. Poor solubility can also
lead to erratic dissolution velocity and, eventually, unpredictable ab-
sorption and poor bioavailability.
Reductions in particle size to micron and submicron levels have
been adopted in the last couple of decades to enhance rate of dissolu-
tion [10]. In comparison with micronization, reduction to the nano-size
has proven to be a more powerful approach because it increases sa-
turation solubility and dissolution velocity. The relationship between
the drug's saturation solubility and the particle size is explained by the
Ostwald–Freundlich eq. [11]:
Cs 2σV
log =
Cα 2.303RTρr
where Cs is the saturation solubility, Cα is the solubility of the solid, σ is
the interfacial tension of the substance, V is the molar volume of the
particle material, R is the gas constant, T is the absolute temperature, ρ
is the density of the solid, and r is the radius.
The effect of particle size on solubility is not likely to be substantial
for larger micron-sized particles, but it is pronounced for nano-sized
materials [12,13]. Furthermore, increasing saturation solubility im-
proves the concentration gradient between the gastrointestinal tract
and blood, boosting the drug's passive absorption.
Reducing particles to nano-size also increases the drug's dissolution
velocity, as explained by the Noyes-Whitney equation [14]:
dx DA
= × (Cs − Ct )
dt hd
where dx/dt is the dissolution velocity, D is the diffusion coefficient, A
is the surface area, hd is the diffusional distance, Cs is the saturation
solubility, and Ct is the concentration around the particles.
2.1. Approaches to prepare nanocrystals for oral drug delivery
Nanocrystal preparations can be broadly categorized into those that
are top down, in which large particles are broken down by attrition or
cavitation, and those that are bottom up, in which nanocrystals are
created by precipitation. Of the two approaches, the top-down ap-
proach using milling is by far the most commonly used.
2.1.1. Top-down approach
In this approach, large drug crystals are reduced to nano-size by a
milling process or high-pressure homogenization. The classical wet-
milling method is NanoCrystal Technology, wherein a ball mill, con-
taining beads made of ceramic, stainless steel, or glass, generate shear
force and impact to reduce the particle size of the drug suspension
[3,15]. To stabilize nano-sized particles, stabilizers such as particle
surface modifiers (surfactants) or hydro-colloids (hydroxyl propyl me-
thyl cellulose) are added, which coat the surface of the nanoparticles to
prevent crystal aggregation. NanoCrystal Technology was first used to
develop sirolimus tablets, which was FDA approved in 1999 [16]. Many
other drug products, such as fenofibrate (Tricor) and aprepitant
(Emend), were subsequently developed using the nano-milling tech-
nology. Table 1 shows the list of products developed with nano-sizing
technologies.
High-pressure homogenization is a high-energy disintegration pro-
cess that involves passing the drug suspension through an orifice under
high pressure. Particle size can be reduced through particle collision
and/or cavitation [17]. Particle size reduction can be achieved by jet
stream homogenizers, such as microfluidization (Microfluidizer, Mi-
crofluidics Inc., USA), or by piston gap homogenization, where nano-
crystals are produced using pressures of up to 1500 bar. In the micro-
fluidization process, frontal collision of fluid streams under high
P. Tyagi, J.A. Subramony
Table 1
List of FDA approved oral drug products that utilize nanosizing technologies.
Drug Trade name/year of approval Indication
Sirolimus Rapamune/1999 prophylaxis of organ rejection in patients aged ≥ 13 years receiving renal
transplants
Aprepitant Emend/2003 Nausea and vomiting associated with cancer chemotherapy
Fenofibrate Tricor/2001 Hypercholesterolemia or mixed dyslipidemia
Megestrol acetate Megace ES/2005 Anorexia, cachexia or significant weight loss in AIDS
Fenofibrate Triglide/2005 Hypercholesterolemia or mixed dyslipidemia
Griseofulvin Gris-PEG/2003 Ring worm infections
Nabilone Cesamet/2006 Nausea and vomiting associated with cancer chemotherapy
pressure inside the interaction chamber reduces particle size by gen-
erating shear forces, particle collision, and cavitation forces. Nekkanti
et al. evaluated the Microfluidics M110-P Microfluidizer for a BCS class
II drug and observed that particle size reduction depends on various
process parameters, such as the number of homogenization cycles,
homogenization pressure, and stabilizer concentration [18]. At a con-
stant homogenization pressure of 30,000 psi, increasing the number of
cycles reduced particle size significantly. Furthermore, particle size
reduction was significantly greater at 30,000 psi homogenization
pressure than 10,000 psi after 60 homogenization cycles. Increasing the
surfactant concentration from 10 to 12 mg/ml also played an important
role in particle size reduction through particle stabilization.
