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ARTICLE IN PRESSG ModelANTOD-724; No. of Pages 14Nano Today xxx (2019) xxx–xxx

Contents lists available at ScienceDirect

Nano Today

jou rn al h om epa ge: www.elsev ier .com/ locate /nanotoday

eview

linical cancer nanomedicine

oy Wolfram a,b,∗, Mauro Ferrari b,c,∗∗

Department of Transplantation/Department of Physiology and Biomedical Engineering, Mayo Clinic, Jacksonville, FL, 32224, USADepartment of Nanomedicine, Houston Methodist Research Institute, Houston, TX, 77030, USADepartment of Medicine, Weill Cornell Medicine, Weill Cornell Medicine, New York, NY, 10065, USA

r t i c l e i n f o

rticle history:eceived 21 November 2018eceived in revised form 22 February 2019ccepted 26 February 2019vailable online xxx

eywords:rug deliveryxtracellular vesicleultifunctionalanomedicineanoparticle

a b s t r a c t

Nanotechnology offers new solutions for the development of cancer therapeutics that display improvedefficacy and safety. Although several nanotherapeutics have received clinical approval, the most promis-ing nanotechnology applications for patients still lie ahead. Nanoparticles display unique transport,biological, optical, magnetic, electronic, and thermal properties that are not apparent on the molecularor macroscale, and can be utilized for therapeutic purposes. These characteristics arise because nanopar-ticles are in the same size range as the wavelength of light and display large surface area to volumeratios. The large size of nanoparticles compared to conventional chemotherapeutic agents or biologicalmacromolecule drugs also enables incorporation of several supportive components in addition to activepharmaceutical ingredients. These components can facilitate solubilization, protection from degrada-tion, sustained release, immunoevasion, tissue penetration, imaging, targeting, and triggered activation.Nanoparticles are also processed differently in the body compared to conventional drugs. Specifically,nanoparticles display unique hemodynamic properties and biodistribution profiles. Notably, the inter-actions that occur at the bio-nano interface can be exploited for improved drug delivery. This reviewdiscusses successful clinically approved cancer nanodrugs as well as promising candidates in the pipeline.

These nanotherapeutics are categorized according to whether they predominantly exploit multifunc-tionality, unique electromagnetic properties, or distinct transport characteristics in the body. Moreover,future directions in nanomedicine such as companion diagnostics, strategies for modifying the microen-vironment, spatiotemporal nanoparticle transitions, and the use of extracellular vesicles for drug deliveryare also explored.

© 2019 Elsevier Ltd. All rights reserved.

ontents

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00Exploiting multifunctionality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

Solubilization and sustained drug release . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00Protection from degradation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00Immunoevasion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00Combination therapy. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .00Targeting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00Triggered activation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

Exploiting electromagnetic properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

Please cite this article in press as: J. Wolfram, M. Ferrari, Clinical cancer nanomedicine, Nano Today (2019),https://doi.org/10.1016/j.nantod.2019.02.005

Exploiting mass transport characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00EPR effect . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00Tumor hemodynamics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

Future directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

∗ Corresponding author at: 4500 San Pablo Rd, Jacksonville, FL, 32224, USA.∗∗ Corresponding author at: 6550 Fannin St, Houston, TX, 77030, USA.

E-mail addresses: wolfram.joy@mayo.edu (J. Wolfram), mferrari@houstonmethodist.org (M. Ferrari).

ttps://doi.org/10.1016/j.nantod.2019.02.005748-0132/© 2019 Elsevier Ltd. All rights reserved.

https://doi.org/10.1016/j.nantod.2019.02.005https://doi.org/10.1016/j.nantod.2019.02.005http://www.sciencedirect.com/science/journal/1748-0132http://www.elsevier.com/locate/nanotodaymailto:wolfram.joy@mayo.edumailto:mferrari@houstonmethodist.orghttps://doi.org/10.1016/j.nantod.2019.02.005

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Engineered extracellular vesicles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00Spatiotemporal nanoparticle transitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00Priming the microenvironment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00Companion diagnostics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00Declaration of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .00References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

I

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Definitions of nanotechnology vary in different countries, whichas implications for the clinical approval of nanodrugs [1]. Nev-rtheless, the common denominator among various definitions ofanotechnology is the use of constructs with nanoscale dimen-ions (Box 1). There are three distinct advantages that arise frompplying nanotechnology to the treatment of disease. The firstdvantage is that several functional components can be incorpo-ated into nanotherapeutics as they are much larger than mostonventional drugs. Each component can be tailored to contributen different ways to overall therapeutic activity and safety. The sec-nd advantage is that materials on the nanoscale display uniquelectromagnetic properties, which can be exploited for therapeuticurposes. The third benefit of using nanotechnology in medicine isn increased ability to differentiate between pathological and nor-al tissues. This ability is based on exploiting distinct interactions

nd processing pathways in the body that are unique to nanopar-icles. Notably, site-specific delivery of systemically injected drugsan be improved by taking advantage of bio-nano interactions.

