nano receptors

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Nanoparticle targeting of anti-cancer drugs that alter intracellular

signaling or inuence the tumor microenvironment☆

Mathumai Kanapathipillai a, Amy Brock b, Donald E. Ingber a,c,d,⁎a Wyss Institute for Biologically Inspired Engineering at Harvard University, Boston, MA, USAb Department of Biomedical Engineering, Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, TX USAc Vascular Biology Program, Boston Children's Hospital and Harvard Medical School, Boston, MA, USAd School of Engineering and Applied Sciences, Harvard University, Cambridge, MA, USA

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

Available online 9 May 2014

Keywords:

Nanoparticle

Tumor

Nanotherapeutics

Delivery

Microenvironment

Drug

Nanoparticle-based therapeutics are poised to become a leading delivery strategy for cancer treatment because

they potentially offerhigher selectivity,reduced toxicity, longerclearancetimes, and increased ef cacy compared

to conventional systemic therapeutic approaches. This article reviews existing nanoparticle technologies and

methods that areused to targetdrugsto treat cancerby altering signaltransductionor modulating thetumor mi-

croenvironment. We also consider the implications of recent advances in the nanotherapeuticseldfor thefuture

of cancer therapy.

© 2014 Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107

2. Targeting signaling networks in cancer cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1092.1. Surface-receptor mediated pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109

2.1.1. Transmembrane delivery vehicles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110

2.2. Targeting intracellular signaling molecules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110

2.2.1. Focused delivery of siRNAs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111

3. Targeting the tumor microenvironment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111

3.1. Vasculature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111

3.1.1. Angiogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111

3.1.2. Hypoxia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112

3.2. Immune response . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112

3.3. Extracellular matrix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112

3.4. Multi-drug resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114

4. Challenges for the future . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114

Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116

1. Introduction

Current cancer chemotherapies often failto improve patient mor-

tality and morbidity due to severe adverse effects on normal tissues.

Targeted nanocarrier systems potentially offer a solution to this

problem becausethey can be designed to deliver cargo preferentially

to the tumor cells, sparing healthy tissue from dose-limiting side

effects. Because their size and shape can be tailored as needed,

nanotherapeutics also enhance bioavailability and plasma solubility,

Advanced Drug Delivery Reviews 79–80 (2014) 107–118

☆ Thisreviewis partof the Advanced Drug Delivery Reviews themeissue on "Engineering

of Tumor Microenvironments".

⁎ Corresponding author at: Wyss Institute for Biologically Inspired Engineering at

Harvard University, Boston, MA, USA.

E-mail address: [email protected] (D.E. Ingber).

http://dx.doi.org/10.1016/j.addr.2014.05.005

0169-409X/© 2014 Elsevier B.V. All rights reserved.

Contents lists available at ScienceDirect

Advanced Drug Delivery Reviews

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decrease systemic toxicity, increase patient compliance and improve

pharmacokinetics of biologics and small molecules that otherwise

have short half-lives in vivo.

The eld of nanotherapeutics has been made possible by the

development of manufacturing methods that enable synthesis, self-

assembly or fabrication of objects with dened size, shape and chemis-

try on the nanometer scale and that are also biocompatible. These are

either composed of multiple molecules or single molecules that are

engineered to contain multiple functional cassettes. Examples of biocompatible nanocarriers include those composed of liposomes,

polymers, micelles, engineered multi-functional antibodies, and nano-

particles that can be composed of metals (e.g., gold, silver), polymer,

quantum dots, dendrimers, fullerenes, ferritin, proteins, DNA, other bio-

logical molecules or combinations of these materials (Fig. 1) [1–4].

Nanomaterials also can be precisely designed to achieve desired

physical and chemical properties necessary to target delivery of drugs

to the dynamic tumor microenvironment with higher therapeutic

ef cacy and lower toxicity. For example, surface to volume ratio, size,

geometry, surface charge, shape and stimuli-responsive properties are

examples of design features that can be controlled when formulating

nanotherapeutics. Once the nanoparticles are created, they also can be

functionalized with additional chemical moieties, such as antibodies

or ligands for cell surface receptors, to target to selective sites, prevent

clearance from blood, and provide other desired functional properties.

To achieve effective targeting to cancerous tissues, systemically de-

livered nanoparticles must rst extravasate from the bloodstream,

pass through the tumor extracellular matrix (ECM), bind to cells, and

then cross the surface membrane to enter the target cells and reach ap-

propriate intracellular sites (Fig. 2) [5–7]. Two targeting strategies, one

active and the other passive, have been utilized to enhance nanoparticle

delivery to tumors. In passive targeting, one capitalizes on the leaky na-

ture of tumor microvessels, which results in enhanced permeability and

retention (EPR) responses that facilitate greater accumulation of nano-

particles compared to individual soluble molecules or drugs in the

perivascular tumor microenvironment. The EPR effect varies depending

on the size and surface properties of the nanocarriers, and hence, nano-

particles must be designed to achieve maximum targeting and thera-

peutic ef cacy. Nanocarriers in the size range of 20–200 nm can easily

extravasate through the walls of poorly formed microvessels in the an-giogenic tumor niche, and poor lymphatic drainage further enhances

their accumulation. Several nanoparticles that utilize this EPR-based

passive targeting strategy are currently in human clinical trials [8,9].

Examples include the doxorubicin-liposome carriers Myocet and Doxil

[10,11], which have increased half-lives in the blood due to the size of

the nanocarrier and in the case of Doxil, chemical coating of the lipo-

some with poly-ethyleneglycol (PEG). Passive targeting polymeric

micelles, nanoparticles, and polymer-drug conjugates are also in clinical

trials for breast, lung, pancreatic and ovarian cancers [9,11].

The creation of targeting antibodies in late1970s led to the develop-

ment of the rst active or targeted nanoparticle delivery strategies [12,

13]. The concept was simple: by coating nanoparticles with antibodies,

they could be selectively directed to sites expressing the target antigen.