In piston-gap homogenizers, the aqueous drug surfactant dispersion
is forced by a piston through a tiny homogenization gap (5–20 μm)
under high pressure. Gas bubbles (known as cavitation) are formed and
imploded when the suspension passes from a cylinder with a larger
diameter than the width of the resulting homogenization gap. The high
shear forces, the turbulent flow, and the powerful shockwaves gener-
ated by cavitation reduce the drug particle's size. PharmaSol's drug
nanocrystal technology is another piston gap homogenizer that is useful
for reducing the particle size of thermolabile drugs because it uses low
vapor-pressure dispersion media (such as mixtures of water and ethanol
or water and glycerol) for homogenization, which helps with processing
at low temperatures because the amount of cavitation in the homo-
genization gap is very small [19–21].
ContraSol™ uses nanotechnology to overcome the limitations of
poorly soluble drugs. ContraSol™ blends a poorly soluble drug within a
water-soluble excipient. On exposure to water, the blend generates
nanoparticles of the drug, which are stabilized by the soluble excipient
[22].
2.1.2. Bottom-up approach
In this approach, the drug is dissolved in a solvent and then pre-
cipitated by the addition of an antisolvent. Drawbacks to the bottom-up
approach include the use of solvents and lack of control of the solid
state of the crystals [23]. For example, the hydrosols technology has the
advantage of producing crystalline drug nanoparticles, but the drug
must be soluble in at least one solvent and the process requires organic
solvents that need to be removed. Auweter et al. developed the pre-
cipitation technology, Nanomorph®, in which amorphous drug nano-
particles are obtained that have higher saturation solubility and a faster
dissolution rate than crystalline nanoparticles [24]. However, conver-
sion to the crystalline state decreases bioavailability. Another category
of bottom-up techniques is based on supercritical fluid technologies, in
which gas antisolvent recrystallization and rapid expansion of super-
critical solution are employed to generate nanocrystals. In gas anti-
solvent recrystallization, drug solution in an organic solvent is satu-
rated with supercritical fluid (such as supercritical carbon dioxide),
causing the drug to precipitate [25]. The other supercritical fluid
technique, rapid expansion of supercritical solution [26], uses the su-
percritical fluid as a solvent. The drug precipitates when the pressure
decreases.
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Journal of Controlled Release 272 (2018) 159–168
Technology used
Top-down milling (NanoCrystal
Technology)
Top-down high-pressure homogenization
Bottom-up precipitation
Bottom-up precipitation
2.1.3. Combination approach
A combination of a pretreatment (bottoms-up) approach followed
by a high-energy (top-down) process is used in NanoEdge technology
[27]. In the first step, crystals are precipitated, and the resulting sus-
pension is subjected to high-pressure homogenization. SmartCrystal
technology combines several different processes based on drug prop-
erties. The advantage of this technology is that it can create particles
smaller than 100 nm. Such small nanocrystals are difficult to produce
with the top-down approach alone. SmartCrystal technology uses spray-
drying, lyophilization, precipitation, or bead milling for the pretreat-
ment step, followed by the main treatment with high-pressure homo-
genization [28].
2.2. Key learnings and future considerations from oral nanocrystal dosage
forms
Nanoparticle approach on the oral side has relied on the ability to
improve the dissolution properties of the API towards enhanced
bioabsorption. Scalability has been easier on the top down nanosizing
approach as evidenced by the number of products prepared by top-
down methods that are commercially available [23]. On the other hand,
the number of products prepared by bottom-up methods is very limited
as evidenced in. Table 1. Part of the reason for the challenges in the
bottom up approach might be due to the fact that controlled crystal-
lization of the actives in the nanodimension due to precipitation
method is very sensitive to a number of variables that are fundamental
to crystal growth.
The number of drug products using the nanocrystal technologies is
continually on the rise over the years. Nanocrystals have accounted
for > 20% of FDA submissions since their invention in the 1970s. This
steady percentage indicates that the nanocrystal platform is versatile,
easy to scale up, safe, and effective. This proportion is expected to re-
main steady because nanocrystals are a promising tool for increasing
the number of poorly soluble drugs in the drug development process In
the five year period from 2010 to 2015, 29% of nanomaterial in-
vestigational new drug applications, and abbreviated new drug appli-
cations received by the FDA were nanocrystals [29]. The only nano-
material drug products for which the FDA received more applications
were liposomes (more about this later), which accounted for 35% of
nanomaterial submissions to the FDA. In the future, development of
targeted nanocrystals with enhanced therapeutic effects and “designer”
nanocrystals that have controlled crystal morphology will be vital to the
advancement of new dosage forms [29]. We would go one step further
with the idea that designer nanocrystals may also help preserve specific
polymorphic form needed for improved bio pharmaceutics in humans.