In 1995, Doxil (liposomal doxorubicin) became the firstanoparticle-based drug for cancer treatment to receive clinicalpproval in the United States [2]. Since then, several other nan-drugs have entered the market for treatment of various diseases.hese nanotherapeutics include topical, local, and systemicallydministered organic and inorganic particles. Clinically approvedanotherapeutics are often categorized based on material com-osition, belonging to the lipid, polymer, protein, or metal-basedanoparticle category [3]. However, an alternative grouping of nan-therapeutics is based on the above-mentioned principles by whichaterials on the nanoscale can improve therapeutic efficacy. This

Please cite this article in press as: J. Wolfram, M. Ferrhttps://doi.org/10.1016/j.nantod.2019.02.005

ype of categorization gives a more complete understanding ofhe diverse benefits of nanotechnology in medicine. Table 1 sum-

arizes the advantages of nanotherapeutics using examples of

Box 1: Definitions of Nanotechnology.Technology that fulfills the following criteria: i) has components in the nanosize

range, ii) is man-made, and iii) has properties that arise due to nanoscaledimensions (Mauro Ferrari, Chief Executive Officer and President of theHouston Methodist Research Institute in the United States) [211]Work at theatomic, molecular, and supramolecular levels in order to understand andcreate materials, devices and systems with fundamentally new propertiesand functions because of their small structure (Robert Langer, InstituteProfessor at the Massachusetts Institute of Technology in the United States)[211]Toolbox that provides nanometer-sized building blocks for the tailoringof new materials, devices, and systems (Jackie Ying, Professor, Agency forScience, Technology and Research in Singapore) [211]

Science, engineering, and technology conducted at the nanoscale, which isabout 1-100 nm (informal definition by the United States NationalNanotechnology Initiative) [212]The use of tiny structures - less than 1000 nmacross - that are designed to have specific properties (informal definition bythe European Medicines Agency) [213]

The branch of technology that deals with dimensions and tolerances of lessthan 100 nm, especially the manipulation of individual atoms and molecules(Oxford Dictionaries) [214]

Fig. 1. Multifunctional properties of nanotherapeutics.

clinical-stage nanoparticles for cancer therapy. For a more com-prehensive list of nanoparticles in the clinic, please refer to reviewsby Wicki et al. [4] and Anselmo et al. [3]. Although certain entriesin Table 1 could fall into several categories, it is clear that the fullpotential of nanotechnology in medicine still lies ahead as nan-odrugs that exploit a comprehensive set of nanoscale benefits haveyet to reach the clinic. In addition to introducing a new systemfor the classification of therapeutic nanoparticles, the focus of thisreview is to discuss clinically available cancer nanodrugs. Somepromising future opportunities in the field of cancer nanomedicineare also reviewed in the closing section.

Exploiting multifunctionality

Nanomedicine enables the design of therapeutics with multiplefunctional elements. Namely, in addition to an active pharma-ceutical ingredient, several other supportive components can beincluded due to the large size of nanoparticles compared to smallmolecules. These supportive components can aid in drug protec-tion, site-specific activation, cellular uptake, and imaging. Fig. 1shows a summary of the various multifunctional properties of nan-otherapeutics.

ari, Clinical cancer nanomedicine, Nano Today (2019),

Solubilization and sustained drug release

The simplest form of multifunctionality in nanomedicine isthe addition of materials that enable drug solubilization. Sev-

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ARTICLE IN PRESSG ModelNANTOD-724; No. of Pages 14J. Wolfram, M. Ferrari / Nano Today xxx (2019) xxx–xxx 3

Table 1Advantages of nanotechnology in medicine with examples of clinical-stage cancer nanotherapeutics.