This active targeted delivery approach was later extended by coating

nanoparticles with targeting ligands, lectin-carbohydrates, or antigen

molecules that recognize specic sites or endogenous antibodies to fa-

cilitate intracellular drug delivery [5,14]; these targeting ligandsinclude

peptides, antibodies, aptamers, small molecules and proteins among

others [5,14,15]. The density of these ligands on the surface of thenano-

particles also can be ne-tuned to optimize binding and targeting using

several physical and chemical immobilization techniques. Examples

Fig. 1. Multi-valent, multi-functional and stimuli-responsive nanocarrier deliverysystems for tumortargeting. Examples of nanocarrierdelivery vehicles include (left to right) liposomes,

polymers, micelles, engineered antibodies and nanoparticles decorated with stimuli-responsive cleavable stealth coatings (blue rectangles), targeting moieties (arrows), and anti-cancer

drugs or imaging agents (stars and suns).

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include technologies that enable precise control of size, shape, composi-

tion and surface modication of nanoparticles [16,17] and a lipid inser-

tion method that is used for targeted functionalization of erythrocyte-cloaked nanoparticles [18]. There are only a few active targeted cancer

nanotherapeutics in clinical trials. The two that are farthest along are

doxorubicin-containing immunoliposomes that are targeted to cancers

using a human monoclonal antibody, and transferrin-targeted oxaliplatin

liposomes; however, mostof the published resultson these nanotechnol-

ogies refer to preclinical studies [11,19,20].

The most common anti-cancer nanotherapeutics in development

target receptors on the surface membranes of tumor cells to enhance

local drug concentrations, facilitate drug entry into the cell, and selec-

tively perturb specic receptor-mediated signaling pathways [5]. How-

ever, as our understanding of cancer biology has advanced, there has

been increased recognition of the importance of intracellular signaling

molecules and particular genes for cancer development, as well as

new appreciation for the key role that the tumor microenvironment(e.g., capillary blood vessels, stroma, ECM composition and mechanics,

immune cells, etc.) contributes to cancer control. Thus, nanocarriers

are also being employed to selectively concentrate drugs at tumor

sites based on the tumor microenvironment, as well as to target specic

intracellular signaling molecules, and genes. Nanoparticles with multi-

valency, multi-functionality, and triggered or stimuli-responsive release

properties (Fig. 1) are all currently being explored for tumor targeting

[6,21–23]. Clearly, there are still many challenges that need to be over-

come before we can design fully effective, targeted, nanoscale, drug

delivery systems that will overcome the complex interactions between

the tumor cell's intracellular information processing network and the

ever-changing tumor microenvironment that govern cancer formation

and expansion. Thus, in this review, we focus on various nanoparticle-

based drug delivery approaches that are being developed to either tar-get intracellular signaling pathways or the tumor microenvironment,

as shown in the schematic in Fig. 3. We also explore the implications

of recent developments in this eld for the future of targeted cancer

therapy.

2. Targeting signaling networks in cancer cells

Cancers are heterogeneous in nature, and their growth and progres-

sion depend on reciprocal interactions between tumor cells and their

surrounding microenvironment [24–26]. Information processing net-

works that govern cancer behavior include both chemical and genetic

signaling pathways inside individual tumor cells, as well as inter-

cellular signaling networks that involve soluble cues transferred back

and forth between tumor cells and surrounding vascular endothelial

cells, cancer-associated broblasts, and cells of the immune system

[24,27,28]. In this section, we examine nanoparticle-based delivery

strategies that target drugs to these diverse information handling net-works that process soluble cues in the tumor.

2.1. Surface-receptor mediated pathways

Detailed molecular characterization of tumor cell signaling networks

has identied changes in growth signaling receptors that characterize

specic tumor types. Active targeting of nanoparticles to these signaling

components couldpotentially increase therapeutic effectiveness and re-

duce collateral damage caused by chemotherapeutic drugs on normal

healthy tissues. Molecular recognition of tumor cells may exploit li-

gand–receptor interactions or antigen–antibody binding to ef ciently

target the nanoparticle drug delivery vehicle to the tumor. Humanized

antibodies are attractive for targeting cell surface receptors due to

their high specicity and low immune reactivity [29]. Active targetingvia antibody interactions can distinguish cancerous cells from normal

tissue and also can target specic subpopulations of cancer cells [30].

Folate is one of the most extensively utilized ligands for targeted

cancer nanotherapeutics due to its high af nity bindingto the folate re-

ceptor (K d ~10−9) and because it is frequently overexpressed in diverse

tumor types including uterine sarcomas, ovarian carcinomas, osteosar-

comas, and non-Hodgkin's lymphomas [30]. Folate recently was used

to target delivery of tamoxifen with albumin and alginate-based nano-

particles [31] and enhanced in vivo targeting of camptothecin to pancre-

atic xenografts was accomplished through the use of folate-coated

mesoporous silica nanoparticles loaded with this drug [32]. Signicant

reduction in tumor volume and enhanced renal clearance were ob-

served compared with uncoated particles in the camptothecin nanopar-

ticle studies.Transferrin is an iron-binding plasma glycoprotein that facilitates

iron uptake by cells through binding to its membrane receptor, TfR

[33], and this receptor is overexpressed by as much as 10-fold in

many tumor cell types [34]. Ligand binding of TfR initiates endocytosis,

which has been exploited for targeted delivery of transferrin-coated

nanoparticles. In vivo examples of this approach include the targeting

of transferrin-conjugated paclitaxel-loaded nanoparticles to prostate

[34], and delivery of transferrin-coated PLGA-nanoparticles to gliomas

[35] in animal studies.