3. Nanocarriers in parenteral drug delivery
It is important to note that several commercial products have been
developed on the oral side based on nanoparticles technologies by
improving absorption aspects of the API. For example, dissolution of
APIs is integral to enhanced absorption and pharmacokinetics and that
is the precise attribute that is being achieved using nanomilling
P. Tyagi, J.A. Subramony
Table 2
List of nanotherapeutics approved by FDA that utilize nanoparticle technologies for parenteral drug delivery. Those in clinical trials can be found in several recent references (see
references [6,39,40]).
Product name Formulation/drug encapsulated Indication
Doxil Liposomes/doxorubicin HIV-associated Kaposi sarcoma, ovarian cancer, multiple myeloma
DaunoXome Liposomes/Daunorubicin HIV-associated Kaposi sarcoma
AmBisome Liposomes/Amphotericin B Systemic fungal infection
DepoCyt Liposomes/Cytarabine Lymphomatous meningitis
Visudyne Liposomes/Verteporfin Classic subfoveal choroidal neovascularization due to age-related macular degeneration, pathologic
myopia, or presumed ocular histoplasmosis
Abraxane Albumin nanoparticles/ Breast cancer
Paclitaxel
Exparel Liposomes/bupivacaine Post-surgical anesthesia
Marqibo Liposomes/vincristine sulfate Philadelphia chromosome–negative acute lymphoblastic leukemia
Onivyde Liposomes/Irinotecan Advanced (metastatic) pancreatic cancer
technologies. The widespread interest of nanoparticle formulations on
the parenteral side came about due to the potential of improved drug
targeting to the site of tumors as well as improved solubility/partition
in the blood stream without having to use several excipients.
Nanocarrier systems have been evaluated for parenteral drug delivery
also due to the fact that they offer enhanced drug protection, controlled
release, extended circulation, compared with their free-drug counter-
parts.
Nanotechnology has been extensively tested for parenteral delivery
of several molecules (Table 2). Some of these molecules have toxicity
because of uncontrolled systemic distribution or non-target effects, or
they require localized delivery through nanoparticle approaches. For
example, doxorubicin, an effective anticancer drug, has serious side
effects, such as cardiotoxicity, and its use is therefore limited [30].
Encapsulation of doxorubicin in nanocarriers, such as liposomal and
polymer-mediated delivery systems, can potentially improve drug tar-
geting and increase serum residence time, and reduce their side effects.
FDA approved the first “nano” particle based delivery system, a 130 nm
albumin-bound paclitaxel nanoparticle, in January 2005 for the treat-
ment of breast cancer. Albumin has several unique features that makes
it particularly appropriate as a vehicle for targeted drug delivery in
oncology. It is a natural carrier of systemic hydrophobic molecules,
such as hormones, and its noncovalent binding characteristics allow it
to transport hydrophobic molecules to the cell surface [31]. Once al-
bumin is in close proximity to the cell surface, it can assist with
transcytosis of albumin-bound molecules. A preclinical study by Desai
et al. showed that endothelial cell surface binding and transcytosis of
paclitaxel were markedly higher for the albumin-bound, nanoparticle
form of paclitaxel compared with cremophor-based paclitaxel [32].
This study also demonstrated that tumor accumulation of paclitaxel
from the albumin-bound, nanoparticle form of paclitaxel was sig-
nificantly higher than with cremophor-based paclitaxel. Albumin
binding can also improve drug substance solubility. Since this study was
published, several nanotherapeutic systems have been under in-
vestigation to explore albumin binding, but none has progressed as a
drug product till now [33]. We will now look at the various nanocarrier
types in the following sections.
3.1. Types of nanocarriers
Nanocarriers have a distinct advantage for drug delivery because of
their unique composition, structure, and surface properties [34,35].
This diversity permits the carriers to be modified for different targets
and achieve different circulation. The most common designs for nano-
carrier drug delivery systems include liposomes, micelles, polymeric
nanoparticles, and dendrimers and Table 2 list examples of FDA-ap-
proved nanocarrier drug products.
Liposomes are spherical vesicles made of lipid layers that can be
unilamellar or multilamellar based on the number of lipid bilayers [36].
Liposomes are an attractive platform for drug delivery because they
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Journal of Controlled Release 272 (2018) 159–168
Year of approval
1995
1996
1997
1997
2000
2007
2011
2012
2015
have a structure and lipid composition similar to cell membranes, and
they are nontoxic and nonimmunogenic because of their natural lipid
composition [37]. Intravenously administered liposomes are cleared
rapidly from the bloodstream because of opsonization. Coating the li-
posomal surface with inert polymers, such as polyethylene glycol, slows
down clearance by allowing liposomes to remain in blood circulation
for several days. Doxil was the first FDA-approved liposomal product
(Table 2), and it features PEGylation (attaching poly ethylene glycol
chains) of liposome containing encapsulated doxorubicin [38].