Name Composition Cancer type Status Refs

MULTIFUNCTIONALITYSolubilization/Sustained ReleaseAbraxaneGenexol-PMLipusuMarqibo

Albumin-bound paclitaxelPolymeric nanoparticles with paclitaxelLiposomal paclitaxelLiposomal vincristine sulfate

Breast, lung, and pancreatic cancerBreast, lung, and ovarian cancerBreast, lung, and ovarian cancerAcute lymphoblastic leukemia

Approved in the US (2005)Approved in Korea (2007)Approved in China (2006)Approved in the US (2012)

[27][11][12][67]

Protection from degradationAtu027ALN-VSP02DCR-MYCMRX34

Liposomal small interfering RNA (siRNA)Liposomal siRNALipid nanoparticle with Dicer-substrate siRNALiposomal micro RNA (miRNA)

Advanced or metastatic pancreaticcancerSolid tumors with liverinvolvementAdvanced solid tumorsAdvanced cancers

Phase I/II completed (2016)Phase I completed (2011)Phase I/II terminated (2016)Phase I terminated (2016)

[27][44][46][215]

ImmunoevasionDoxilOnivyde

Liposomal doxorubicin (pegylated)Liposoml irinotecan (pegylated)

HIV-related Kaposi sarcoma,ovarian cancer, and multiplemyelomaAdvanced pancreatic cancer

Approved in the US (1995)Approved in the US (2015)

[27][54]

Combination therapyVyxeos Liposomal cytarabine and daunorubicin High-risk acute myeloid leukemia Approved in the US (2017) [27]Targeted therapyMM310 Ephrin type-A receptor 2-targeted liposomal

docetaxelSolid tumors Phase I ongoing [82]

Triggered activationCX-2009CX-072ThermoDox

CD166 probody- maytansinoid conjugateAnti-programmed death-ligand 1 (PD-L1) ProbodyThermosensitive liposomal doxorubicin

Solid tumorsSolid tumors and lymphomaHepatocellular carcinoma

Phase I/II ongoingPhase I/II ongoingPhase III completed (2016)

[107][105][108]

UNIQUE ELECTROMAGNETIC PROPERTIESNanoThermAuroShell

Iron oxide NPGold nanoshells

Brain tumorsProstate cancer

Approved in Europe (2011)Phase I ongoing

[27][113]

UNIQUE TRANSPORT PROPERTIESEnhanced permeability and retention (EPR) effectSMANCSDaunoXomeMyocetMEPACT

Polymer-neocarzinostatin conjugateLiposomal daunorubicinLiposomal doxorubicinLiposomal muramyl tripeptidephophatidyl-ethanolamine

Liver and renal cancerHIV-associated Kaposi’s sarcomaMetastatic breast cancerOsteosarcoma

Approved in Japan (1993)Approved in the US (1996)Approved in Europe/Canada (2000)Approved in Europe (2009)

[27][65][66][68]

Breas

enebEtooAcAcirhdsMtrAafmasui

HemodynamicsNanoparticle generator Porous silicon microparticle with polymeric

doxorubicin

ral chemotherapeutic agents are poorly water soluble, whichecessitates the use of toxic solvents for administration. Forxample, docetaxel and paclitaxel are administered with polysor-ate 80 (Tween 80) and polyethoxylated castor oil (KolliphorL)/dehydrated ethanol, respectively [5], Accordingly, corticos-eroids and antihistamines are prescribed to cancer patients inrder to avoid the side effects of these solvents [6]. Nanomedicineffers a safer alternative to harmful formulations. For instance,braxane is an albumin-based paclitaxel-containing nanoparti-le that has been approved by the United Stated Food and Drugdministration (FDA) for the treatment of several types of can-er [7]. Albumin is suitable for nanomedicine applications as its the most abundant protein in the blood and serves as a car-ier for endogenous hydrophobic molecules [8]. Clinical studiesave shown that Abraxane administered at a 50% higher paclitaxelose than the conventional formulation results in fewer and lessevere cases of neutropenia, a common side effect of therapy [9].oreover, Abraxane can be administered over a shorter infusion

ime (30 min) than paclitaxel due to less risk of hypersensitivityeactions [10]. It is thought that the enhanced safety profile ofbraxane is potentially due to improved pharmacokinetics and thebsence of Kolliphor EL [9]. Other examples of avoiding side effectsrom toxic solvents is the use of paclitaxel-containing polymeric

icelles composed of polyethylene glycol (PEG) and poly-DL-lactic

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cid copolymers (Genexol-PM, approved in Korea [11]) and lipo-omes (Lipusu, approved in China [12]). Although nanomedicinesually enables higher drug doses to be administered, studies