Epidermal growth factor receptor (EGFR) is over-expressed in many

cancers and can serve as a target for nanotherapeutics through binding

of one of its two natural ligands: epidermal growth factor (EGF) or

transforming growth factor-alpha (TGF-α). EGF-conjugation of stearoyl

gemcitabinenanoparticles (GemC18-NPs) has been shown to result in a

Fig. 2. A schematic depicting the mechanism by which nanoparticles are transported to the tumor and its surrounding microenvironment. Nanoparticles that escape reticuloendothelial

system (RES)during circulation,reach tumorblood vessels and subsequentlyextravasate into the tumorinterstitium. After overcoming transport barriers and diffusion limitations, nano-

particles reach the targeted tumor cells, where they deliver their cargo or imaging agents.

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2-fold improvement in its targeting to tumor cells that over-express

EGFR [36]. Human epidermal growth factor receptor 2 (HER2) is over-

expressed in breast, ovarian, gastricand uterine cancers and it is the tar-

get of the monoclonal antibody trastuzumab (marketed as Herceptin).

Human serum albumin nanoparticles that were surface-coated with

trastuzumab and loaded with the cytostatic drug doxorubicin displayed

enhanced cell uptake and biological activity [37]. Such targetedapproaches may be key to reducing clinical issues of cardiotoxicity

that can be a major source of morbidity in patients receiving doxorubi-

cin. The challenge of doxorubicin resistance also may be overcome, or

at least reduced, using nanoparticles that are not recognized by

P-glycoprotein, which is one of the main protein pumps that mediates

the multidrug resistance response.

2.1.1. Transmembrane delivery vehicles

Several transmembrane nanoparticle-based delivery strategies have

been reported to permit the delivery of small molecules, proteins, small

interfering ribonucleic acids (siRNAs) and imaging agents to intracellu-

lar compartments of tumor cells. Smallmolecule-mediated nanoparticle

delivery of small molecules is commonly accomplished by utilizing

mechanisms of endosomal escape and cytosolic release of nanoparticles.Delivery of doxorubicin by mesoporous silica nanoparticles coated with

polyethylenimine (PEI) [38] and gold nanoparticles coated with doxo-

rubicin conjugated to an acid cleavable PEG linker [39] have both been

shown to effectivelydeliver small molecules into cytosol. More recently,

it was shown to be possible to target chemotherapeutic drugs to mito-

chondria using poly(lactic-co glycolic acid) (PLGA)–PEG nanoparticles

blended with triphenylphosphonium, a lipophilic cation known to

cross mitochondrial membranes [40,41].

For intracellular protein delivery, silica nanoparticles that target

phospho-Akt (protein kinase B) to inhibit its kinase activity have been

developed [42]. The nanoparticles enter cells through the endosomal

pathway and subsequent escape into the cytosol, causing protein deliv-

ery and eventually, cell death. Intracellular protein delivery also has

been accomplished by targeting the ribosome inhibiting proteins (RIP)

that suppress translation [43]. Several “cell penetrating peptides,” such

as the TAT (transactivated-transcription) sequence, have been utilized

to mediate transfer of peptides and proteins across cell membranes and

directly intothe cytosol [44]. These havebeen used to create nanocarriers

that can enter the cytoplasm as well. Examples include TAT-conjugated

PLGA nanoparticles to deliver K237 peptides that target the kinase insert

domain receptor for brain drug delivery [45], and intracellular delivery of quantum dot-mediated protein delivery and imaging [46].

The physical properties of nanoparticles also have been leveraged to

enhance cellular internalization.For example, the specicity of targeting

ligand-coated nanoparticles can be augmented by engineering their

shape to exhibit distinct 3D geometries. PEG-hydrogel rods with a

high aspect ratio of 150 × 450 nm exhibit higher cellular uptake com-

pared with cylindrical particles with 200 × 200 nm dimensions [47].

Uptake and internalization of trastuzumab-coated nanoparticles by

breast cancer cells also were found to be higherfor rod-shapedparticles,

followed by disks, and then spheres [48]. Interestingly, rod-shaped par-

ticles were also found to provide improved specicity of endothelial

targeting in lung and brain compared with other geometries [49].

Thus, the abilityto shape nanocarriersprovides an alternative way to af-

fect drug delivery, independently of the drug being delivered or theirsurface coating.

2.2. Targeting intracellular signaling molecules

Intracellular signaling molecules control cell proliferation, differenti-

ation, andsurvival, as well as myriadmetabolic functionsin cells.During

cancer progression, the signaling networks undergo severe alteration

and deregulation. Therapeutic targeting of specic molecules within

these complex tumor signaling networks remains a challenge due to

poor drug penetration into intracellular compartments. Nanocarriers

offer a new way to overcome this challenge because they can be tuned

to deliver the cargo by receptor-mediated internalization as well as

organelle- or intracellular molecule-specic targeting. Below, we out-

line some recent developments in this area.

Fig. 3. Schematic of potential strategies forselectively targeting nanoparticles to various key structures and features of the tumormicroenvironment, including the vasculature (1), hypoxic

regions (2), and intracellular signaling networks (3, a) and surface receptors (3, b) on tumor cells, extracellular matrix (4) and the immune response (5). Insert at left shows a multi-

functional nanoparticle decorated with targeting moieties and loaded with drugs.


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2.2.1. Focused delivery of siRNAs

The recent development of siRNAs, which direct sequence-specic

degradation of target mRNA transcripts, has led to the development of

an entirely new class of therapeutics for cancer. As the siRNA molecule

itself is strongly negatively charged, delivery to cells is typically accom-

plished by complexing it with a cationic carrier or lipid-based nanopar-

ticle. In the rst-in-human trials studies, siRNA was targeted to the M2

subunit of ribonucleotide reductase via cyclodextrin–PEG nanoparticles

coated with human transferrin [50]. TfR receptor is expressed at highlevels on malignant cells as described above, and thus this enhanced

accumulation of the nanoparticles at tumor sites in patients with refrac-

tory melanoma.