Micelles are 5–100 nm colloidal particles, often consisting of am-
phiphilic surface-active agents, which have two distinct regions, a hy-
drophilic head group and a hydrophobic tail. At concentrations above
the critical micelle concentration of the surface-active agents, ag-
gregation and self-assembly occur, and micelles are formed [41]. Above
the critical micelle concentration, hydrophobic ends tend to arrange
themselves in an entropically favorable state and establish hydrogen
bond networks with the surrounding water [41]. Many micellar for-
mulations are being tested in clinical trials.
Polymeric nanocarriers consist of a spherical polymeric matrix that
encapsulates the drug. A typical example is poly (lactic acid-co-glycolic
acid) polymer based particles. However, other forms of nanoparticles
have also been considered for drug targeting [42]. such as dendrimers
are also under development [43–45].
The release of drug particles from nanocarrier upon parenteral in-
jection can be explained as follows and is depicted pictorially in Fig. 2.
In most cases, the drug is evenly distributed throughout the nanocarrier
matrix and slowly released by diffusion and/or erosion of the nano-
carrier [46]. Diffusion-controlled release through porous structure can
be explained by extending the well-known Higuchi equation
1/2
Dε
Q = ⎡ (2CT − εCS )CS t⎤
⎣ τ ⎦
Where Q is the amount of drug released at time t, D is the diffusion
coefficient of the drug in the polymer matrix, CT is the total con-
centration and CS the solubility of the drug in the polymer matrix, ε is
the porosity of the matrix and τ-the tortuosity of the system. However,
most of the current polymeric nanocarriers are degradable in nature
and have a decreasing surface area and thus some modifications to the
above mentioned Higuchi equation is required [47]. Furthermore, sol-
vation of the drug therapeutic (once on the surface or out of the na-
noparticle) has been suggested to be more important than diffusion
[48]. Single dose of a sustained release particle formulation can release
therapeutics for longer duration and hence reduce the frequency of
injections via the process of surface and bulk erosion eventually leading
to complete degradation of the particles.
3.2. Passive tumor targeting with nanocarriers
Passive targeting of nanocarriers containing drug molecules to
tumor tissues, by exploitation of abnormal tumor physiology and
P. Tyagi, J.A. Subramony
vasculature, has been explored in preclinical models wherein pre-
ferential accumulation of nanoparticles has been observed in tumors
[49]. In an early study, Matsumura and Maeda showed that proteins in
the molecular weight range of 15,000–70,000 g/mol can effectively
accumulate in a solid tumor [50]. Size is a significant factor in tumor
accumulation and preventing diffusion back into the systemic circula-
tion. In general, particles of around 400 nm are removed more slowly
from the circulation than larger ones [51]. However, along with particle
size, other characteristics of nanocarriers must be taken in to account to
achieve targeting such as surface charge/zeta potential, circulation time
etc. For example, instability of polymeric micelles in blood results in
their complete release within 15 min [52].
Preferential accumulation of nanocarriers in tumors tissues (also
known as passive targeting or enhanced permeation and retention, EPR
effect) is attributed to the leaky vasculature of the tumor tissues [53],
which in turn results from imbalance of angiogenic factors at the tumor
site [54]. Discrepancy in angiogenic factors results in porous, dis-
organized vessels that lack tight junctions. In addition to a leaky vas-
culature, tumors do not have a well-developed lymphatic system, which
leads to nanocarrier accumulation within the tumor tissue [55]. This
leaky vasculature and impaired lymphatic clearance mechanism is the
supposed primary cause of passive targeting of nanocarriers in human
tumors [54]. However, this mechanism alone cannot be used to develop
robust drug products because the vascularity index can vary, depending
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Journal of Controlled Release 272 (2018) 159–168
Fig. 2. Comparison of pharmacokinetic profiles of con-
ventional immediate release (red) and sustained release
formulation (blue). Arrows in red and blue indicate the
time of dosing for conventional and sustained release for-
mulations, respectively. The enlarged section depicts the
interfacial diffusion limitations during release of active
from nanoparticle formulations. Cs, Ci, and Cb represent the
drug concentration at surface, interface, and boundary
layer. ks and kd are solvation and diffusion constants, re-
spectively. (For interpretation of the references to colour in
this figure legend, the reader is referred to the web version
of this article.)
on the age of the tumor and variability between preclinical species
during the drug development process. Recently there was an editorial
[56] article that further questioned our understanding of “enhanced
permeability” wherein they highlight other extravasation mechanism as
a key contributor to nanoparticle accumulation in tumors. Wong et al.