nvestigating optimal nanoparticle dosing, scheduling, and infusion

t cancer lung metastasis Planning of phase I [91,161]

rates are lacking. Additionally, due to the heterogeneity of nan-otherapeutics the development of standardized methods for dosecalculations is challenging. Notably, for breast cancer, Abraxanewas shown to outperform solvent-based paclitaxel when admin-istered every three weeks [9], while weekly injection scheduleshave revealed contradictory results in regards to the superiorityof albumin-bound paclitaxel. Specifically, one study demonstratedthat Abraxane was superior to solvent-based paclitaxel in regardsto treatment response [13], while another study demonstrated atrend toward longer progression-free survival and less side effectswith solvent-based paclitaxel [14]. The latter study suggests thatnanoparticle-based therapy would primarily be advantageous formaintaining drug concentrations within the therapeutic windowwhen doses are administered less frequently. However, it is worthnoting that in this study, Bevacizumab was also administered inboth treatment arms [14]. It is possible that Bevacizumab has differ-ent effects on free and nanoparticle-bound paclitaxel, especially asintratumoral accumulation of Abraxane is more dependent on vas-culature properties. In fact, preclinical studies have demonstratedthat antiangiogenic therapy lowers intratumoral drug accumula-tion [15].

Another important consideration for nanotherapeutics is highdrug loading capacity to reduce exposure to excess materials thatlack therapeutic efficacy. In particular, high levels of drug load-

ari, Clinical cancer nanomedicine, Nano Today (2019),

ing can be achieved with nanocrystals [16] or therapeutic agentsthat self-assemble into nanoparticles [17]. In addition to improvingsolubility, nanoparticles enable sustained drug release, which per-mits drug concentrations to stay within the therapeutic window

https://doi.org/10.1016/j.nantod.2019.02.005

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nstead of fluctuating between toxic and sub-therapeutic levels,hich is usually the case for small molecules [18]. In a mouse studyhere liposomes with various drug release kinetics were com-

ared, slower release rates, similar to that of clinically approvedherapeutics, were found to lead to increased drug accumulationn tumors and improved therapeutic efficacy [19]. Notably, slowerelease kinetics also led to increased drug deposition in cutaneousissues [19]. However, preclinical studies have also demonstratedhat therapeutic efficacy is compromised with excessively slowelease rates, as cancer cells have minimal exposure to bioavailablerugs [20].

rotection from degradation

Another supportive function of nanoparticles is to provide pro-ection from enzymatic and mechanical degradation of drugs. Thisroperty is particularly important when it comes to drugs thatre sensitive to degradation, such as various gene-silencing agents,ncluding antisense oligonucleotides (ASOs), small interfering RNAssiRNAs), and microRNAs (miRNAs). Gene-silencing agents pro-ide new ways to modulate disease-associated signaling pathwayshat are difficult to target using contemporary approaches. How-ver, the blood, extracellular space, and lysosome contain highevels of ribonucleases and deoxyribonucleases that rapidly destroyligonucleotides [21–23]. Therefore, chemical modifications andrug delivery systems are necessary to ensure the stability ofhese therapeutic agents [24]. Moreover, the negative charge andelatively large size of oligonucleotides prevent efficient cellularnternalization [25]. In addition to protecting oligonucleotides fromnzymes in the circulation and extracellular space, nanoparticlesan aid in cellular uptake and lysosomal escape [24]. Notably, initro-in vivo correlations of therapeutic efficacy are difficult to makes most cell culture models lack key aspects of the biological envi-onment. In a study investigating the ability of in vitro assays toredict the effect of hepatocellular siRNA nanodelivery in animalodels, it was found that cell type and siRNA entrapment efficiency

layed a role, while nanoparticle size and zeta potential did not26]. In particular, freshly isolated primary hepatocytes were moreredictive of in vivo gene silencing efficiency compared to cancerells. Interestingly, the use of HeLa cervical cancer cells lead to moreredictive results than Hepa1-6 liver cancer cells. Overall, there

s a need to evaluate and optimize in vitro assays for improvingranslation of nanodrugs.