A new class of nanoparticle materials, termed “lipidoids,” has been

developed to facilitate siRNA delivery. Lipidoids are derived from an

amine-containing backbone from which aliphatic chains extend; the

amines appear to interact electrostatically with the nucleic acid phos-

phate backbone and facilitate endosomal escape as the compartment

becomes acidied. The ef cacy of lipidoid nanoparticle-siRNA formula-

tions has been demonstrated in the silencing of tight junction protein

claudin-3 in animals models of ovarian cancer [51] as well as Parp1 in

Brca1-decient ovarian tumors [52] and glycolytic enzyme pyruvate ki-

nase in several xenograft models [53]. Injection of lipidoid nanoparticles

carrying siHoxA1 through the nipple and directly into the mammary

duct prevented the progression of mammary tumors in the C3(1)

SV40TAg transgenic mice [54].

Although siRNA delivery by nanoparticles is able to delivery micro-

grams of drug per kilogram of animal body weight, there remains

room for additional improvement. For example, better understanding

of the endocytotic pathways of particular siRNA–nanoparticle formula-

tions may improve their ef ciency of delivery. After lipid nanoparticles

enter the cell, a signicant amount of siRNA remains trapped in endocy-

totic vesicles and does not reach its intracellular target. In parallel, vesicle

recycling continues to rapidly clear the siRNA–nanoparticles from the cell

before the payload is delivered. One recent study identied Niemann–

Pick type C1 (NPC1) as a critical regulator of the major recycling path-

ways of lipid nanoparticle-formulated siRNAs. In cells lacking NPC1,

there was enhanced retention of nanoparticles inside the endosome

and lysosomes of the recycling pathway, and the level of gene silencingachieved with RNAinterference was 10 to 15 times greaterthan that pro-

duced in normal cells [55]. Strategies to target specic molecular compo-

nents of endosome production, traf cking and recycling may be

incorporated into the design of the next generation of nanocarriers for

siRNA delivery. Another challenge is that immune stimulation through

α / β interferons, RNA-dependent kinase effects, and Toll-like immunity

couldlimit the safety proles of siRNAs. However, if this immune activat-

ing activity of the siRNAs can be controlled or even exploited for use

against the tumor, this sideeffectcould potentially improve treatment ef-

cacy. The current status of nanoparticle-based RNAi therapies in clinical

trials for oncology applications is shown in Table 1.

3. Targeting the tumor microenvironment

Over the past few decades, it has become recognized that tumor

growth and progression are not solely the result of cumulative gene

mutations, but are also signicantly inuenced by the surrounding

tumor microenvironment. In addition to cancer cells, the dynamic

tumor microenvironment consists of ECM, stromal cells, endothelial

cells, and immune cells that are recruited from surrounding tissues as

well as from the bone marrow [25,56]. Interestingly, the chemistry,

structure and mechanical properties of the tumor microenvironment

all inuence the cellular information processing networks that drive

phenotypic changes leading to tumor malignancy [24,56–59]. Hence, it

is important to consider both cellular and non-cellular components of tumor microenvironment for cancer therapy as their complex interac-

tions play a critical role in cancer progression. In this section we discuss

nanoparticle-based delivery systems that have been reported to target

various components of the tumor microenvironment.

3.1. Vasculature

In contrast to normal tissues, the vasculature of tumors is marked by

a heterogeneous distribution of vessel sizes and shapes, generally larger

vessel diameters, higher vascular density, and enhanced permeability.

This inherently leaky vasculature promotes preferential accumulation

of nanoparticles in the tumor interstitial space, and when coupled

with impaired lymphatic drainage of macromolecules, this results in

the EPR effect characterized by increased accumulation of nanoparticles

that is observed in many solid tumors [60], as described above. The EPR

effect has been successfully exploited for passive targeting of drugs to

solid tumors in animals that areencapsulatedin a variety of high molec-

ular weight carrier systems, including polymeric drug conjugates, lipo-

somes, polymeric NPs and micelles [61–64]. However, below we focus

on strategies for active targeting of nanotherapeutics to the tumor

vasculature.

3.1.1. Angiogenesis

One of the major angiogenic factors elaborated by tumors– vascular

endothelial growth factor (VEGF) – both stimulates new capillary in-

growth and increases the permeability of blood vessels [65]. Targeting

the VEGF receptor on endothelial cells is therefore an effective method

to prevent cancer growth by blocking tumor angiogenesis. Targeting

the VEGF receptor-2 (VEGFR-2) has been investigated as a way to en-hance delivery of variousnanoparticle systems to tumor vessels, includ-

ing boronated dendrimers and radioisotype-labeled polymerized

liposomes [66]. VEGF targeting also was accomplished in an animal

model of hepatic carcinoma using dextran magnetic nanoparticles

(DMNs) conjugated to radiolabeled 131I-anti-VEGF monoclonal anti-

bodies [67].

One of the rst angiogenic inhibitors TNP-470 demonstrated broad

spectrum activity in early trials, including ef cacy in metastatic disease

in human patients, but then stalled in part due to neurotoxicities. A

reformulation of this angiogenesis inhibitor composed of polymeric

PEG–PLGA nanoparticles surrounding a TNP-470 core, called Lodamin,

was shown to prevent the compound from crossing the blood–brain

barrier, eliminating its neurotoxicity, while retaining its potency and

broad-spectrum activity [68–70]. The new Lodamin formulation alsogreatly improved oral availability as the polymeric PEG and PLA protect

the inner active compound from degradation by the acidic stomach

Table 1

Nanocarrier-mediated siRNA delivery systems in clinical trials.

Drug Disease Target Nanoparticle Company Stage Status

FANG Ovarian tumors FURIN Liposome Gradalis, Inc. Phase II Active

CALAA-01 Solid tumors RRM Cyclodextrin/PEG, transferrin Calando Pharm Phase I Ongoing

ALN-VSP02 Solid tumors with liver involvement VEGF, KSP SNALP Alnylam Pharm. Phase I/II Completed

Atu027 Advanced or metastatic solid tumors PKN3 Liposome Silence Therapeutics Phase I Completed

siG12D LODER Pancreatic ductal adenocarcinoma KRASG12D Polymer matrix Silenseed Ltd. Phase I Ongoing

TKM 080301 Solid tumors PLK1 SNALP Tekmira Pharma Phase I Recruiting

siRNA–EphA2–DOPC Solid tumors EphA2 Liposome M.D. Anderson Cancer Center Phase I Recruiting



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environment. Lodamin is also under investigation as a therapeutic for

ophthalmologic diseases of the vasculature including diabetic retinopa-

thy and age-related macular degeneration.