[57] and Ulmschneider and Searson [53] addressed the varying vas-
culator issue by quantifying the vascularity. However, the vascularity
index can vary between patients during a trial, and, hence, it is very
difficult to develop accurate correlations between experimental in-
vestigative systems towards developing a controlled drug product/for-
mulation that can deliver therapeutic quantities to the tumor site re-
producibly to achieve a label claim for the product.
4. Actively targeted nanoparticles and gene delivery
Active targeting of nanocarriers [58] has been attempted with the
use of targeting ligands (peptides, small molecules or antibodies) that
interacts with receptors on the surfaces of tumor cells based on the idea
that targeting ligands will serve as “zip codes” to send the drugs to the
appropriate tissue site. Once the nanocarrier containing the active
molecule reaches the tumor site by systemic circulation, the receptor
and ligand interact with one another to enable uptake via endocytosis.
In oncology, particularly for the treatment of solid tumors, active tar-
geting is advantageous because it can lead to reductions in side effects
P. Tyagi, J.A. Subramony
involving nontumor cells [59]. Several studies have evaluated active
targeting in preclinical models containing overexpressed tumor cell
receptors for folate and transferrin [34]. These targeted nanocarriers
have shown significant improvements in tumor cell cytotoxicity com-
pared with nontargeted nanocarriers [60]. Beletskii et al. [42] delivered
folate-conjugated doxorubicin loaded liposomes and observed high
accumulation in murine lung carcinoma (M109R-HiFR) cell line [61].
In contrast, free doxorubicin diffused out of the cells quickly. This ac-
cumulation is especially important because many tumor cells exhibit
multidrug resistance by rapid efflux of the drugs.
However, the extent of targeting depends on various factors, in-
cluding the specificity of the targeted receptor to tumor cells versus
non-tumor cells and availability of the receptor on the tumor cells. The
general concentration distribution of the receptor on the tumor site vs
non tumor location is also important to promote active targeting.
Furthermore, cancer cells can also alter receptor expression over time,
resulting in lack of targeting [62]. Even if the nanocarriers accumulate
preferentially at the tumor site, delivering the drug might be challen-
ging because endosomes (vesicles pinched off from the cell membrane
into the cytoplasm) play a role in breaking down entrapped materials
once the tumor internalizes the nanocarrier [60]. For nanotherapeutics,
understanding the translational aspects during drug development (from
in vitro, ex vivo, to preclinical studies to studies in humans) as well as
biology of the membranes/cells such as receptor density, volume of
distribution of the particle, stability of the active molecule, are im-
portant for successful development as drug products. The configuration
of the ligand with respect to the active molecule in terms of binding and
interaction of the ligand with the receptor due to conformational ro-
tations and docking can also play a significant role in internalization.
Nanoparticles have shown great potential for delivery of genetic
material including DNA and mRNA. Nanoparticles can protect genetic
material from degradation in vivo and can also help in endocytosis and
endosomal escape of the material. Many nanoparticle based systems
have been assessed for gene delivery [63]. In 2008, CALAA-01 was the
first nanoparticle to be approved for cancer clinical trials to treat pa-
tients with solid tumors that were refractory to standard-of-care
therapies [63]. Since then different platforms including lipid nano-
carriers, polyethyleneimine nanoparticles, and cyclodextrin nano-
particles have been tested in humans to evaluate the safety of gene
delivery.
With regard to some new entrants in the nanoparticle field, various
nanostructured materials, such as carbon nanotubes [64], silica nano-
particles [65], and 3DNA nanostructures [66] of different shapes, are
currently under evaluation for drug delivery and other nanomedicinal
uses, and they can serve as future flag bearers for nanomaterials. These
promising nanostructured materials are worthy of future scientific at-
tention because of their scalability.
5. Nanomedicines for immunotherapy
The recent trends in the treatment of cancer is moving from a tra-
ditional chemotherapy towards cancer immunotherapy, whereby we
are maximizing the power of the immune system to fight cancer.
Therefore, we need to look at the nanoparticle opportunity in a dif-
ferent light for cancer immunotherapy and possibility to utilize and
leverage what we know about their intrinsic properties particularly,
their ADME. In order to improve the success rate of nanomedicine in
oncology, we must first adapt the utility of nanoparticles to the un-
derlying requirements of tumor biology and address what are those
aspects that we can positively influence matching the intrinsic physi-
cochemical aspects of nanoparticles.
For instance, in traditional chemotherapy, high concentrations of
the active is required to inhibit tumorigenesis with potential side effects
(both targeted and non-targeted). In the new immuno-oncology ap-
proach, the role of nanoparticles would be to effectively modulate the
immune system to elicit anti-tumor response while using smaller
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Journal of Controlled Release 272 (2018) 159–168
amount of materials and hence with lesser side effect potential. The
subsequent sections would highlight some of the key mechanistic dif-
ferences between the two approaches.