Currently, there are several clinical trials for oligonucleotide-ontaining nanoparticles, and one has received clinical approval27,28]. A few chemically modified ASOs have also reachedhe market. For instance, fomivirsen, mipomersen, nusinersen,nd eteplirsen are clinically approved in the United States forhe treatment of cytomegalovirus retinitis (intraocular injec-ion), homozygous familial hypercholesterolemia (subcutaneousnjection), spinal muscular atrophy (intrathecal injection), anduchenne muscular dystrophy (subcutaneous injection) respec-

ively [29,30]. A major challenge for the clinical translation ofene silencing agents is safety. In a pilot study of an intravenouslynjected lipid-conjugated ASO (Imetelstat) for myelofibrosis, 18%f patients experienced thrombocytopenia, which lead to intracra-ial hemorrhage and death in one patient [31]. Similarly, ahase II clinical trial of Imetelstat for pediatric brain tumorsas prematurely terminated due to two deaths caused by

hrombocytopenia-induced intratumoral hemorrhage [32]. How-ver, platelet deficiency does not seem to be a class effect of ASOs33] and may be due to specific chemical modifications that lead to

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inding to platelet-specific glycoprotein-VI receptors [34].In 2016, the field of RNA interference (RNAi) faced a major

etback as the development of revusiran, a promising siRNA--acetylgalactosamine (GalNAc) ligand conjugate for hereditary

PRESSday xxx (2019) xxx–xxx

transthyretin amyloidosis with cardiomyopathy, was discontin-ued due to higher mortality than the placebo group in a phaseIII trial [35]. Potential reasons for increased mortality in responseto revusiran are off-target effects, unexpected on-target effects,immunological activation, and poor study design in regards tostatistical analysis. Mice studies have demonstrated that siRNAscan induce vascular and immune effects through activation ofToll-like receptor 3 (TLR3) [36], which is a sensor for foreigndouble-stranded RNA [37]. However, chemical modifications, suchas those used for revusiran [38], have been shown to preventTLR activation [39]. Moreover, design algorithms were used in thedevelopment of revusiran in an attempt to avoid off-target effects[35]. It is possible that on-target effects arising from suppressionof wild type transthyretin could lead to side effects, as this pro-tein plays an important role in several physiological processes.For instance, thransthyretin is a carrier for thyroid proteins andretinol-binding proteins in the blood, protects pancreatic �-cellsfrom apoptosis, and displays proteolytic activity against amyloid�, apolipoprotein A-I, and amidated neuropeptide Y [40]. With sev-eral oligonucleotide nanomedicines in clinical trials, it remains tobe seen whether toxicity will be common for this type of drugs orlimited to specific therapeutic agents/formulations. In fact, encour-aging safety results were recently reported for a phase III trial ofpatisiran [41], a liposome-based version of revusiran [42], whichbecame the first siRNA-based therapy to receive clinical approval[28], marking a major breakthrough in this field. In regards to cancertreatments, RNAi nanotherapeutics are in earlier stages of clinicaldevelopment. Specifically, lipid, cyclodextrin, and bacterial mini-cells have been utilized in phase I/II clinical trials for delivery ofsiRNAs and miRNAs [43]. Although the treatments were gener-ally well tolerated with some mild inflammatory reactions, a smallnumber of grade 3–4 adverse events [44–46] and one potentialtherapy-related death due to liver failure [44] were reported. Themost favorable outcome of these trials was stable disease withsome partial responses [43]. Accordingly, the clinical developmentof several siRNA nanotherapeutics for cancer, such as the lipidnanoparticle DCR-MYC, has been discontinued due to insufficientpatient outcomes [47]. On the contrary to hereditary conditionsthat are caused by a single genetic defect, most cancers have sev-eral genome abnormalities that together contribute to malignancy.Therefore, clinical trials should be designed as combination ther-apies, as siRNA-based treatments for cancer are unlikely to beeffective unless coupled with other therapeutic agents. Anotherpromising application of nanomedicine is gene editing based ondelivery of components of clustered regularly interspaced shortpalindromic repeats (CRISPR)/associated protein 9 (CRISPR/Cas9)technology. Although genome editing therapeutic agents are inearly stages of development, nanoparticles offer an alternative toviral vectors, which pose various safety concerns [48].

Immunoevasion

Another example of multifunctionality in nanomedicine isthe use of components that facilitate immunoevasion. Commonstrategies for avoiding immunological recognition and clearanceof nanoparticles include surface modifications with anti-foulingpolymers [49], self peptides [50], and cell coatings [51,52]. A clini-cally approved strategy for decreasing nanoparticle uptake by theimmune system relies on the stealth polymer, PEG. Nanoparticlepegylation creates a steric barrier that reduces binding of plasmaopsonins and prevents interactions with cells [53]. Clinicallyapproved examples of pegylated nanoparticles include Doxil [2]

ari, Clinical cancer nanomedicine, Nano Today (2019),

and Onivyde [54], which are liposomal formulations of chemother-apy drugs. In many cases, the altered pharmacokinetics of drugsas a result of nanodelivery reduces side effect. In fact, Doxil wasapproved for multiple myeloma based an equivalent efficacy but

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mproved safety compared to free doxorubicin [2]. However, it ismportant to note that while nanotherapeutics generally reducerug toxicity, they may cause new types of side effects to arise. For

nstance, although Doxil decreases the risk of cardiotoxicity, theccurrence of palmar-plantar erythrodysesthesia increases [55].