Vascular cell adhesion molecule-1 (VCAM-1) is an immunoglobulin-

like transmembrane glycoprotein receptor whichmediates immune cell

adhesion that is expressed at high levels on the surfaces of endothelial

cells in tumor blood vessels. Liposome nanoparticles coated with anti-

VCAM-1 antibodies selectively target the human tumor vasculature

in vivo in xenograft studies [71]. Thus, VCAM-1-targeted nanocarrierscould provide a way to both enhance delivery of drugs to growing

tumor vessels and alter endothelial function.

Knowledge of the mechanismby which nanoparticles enter vascular

endothelial cells also has given rise to modications that further en-

hance targeting. Caveolae-mediated endocytosis is the most prominent

transendothelial delivery pathway and has attracted interest because it

has the capability to bypass lysosomes. Surface modications that

enhance caveolae targeting, such as coating gold nanoparticles with

an antibody to the caveolae-enriched protein aminopeptidase P, have

been shown to be effective in concentrating the nanomedicine in lung

tumor microvasculature [72].

3.1.2. Hypoxia

Hypoxia is a hallmark of cancer, which results from rapid tumor cell

growth that overcomes the rate of vascular ingrowth. Hypoxic regions

in tumors experience high interstitialpressurebuild up dueto compres-

sion of blood vessels, poor lymphatic drainage and deterioration of

structural features of tissues that support diffusion. Hypoxia is also com-

monly associated with poor circulation, tumor progression, malignancy

and multi-drug resistance and hence, the delivery of drugs to hypoxic

microenvironments remains a signicant clinical challenge in cancer

therapy [73–75].

Several nanotherapeutic strategies have been used to develop

theranostics (combined therapeutics and diagnostics) for hypoxic envi-

ronments within tumors. For example, a layer-by-layer assembled poly-

electrolyte nanoparticle has been shown to selective target to hypoxic

regions of tumors [76]. Targeting is based on the low pH in the hypoxic

microenvironment, which triggers shedding of the outer layer of the

nanoparticle, revealing a charged nanoparticle surface that is subse-quently taken up by cells. Hypoxia-activated, near-infrared (NIR)

light-triggered chitosan nanoparticles also have been developed to

deliver drugs to cancer cells [77]. This smart nanosystem consists of a

hypoxic sensor, a photoactivatable trigger, and a caged drug; as it is

highly specic to the hypoxic tumor microenvironment, its selectivity

is greatly increased. Polystyrene nanoparticles doped with an oxygen-

sensitive NIR-emissive palladium meso-tetraphenylporphyrin that se-

lectively activate and emit optical signals when they experience hypoxic

conditions also have been developed [78]. In vitro, the nanoparticles

were easily phagocytosed by murinealveolar macrophages and exhibit-

ed sensitivity to low oxygen concentrations resulting from induction of

hypoxia inducible factor-1α (HIF-1α). Experiments performed in vivo

with tumor-bearing mice conrmed that these nanoparticles can be

used as imaging probesfor tumor hypoxia; however, thesame approachpotentially could be leveraged to develop therapeutics.

3.2. Immune response

Renewed recognition of the critical role of the immune system in

cancer has driven the development of immunotherapies that target

existing tumors and enhance long-term immune surveillance. Nanopar-

ticle carriers improve specicity of immune-cell targeting, permit

simultaneous delivery of antigens and immune agents, and enhance

sustained release.

Human interleukin 12 (IL-12) induces a potent anti-tumor immune

response, but systemic administration of IL-12 in early clinical trials

resulted in serious dose-dependent toxicities, including death. In xeno-

graft models, intratumoral injection of IL-12 loaded liposomes resulted

in an enhanced memory T cell response [79]. Importantly, the drug

did not enter the peripheral blood circulation, suggesting that this

could help to improve its safety prole [79].

Biodegradable nanoparticles are emerging as tools for targeting

tumor antigens to dendritic cells, which is a critical step in eliciting cyto-

toxic T cell activation for antitumor vaccine ef cacy [80]. Several studies

have demonstrated the successful use of mannose-grafted nanocarriers

and an increase in the uptake of these carriers by dendritic cells. For ex-

ample, PLGA nanoparticles coated with mannan, a natural polymannoseisolated from the cell wall of Saccharomyces cerevisae, have been shown

to have a stronger binding af nity and produce increased CD4+ and

CD8+ T cell responses compared to unmodied nanoparticle surfaces

[81]. Other particulate vaccines have been successfully developed to spe-

cically target the mannose receptor, such as mannose–PEG3–NH2-dec-

orated PLGA mannose [82], mannosylated-gelatin nanoparticles [83] and

liposomes containing mannosyl lipid derivatives [84]. Targeting antigens

to C-type lectin receptors also has been shown to result in enhanced hu-

moral and cellular responses which are suf cient to induce immunity

against melanomas tumors in vivo [85]. Moreover, similar targeting can

be accomplished using anti-lectin antibodies instead of lectin ligands

[86].

Another attractive property of nanoparticle carriers is thatthey offer

the potential to deliver both antigenand adjuvant simultaneously to the

same cell, thus improving the sustained immune response. Several Toll-

like receptor (TLR) ligands have been tested as adjuvants of cancer vac-

cines in clinical trials for various cancers, including melanoma, lympho-

ma, glioblastoma, breast, prostate, ovarian and lung. As some TLR

ligands have severe systemic side effects, nanoparticle delivery to the

desired site of action can greatly improve their safety prole and permit

use of lower doses. In addition, nanocarriers can improve intracellular

uptake, thus expanding the range of targeting options to intracellular

receptor family members such as TLR3, 7, 8 and 9 [87].