Immunotherapy treats diseases by inducing, enhancing, or sup-
pressing an immune response. Cancer utilizes complex regulatory net-
works to suppress immunity. Hence a multipronged approach is re-
quired to overcome immune suppression. In contrast to conventional
tumor targeted nanoparticles, which require targeting to tumors by
using receptors (such as αVβ3 integrin) overexpressed on tumors, na-
nomedicine for immuno-oncology will be used to effectively target the
immune cascade [67] in bringing about antitumor response either by
amplifying immunoresponse or by improving costimulatory signals.
Nanoparticles have the potential to improve immune response
[68,69] and modulate receptor clustering [55]. In recent years, im-
munotherapy has become great interest due to its promise to treat
various forms of cancer [70]. Results from anti CTLA-4 and anti-PD-1/
PDL-1 antibody therapies have brought forward the power of im-
munotherapy through immune targeting and T Cell activation [71]. Key
differences between immune targeting and tumor targeting is that in
the former immune systems is primed through a number of mechanistic
pathways. Mechanisms by which one can prime and fine tune activation
of T cells and antigen presenting cells (APC) are therefore very im-
portant. Approaches to augment immune response will involve ampli-
fying costimulatory signals, block immune suppression as well as op-
timize output from primed T cells. Furthermore, sustained activation
from T cells can also be obtained by using co-stimulatory targets that
can prolong the activation. In the literature, researchers have used
OX40 [72] and GITR [73] agonists for costimulatory effects.
5.1. Methodology for immune targeting by nanotherapeutics
While creating nanomedicines for immuno-oncology, the nano-
particles containing immune activation agents will influence and
trigger various components of the immune cascade to bring about
collective antitumor activity from various locations. This mechanism of
priming immune cells that are far from the disease site/tumor tissue to
bring about antitumor response is a new approach that can fit well with
the increased volume of distribution of nanoparticles and their het-
erogenous bio-distribution upon injection which is an intrinsic property
of nanoparticulate systems. Choosing the right immuno- oncology agent
is key to bring positive aspects of this approach. In the immune cascade
routine, nanoparticles would trigger various cell types such as T-cells,
memory cells or dendritic cells to provide collective antitumor re-
sponse. Nanoparticles are also known to have a propensity to be taken
up by macrophages, get deposited in bone marrow and spleen to elicit
immune production and antitumor immunity. This phenomenon of
bringing specific immune-activation by nanotherapeutics to bring about
cancer immunity is akin to “cooperative action from different locations”
or nanoparticles working as catalysts from various organs/locations to
trigger immune response. Nanoparticles have the potential to bring
about these effects when used with the right molecules and hence ef-
fectively address the complex cancer regulatory network. Both innate
and acquired immune responses can be improved in this mechanism.
The tunability aspects of drug release from nanoparticles, ability to load
different type of agonists/antagonists, and co-stimulatory molecules
makes nanoparticles as ideal tools for immune targeting.
Another aspect of the nanoparticle approach for immuno-oncology
is that specific combinations and “fine tuning” of the composition can
enable a rapid high throughput screening using appropriate ex vivo cell
lines to measure and quantify various immune cascade signals (im-
munomonitoring). Since the focus of such studies would be to better
understand cellular uptake and understanding immune stimulatory
signals, the methodology is very different than the previous studies
required for passive targeting and tumor pharmacokinetics that relied
on in vivo models most of them without a common baseline and hence
leading to clinical translational challenges. Thus, immuno-
P. Tyagi, J.A. Subramony
nanomedicines can be designed to target components of immune
system and help the immune system to effectively destroy cancer cells.
Also, once activated, a continuous production of T cells can be achieved
by memory of the immune system [74]. The approach bears a different
outlook to treating cancer and can be advantageous in the future as our
understanding of immune system and its responses to antitumor treat-
ments are progressing rapidly.
Many strategies to target cancers via immune system are under in-
vestigation currently. Li et al. [75] have developed a vaccine that
combines Toll-like receptor (TLR) agonists and antigen-carrying lipid
nanocapsules. The combination vaccine elicited a long-lived memory T
cell response in the lung mediated by the APC. Cho et al. [76] de-
monstrated that dendritic cells can take up superparamagnetic iron
oxide nanoparticles covered with ZnO shell and elicit an anti-tumor
immunity. Li et al.’s [77] work with alpha-alumina (α-Al2O3) nano-
particles indicated that α-Al2O3 based cancer therapeutic vaccine can be
used for the delivery of antigens to the APCs. Their findings also sug-
gested that α-Al2O3 can boost the antitumour efficacy of tumor-derived
autophagosomes to deliver antigens to T cells. Min et al. delivered
tumor-specific proteins to APCs and demonstrated a significant im-
provement in the cytotoxic T cell response [78]. The utility of nano-
particles for immune targeting has come from the ability of nano-
particles for signal transduction which is described in the next section.