Another approach uses programmable macroporous 3D PLGA scaf-

folds containing nanoparticles to induce endogenous dendritic cells to

recognize tumor antigens placed within the scaffold, and to mount a

systemic anti-cancer response in vivo [88]. The scaffolds contain tumor

antigens from biopsy samples along with PLGA microspheres that

release the chemokine GM-CSF to recruit the dendritic cells, andpolyethylenimine (PEI)–CpG cationic nanoparticles containing the im-

mune adjuvant CpG to stimulate dendritic cell maturation. When im-

planted in a mice tumor melanoma model, this nanoparticle-enabled

vaccine resulted in survival of 90% of tumor-bearing animals [88] and

intracranial regression of glioma has been demonstrated in rats [89].

These programmable scaffolds comprising micro and nanoparticles

show potential in future cancer immune therapy, and human Phase I

clinical trials were recently initiated with this technology.

Onenew approach to stimulating the host immuneresponseis using

light-activated nanoparticles: biodegradable polymer and zinc phthalo-

cyanine (ZnPc) photosensitizer nanoparticles target the mitochondria

of cancer cells and subsequent light activation elicits the production of

tumor antigen, triggering dendritic cell activation [90]. These ex vivo

studies suggest that secreted compounds from mitochondria-targetedand light-activated cancer cells also might be useful as cancer vaccines.

3.3. Extracellular matrix

Tumors have a dense ECM consistingof highly interconnected cross-

linked collagen bers and associated large glycoproteins and proteogly-

cans. Changes in the structure and mechanical properties of the ECM

can alter cellular mechanical signaling pathways and thereby inuence

subsequent tumor progression and metastasis [57,91,92]. For example,

during tumor progression, overexpression of the ECM-modifying en-

zyme lysyl oxidase (LOX) results increased collagen cross-linking,

thereby increasing ECM rigidity and tissue tension [93,94]. Moreover,

experimental inhibition of LOX enzyme activity suppressestumor growth

and prevents cancer progression, and this is mediated by decreasing



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collagen cross-linking and restoring more normal-like tissue compliance

[91,95]. Importantly, when blocking LOX antibodies were delivered on

biodegradable PLGA-based polymer nanoparticles (~200 nm diameter)

created using self-assembly, they produced more effective tumor inhibi-

tion in animals using lower doses, and they had a higher therapeutic

index (Fig. 4) [96]. Compared to systemic injection of free LOX antibody,

this LOX antibody-coated nanotherapeutic exhibited signicant

tumor inhibition with only ~ 1/30th dose normally used for systemic

administration.Proteoglycans are also a major contributor to ECM mechanics and in

particular, studies have focused on the roles of hyaluronic acid (HA)and

the proteoglycan versican in tumor ECM mechanics. Interaction

between versican with HA and its cell surface receptor, CD44, serves

to modulate the ECM mechanical environment, thereby altering cancer

cell proliferation and migration [97,98]. Several studies have utilized

HA-conjugated nanocarriers to target CD44 over-expressing cancer

cells and to deliver anti-cancer drug to the tumor site. For example,

intelligent core-corona HA–PEG–PCL nanoparticles have been used

to deliver doxorubicin in Ehrlich ascites tumor-bearing mice, and

HA-decorated PLGA nanoparticles enhance doxorubicin uptake by cul-

tured human breast adenocarcinoma cells [99,100]. Ef cient CD44

targeting also has been accomplished using HA-modied magnetic

nanocrystals for MRI imaging of breast cancer cells [101]. Importantly,

HA is also a viable target for cancer delivery (independently of CD44)

because it is often over-expressed during tumor progression. Hyaluron-

idase liposomes disrupt the HA matrix when injected intravenously in a

human osteosarcoma solid tumor model [102] and silica nanoparticles

coated with hyaluronidase enzyme have been used to deliver adjuvantdrugs to treat skin cancer [103].

Apart from activation of chemical signaling, the tumor stroma phys-

ically limits the penetration of nanotherapeutics and molecular drugs

into the tumor. For this reason, co-delivery of proteases, such as collage-

nase, gelatinase and hyaluronidase, signicantly aids in breakdown of

the dense ECM barrier and thereby, increases transport of nanoparticles

into the tumor and enhances targeted delivery [1,104,105]. Other en-

zymes (e.g., transglutaminase) and proteins (e.g., the proteoglycans

lumican, decorin and biglycan) also may play a role in modifying tumor

ECM properties and inuencing cancer development [106–111]. For

Self-assembly in

aqueous solutionPLGA-b-PEG-COOH

A

LOXAb

EDC/NHS

PLGA-b-PEG-COOHNanoparticle (NP)

O OH

O

r

OO H

O

r

LOXAbNP

LOX

Untreated

AbNP

O

OOr N

O

OO

OOr N

O

O

Or

O

LOX AbNH

O O H

O

rPLGA

500 nm

1

2

0

T u m o r S i z e ( x 1 0 3 m m

3 )

Days

5 8 122

Untreated Tumor

LOXAbNP treated Tumor

0 100 10000

10

20

30

I n t e n s i t y %

Size (nm)

B

C

Fig. 4. A) Schematic of the process by which lysyl oxidase-antibody (LOX-ab) coated PLGA nanoparticles are formed by solvent displacement, aqueous self-assembly and surface conju-

gation. B) Size characterization of the LOX-ab nanoparticles using transmission electron microscopy (TEM) (left) and dynamic light scattering (DLS) (right) con rming that their size is

~200nm indiameter andthatthey aresphericalin form.C) Thetherapeuticpotential ofthese nanoparticleswas demonstratedby injectingthe LOX-abcoated nanoparticlesintravenously

in a mammary tumor xenograft model; this resulted in signicant inhibition of tumor growth when analyzed over time (left) or by visual inspection at the end of the experiment after

removal of the tumors (right) (adapted with permission from Kanapathipillai et al. [96]).