5.2. Nanoparticles for signal transduction activation
Nanotechnology is being explored for its ability to activate signal
transduction in many receptors. Receptor clustering is required for the
activation of many receptors that play important roles in the develop-
mental process by binding to proteins such as cytokines and growth
factors [79]. Such receptors can be activated by applying mechanical
forces to physically promote clustering and lead to signal transduction
[80].
Using nanotechnology to manipulate individual receptors, Mannix
et al. [80] demonstrated that nanoparticles can activate signal trans-
duction in cells. Mannix et al. triggered the receptor clustering of FcεRI
– IgE receptor-antibody complexes present on mast cells to induce
calcium secretion by using superparamagnetic nanoparticles (30 nm)
covalently conjugated to dinitrophenyl-lysine ligands. Activation of
such nanoparticle mediated receptor clustering can open up new ave-
nues to control cellular responses and control cell metabolism [80].
Development of such nano-technologies also have potential to be
combined with other segments such as tumor diagnosis [81]. Seo et al.
[82] used magnetoplasmonic nanoparticles to activate Notch receptor
Fig. 3. Schematic showing possible mechanism by which nanorobots trigger priming of T cell towards tumor destruction. Upon systemic circulation, nanoparticles can interact with
macrophages and dendritic cells to activate T cells and also can unleash natural killer cells based on their biodistribution in the bone marrow. Some of nanoparticles previously localized
in tumors during biodistribution may pick up tumor antigens and can further accumulate in lymph nodes to continually improve immune response via dendritic cell uptake. Nanorobots
can also trigger immune response by identifying tumor antigens and stimulating immune cells systemically.
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Journal of Controlled Release 272 (2018) 159–168
signaling pathway. Magnetoplasmonic nanoparticles combine the use
of magnetism to deliver a small amount of force and plasmonic gold
shell that can be used for imaging. Seo et al. have also shown that by
force loading, Notch receptors can be unfolded, resulting in the ex-
posure of the buried proteolytic site. Unfolding of the Notch receptor
and exposure of the proteolytic site is an important step in activation of
Notch. These studies depict the potential of using nanoscale force to
alter and evaluate cell signaling pathways. Furthermore, studies such as
by Seo et al. [82] point to multiple innovative applications of magnetic
nanoparticles. Although these technologies are in their infancy, they
hold immense potential for the future of nanotechnology in cancer
immunotherapy.
5.3. Nanorobots and molecular machines
Advances in microprocessing to submicron scale have enabled re-
searchers to consider designing molecular machines at a nanoscale.
Although molecular machines are conceptual at this stage, they have
potential medical applications in early detection and treatment of dis-
eases via imaging, sensing, and drug delivery. These molecular ma-
chines in the nano dimensions have the potential to bring innovative
solutions to healthcare because of their intrinsic biodistribution, disease
recognition and signal transduction properties.
Douglas et al. have developed a nanorobot using DNA strands that
rearrange their conformation and release the drug load when they reach
the target cells [83]. Freitas et al. use a synthetic material to prepare
nanorobots (which they call “pharmacytes”) that can target selected
cells and travel through cell membranes [84]. Wu et al. developed a
tubular nanorocket made of chitosan and sodium alginate polymers
with highly efficient driving force [85]. The motion and direction of the
nanorocket can be adjusted with an external magnetic field. Ma et al.
have developed a Janus nanomotor using mesoporous silica nano-
particles smaller than 100 nm [86]. The nanoparticles' high porosity is
used for drug loading.
Nanorobots can help trigger the immune system to recognize cancer
cells and destroy them. Immune targeting nanocarriers can be more
effective because the intrinsic immune cell cytotoxicity tend to be tar-
geted to signals from the tumor specific antigens. In this way nanor-
obots could both sense and eliminate cancer cells. Immune targeting
based therapy is not expected to be repetitive as traditional che-
motherapy since the immune system has memory effects to sustain the
effect via continued generation of T cells that can prevent tumor re-
occurrence [74]. The toxicity to healthy cells from chemotherapy can
be overcome by development of a nanorobot that can distinguish cancer
P. Tyagi, J.A. Subramony
Fig. 4. Schematic showing flexibility for developing immunotherapy combinations based
on patient parameters such as tumor phenotype and tumor biomarker information.
cells from healthy cells or trigger improved immune response in col-
lective ways as described in the next paragraph.