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example, macromolecular drugs (e.g., FITC-dextran, FITC-albumin) pene-

trate more deeply into tumor tissue after degradation of collagen and

decorin in the ECM [112]. Thus, nanoparticle-based strategies to modu-

late these ECM enzymes and protein functions, will likely improve cancer

targeting specicity, drug diffusion and accumulation, as well as drug re-

tention at the tumor site.

The ECM is a dynamic structure as it is maintained through ongoing

coupling between synthetic and degradative mechanisms; however,

ECM remodeling becomes deregulated during tumor progression [57]and this is mediated by elevated expression and activity of various pro-

teolytic enzymes, including matrix metalloproteinases (MMPs) and

urokinase-based plasminogen activator systems [113,114]. Interesting-

ly, several nanoparticle-based theranostics have been designed to both

monitor MMP expression and inhibit their activity in the tumor micro-

environment. For example, liposomes modied with the GPLPLR pep-

tide sequence to target MT1-MMP showed higher binding ef ciency

and higher therapeutic ef ciency in tumors than uncoated liposomes

[115]. Nanoparticle-based prodrug strategies also have been developed

by taking advantage of MMP-sensitive activation mechanisms which

are selectively triggered in the tumor microenvironment. An albumin-

binding doxorubicin prodrug that is sensitive to MMP2 has been

shown to be effective in malignant melanoma [116]. Gold nanoparticles

conjugated to MMP-sensitive peptides also have been used in tumor

imaging and drug delivery [117,118]. Recently, MMP-2-sensitive

denuding of gold nanoparticles in human breast adenocarcinoma cells

has been shown to improve their uptake and hence, enhance targeted

delivery to tumor cells [119]. Tumor-triggered release of doxorubicin

from MMP-sensitive gold nanoparticles also has been demonstrated in

a SSC-7 subcutaneous tumor model [120].

The serine protease inhibitor, urokinase (uPA), plays a major role in

tumor cell invasion and metastasis and uPA-targeting nanoparticles

have been developed for tumor targeting and imaging. Magnetic nano-

particles coated with uPA receptor (uPAR)-targeting peptides have

higher rates of uptake in uPAR positive cancer cells compared with un-

coated particles [121], and uPA-targeting iron oxide nanoparticles have

been used for in vivo imaging of breastcancer [122]. Thesenanoparticles

were coated with a recombinant peptide of the amino terminal frag-

ment of uPA, which targets the uPAR that is overly expressed in thebreast cancer tissue microenvironment.

Nanoparticles or small molecule drug carriers also have been de-

veloped to target tumor support structures. The cyclic decapeptide,

CGLIIQKNEC, which is known to bind bronectin–brin complexes

in the tumor ECM, has been conjugated to gadolinium-containing

dendrimers for targeting breast tumor xenografts [123]. The tumor

stromaalso containsan abnormal meshwork of clotted plasma proteins,

such as brin. The plasma clot-binding peptide, CREKA, which was se-

lected using phage display [124] has been used for targeted delivery

to the brin mesh in tumor stroma [125]. Nanoparticles and liposomes

were conjugated to the CREKA peptide and shown to both target to the

tumor stroma and also to induce self-triggered clotting.

3.4. Multi-drug resistance

Nanoparticles also have the potential to overcome multi-drug resis-

tance (MDR) that can severely restrict the effectiveness of conventional

chemotherapeutic drugs in the tumor microenvironment [126]. Due to

non-specic and ligand receptor-mediated targeted uptake, nano-

particles are able to bypass ef ux by ABC transporter proteins and

accumulate intracellularly; this is in contrast to free drugs which

can be highly susceptible to ABC transporter P-glycoprotein (P-gp)

capture and ef ux [127,128]. For instance, P-gp ef ux and multi-

drug resistance could be overcome in cultured P388 leukemia cells

using doxorubicin-loaded poly(alkyl-cyanoacrylate) nanoparticles

[129,130]. In murine cancer models, these nanoparticles circulated

longer and displayed increased accumulation in tumor tissue when

coated with PEG-modied drug.

Another strategy to overcome MDR is to exploit the cell-surface re-

ceptor binding strategies of nanoparticles. Studies using transferrin-

conjugated nanoparticles or liposomes-containing paclitaxel and

oxaliplatin, as well as pH-stimuli-responsive polymeric micelles contain-

ing doxorubicin, have all shown less development of drug-resistance in

tumor mouse models compared to free drug [20,34,131]. Other recent

studies on MDR-modifying nanoparticles with increased therapeutic ef-

cacy include doxorubicin-loaded lipid nanoparticles in human breast

and ovarian cancer cells [132], anti-P-glycoprotein-conjugated PEG–

PLGA, and poloxamer-modied PLGA nanoparticles for the targeted de-

livery drugs to cervical carcinoma cells over-expressing P-gp [133].

4. Challenges for the future

While there have many great advances in the area of nanocarrier-

based drug delivery over the past few decades, many challenges remain

for the design of effective nanotherapeutics for clinical cancer therapy.

Nanomaterials that target specically to the cancer cell or the tumor

microenvironment, but do not exhibit long-term toxicity and can be

effectively cleared from the circulation and removed from the body,

without compromising circulation time or bioavailability must be de-

veloped [134]. A deeper understanding of the complexities involved in

cancer cell physiology and the tumor microenvironment, as well as

drug and particle pharmacokinetics are key for the development of

successful new cancer therapeutics. It will be crucial to develop smart

nanomaterials that improve stability, extend circulation time, enhance

targeting and improve retention at the tumor site, while minimizing

short- and long-term systemic toxicities. Emerging nanoparticle

technologies for the delivery of siRNA, anti-metastatic agents, and

multi-resistant drugs [8,135,136] show promise, and demonstrate

the ability to overcome many of the roadblocks that have limited

the effectiveness of conventional drug delivery approaches. More re-

cent identication of CD47 as a “marker of self ” whichhelps to evade

phagocytosis uptake of nanoparticles by macrophages, and is also

overexpressed in several solid tumors [137,138], raises the possibil-

ity of further increasing the effectiveness of nanocarrier drug deliv-

ery systems. In fact, nanobead circulation times and therapeutic

ef cacy were shown to be signicantly enhanced in lung carcinoma-bearing mice when the nanoparticles were decorated with CD47 or

anti-CD47 antibodies [138].