Immune targeted nanotherapeutics (nanorobts) (Fig. 3) would
comprise nanoparticles targeting the antigen presenting cells (such as
dendritic cells), which are predominantly distributed in the body in
unleashing attack against cancer cells through antigen presentations.
Nanorobots containing antibodies intended for tumor cell receptors can
be targeted through dendritic cells. In a parallel event, sensing moieties
on nanorobots can identify tumor specific antigens and trigger the na-
norobot to interact immune cells to trigger a response. In this way
nanorobot containing sensors can help to act as GPS (global positioning
system) enabled particle for optimum immune modulation and also act
in a closed loop fashion with sensing and delivering a therapeutic (see
graphical abstract).
Natural killer (NK) cells originate in the bone marrow and they are
body's defense mechanisms against cancer cells. Immune targeting na-
noparticles may also end up in bone marrow which can then trigger the
release of NK cells for destroying the cancer cells in a collective me-
chanism. Jang et al. [87] reported the use of iron oxide (Fe3O4) mag-
netic nanoparticles to trigger natural killer cells to target tumor sites in
an early demonstration of a nanorobot.
6. Future outlook
The key to designing future nano delivery systems from a precision
medicine perspective involves understanding specific individual tumor
pathophysiology and how nanoparticle based drug delivery systems
would interact with the biology and create immune stimulatory re-
sponses. Using a disease driven evaluation approach helps to better
bring about the clinical translational needs of the delivery system.
Understanding the tumor phenotype and variability is critical for better
translational results. Biomarker development and use of companion
diagnostics are important for patient stratification and to assess the
right outcome parameters. Circulating tumor DNA [88] and tumor cells
[89] are two such tools that can help with patient stratification. Digi-
tally enhanced immuno monitoring of data at the RNA, protein, and
cellular level together with data collection and mining will help re-
searches to pick and choose which combinations to adopt, test, and
develop predicitive algorithm to support future clinical and transla-
tional studies [90].
Due to the heterogeneity in the mechanisms of cancer, future
treatments in oncology would heavily involve combination therapies.
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Journal of Controlled Release 272 (2018) 159–168
Similar to fixed dose combinations in small molecule oral therapy,
particularly for cardiovascular and metabolic disorders, cancer im-
munotherapy in the near future would include mixture of various
combinations including antibodies, small molecules, and cancer vac-
cines that would bring about synergistic effects and improved immune
response towards tumor eradication. Nanorobots with unique surface
functionalization capabilities will have the ability to support multiple
drug combinations. The use of nanoparticles in obtaining antigen pre-
sentation as well as priming of a T-cell towards producing adaptive
immune response will be the key. Ability to mix and match various
receptor targeted or antitumor response inducing nanoparticles will
further provide flexibility to the utility of nanomedicine for immuno-
oncology (Fig. 4). Genetic test including biomarker and tumor imaging
will be used towards carrying out designing of the right proportion of
nanoparticles for optimum response. Precision medicine and digital
data analytic tools will help in designing the right combination and
dose for patients. Nanorobots, as tiny devices, can couple important
aspects of sensing/diagnosis to trigger the release of the right immune
function entity and bring about the best mechanism needed for anti-
tumor response. Versatile nanoparticles systems with these features that
are scalable will certainly have an advantage towards commercializa-
tion.
Developability and scale up of the nanotherapeutic system while
paying attention to critical quality attributes, content uniformity in
loading/attaching of the therapeutics in the nanosystem, and stability
of the nanotherapeutics are other important aspects to consider in
bringing nanomedicines to patient from a commercial perspective.
7. Conclusions
With the advent of nanotechnology in the last few decades, the
projected future of new applications towards healthcare is expected to
offer many improvements for human life and drug delivery in parti-
cular. However, if nanomedicines are to realize their full potential, a
few impediments must be addressed right now. For drug delivery, for
example, a deeper understanding of the biology and development of
appropriate disease models and the interaction of cells with nano-
carriers is needed, PK characteristics and the fate of nanocarrier in vivo
is also critical to develop PK/PD correlations for immunotherapy while
using nanoparticles.
The winner of the 2016 Nobel Prize in chemistry, Dr. Bernard L.
Feringa, envisioned many possible biomedical applications for mole-
cular machines, saying “It's a bit early days, of course, but once you are
able to control movement, all sort of things are possible. Think about
tiny little robots that the doctor would inject into your bloodstream,
and that would go search for a cancer cell or deliver a drug” [91]. We
hope that nanotechnology will disrupt cancer immunotherapy with new
treatment possibilities very soon in the near future….
Author contribution
Anand Subramony conceived the manuscript idea. Puneet Tyagi and
Anand Subramony wrote the manuscript.
Competing financial interest statement
The authors declare no competing financial interests.
Acknowledgment
Editorial support was provided by Deborah Berlyne.
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