Another emerging technology in nanotherapeutics is the develop-

ment of DNA-based nanostructures that form through programmable

self-assembly [139,140]. The ability to create virtually any 3D shape

using this approach provides a potentially powerful new way to

tailor nanoparticle design to optimize its function. For example, self-

programmable DNA nanorobots that are capable of selectively deliver-

ing anti-cancer payloads (ligands for apoptosis-inducing receptors)

only when they bind to specic types of cancer cells (Fig. 5) were re-

cently developed [141]. Exploration of the value of this approach for

treatment of human cancer will require demonstration of biocompati-

bility and stability of these materials when injected in vivo. However,

the range of design control and the programmability of these nanoscalerobots are huge, and hence, this area will be interesting to watch over

the next decade.

Further, in vitro tumor engineering and tumor organ on a chip tech-

nologies could serve in optimal design and screening of nanoparticles

prior to preclinical studies. For example, the potential of using tumor

spheroids for drugscreening and delivery to pathological tissues has re-

cently been reported [142]. Spheroids resemble the 3D architecture of

tumor tissues, mimicking cell–cell interactions and diffusional limita-

tions of mass anduid transport, and therefore represent a moresuitable

platform for testing drugs and nanotherapeutics than conventional pla-

nar culture substrates [142,143]. A microuidic tumor culture device or

“tumor-on-a-chip”model, alsohas beendeveloped to study nanoparticle

transport in tissues [144]. Tumor-like spheroids were incorporated

into the microuidic channels and accumulation of nanoparticles was



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measured in real-time at physiological blood ow rates. In a further ad-

vancement of this organ-on-a-chip approach, the in vivo microenviron-ment of a breathing human lung alveolar-capillary interface was

recreated in a microuidic chip [145], and this platform was shown to

be effective at detecting both physiological nanoparticle absorption and

relevant toxicities in vitro [146]. Hence, these organ-on-chip devices

might be used in the future as a replacement for animal studies for

rapid testing nanotherapeutic ef cacy as well as toxicity. In this manner,

emerging tumor engineering technologies may synergize with new ad-

vances in nanobiotechnology to accelerate the development of more ef-

fective nanocarriers while reducing both the time and cost of

investigations required to meet these goals.

However, to develop truly effective cancer nanotherapeutics, we

have to focus beyond the design of the nanoparticle itself and instead,

learn more about the process of cancer formation and progression as

well as how we might more selectively interfere with, or prevent,these critical processes. For example, as it is becoming increasingly

clear that cancer is not merely a disease of uncontrolled cell growth,

and instead that it is a disease of normal tissue development [26],

then we might think to explore ways to harness knowledge about de-

velopment control to create new cancer therapeutics. In this context,

it is interesting that developmental biology studies have shown that

when ECM isolated from embryonic tissue is combined with adult epi-

thelial cancers, it is capable of normalizing the cancer by suppressing

growth and stimulating tissue-specic differentiation [147,148]. Thus,

development of biomimetic nanomaterials that mimic the inductive

cues found within embryonic ECM could lead to the development of

novel nanotherapeutics that might normalize the cancer tissue micro-

environment rather than simply kill all growing cancer cells [26]. Inter-

estingly, molecules within embryonic ECM that are responsible for this

type of cancer-normalizing activity (e.g., biglycan) are beginning to be

identied [111]. Given the key role of ECM mechanics in cancer devel-opment, it also might be possible to design nanotherapeutics that pre-

vent tumor growth or normalize the cancer phenotype by selectively

modulating the mechanical or structural properties of the tumor micro-

environment [26]. This could be facilitated by development of smart

nanotechnologies that have programmable mechanical properties, or

that can be triggered to alter their physical properties by microenviron-

mentalcues, as is possible,for example, with self assemblingDNA nano-

structures [149].

In conclusion, it is clear that simultaneously advancements in geno-

mic, proteomic and nanotechnology elds have enabled many types of

new nanotherapeutics to be developed that offer the potential to trans-

form cancer therapy. The newest wave of nanomaterials are multi-

functional, programmable, and even self-assembling; they also offer

ways to provide longer circulation times, higher selectivity, and moreeffective targeting to tumor sites. The recent clinical approval of the

liposomal-doxorubicin nanoparticles, Doxil and Abraxane [1,11], has

validated the value of nanotechnology for cancer treatment. However,

the future is even brighter, and the continuing exciting advances in de-

velopment nanoparticle-based therapeutics will hopefully attract many

new investigators in various disciplines into this exciting interdisciplin-

ary eld.

Acknowledgments

We thank K. Johnson for help with the graphics. This work was sup-

ported by the Wyss Institute for Biologically Inspired Engineering at

Harvard University, and grants from the Department of Defense

(W81XWH-08-1-0659 and W81XWH-10-1-0565).

Fig. 5. A) Schematic depicting self-programmable DNAnanorobots loadedwith antibody payload, in lockedposition(left) andthe ligandlocking mechanism(middle) andopen position of

nanorobot afterligand–receptor interactionexposingthe payload. B) Eightdifferentnanorobots were loadedwithuorescentlylabeled anti-HLA-A/B/C antibody fragments andtheir bind-

ing specicity was tested in 6 different cancercell types expressing various combinations of apoptosis-inducing receptors. The histograms show count of cells versus uorescence due to

anti-HLA-A/BC labeling (reproduced with permission from Ref. [141]).



10/12

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