Notably, pegylation does not necessarily result in substantialmprovements in tumor accumulation, although this strategy dra-

atically prolongs circulation times [56,57]. The PEG dilemmas based on the inability of this polymer to specifically preventnteractions with immune cells, leading to decreased nanoparti-le uptake in cancer cells as well [58]. Additionally, pegylationredominantly delays immunological clearance as opposed to pre-enting this process, as evidenced by studies demonstrating thathospholipid-PEG conjugates gradually detach from lipid nanopar-icles in the circulation [59,60]. Furthermore, in some patients,egylated nanoparticles have been found to activate the com-lement system, which can increase immunological removal andause hypersensitivity reactions [61,62]. Frequent infusion reac-ions have also occurred when Doxil was administered over a

h period, causing longer infusion times (4 h) to be used [63].n animal models, repeated injections have resulted in the pro-uction of PEG antibodies that are responsible for the acceleratedlood clearance (ABC) phenomenon, in which pegylated nanopar-icles are rapidly cleared by the immune system, paradoxicallyeading to much shorter circulation times [64]. However, it is ques-ionable whether the ABC phenomenon is clinically relevant, asxcessively high doses of pegylated liposomes have been usedn animal studies. Notably, several clinically approved liposomesor cancer therapy are non-pegylated, including DaunoXome [65],

yocet [66], Marqibo [67], and MEPACT [68]. Taken together, moreffective and specific strategies are necessary to avoid immuno-ogical clearance of nanoparticles. Efficient immunoevasion willikely require the concurrent use of microenvironmental modifi-ation strategies and innovative nanoparticle design approacheshat avoid immunological triggers and incorporate markers ofelf, such as CD47 [50]. Various strategies for priming the innatemmune system for reduced nanoparticle uptake will be discussedn the future directions section of this review. A major exceptiono the aforementioned approaches is immunotherapy, where thebility of nanoparticles to activate the immune system is advan-ageous. In fact, a few nanovaccines for the treatment of cancerave already reached clinical trials. For instance, a liposome-basedaccine that incorporates patient-specific cancer cell membraneroteins as well as interleukin-2 (IL-2) has shown promise for

nducing sustained tumor-specific T cell responses in patients withymphoma [69]. Indeed, one of the main advantages of nanoscaleaccines is the ability to incorporate multiple components, suchs various antigens and adjuvants, in the same platform. Addition-lly, nanoparticles enable the use of safe and broadly applicableessenger RNA (mRNA)-based vaccines by providing protection

rom degradation and inducing cellular internalization. For exam-le, in a small cohort of melanoma patients, antigen-specific-cell responses occurred after systemic treatment with a lipid-ased mRNA nanovaccine designed to target dendritic cells [70].lternatively, dendritic cells can be exposed to antigen-loadedanoparticles ex vivo prior to injection into the body as cell-basedaccines [71].

ombination therapy

Combination treatment regimens are considered standard ofare for most cancer types [72]. In fact, concurrent targeting of mul-

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iple malignant processes or different elements of the same processsually leads to improved outcomes [73]. Studies have also demon-trated that it is important to consider drug ratios to obtain optimalherapeutic synergy [74]. A major advantage of nanomedicine is the

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ability to deliver several drugs in the same nanoparticle. Indeed, itis easier to achieve ideal intratumoral and intracellular drug ratiosby co-encapsulation in nanoparticles than taking into account thepharmacokinetics of individual therapeutic agents. In 2017, Vyx-eos became the first nanomedicine for combination therapy toenter the market. This nanodrug is a liposomal formulation ofcytarabine and daunorubicin [75] approved for the treatment ofhigh-risk acute myeloid leukemia (AML). Vyxeos has a cytarabineto daunorubicin molar ratio of 5:1, as this ratio was found to dis-play the highest synergistic therapeutic activity in cell culture andanimal studies [76]. Importantly, it was also shown that the ratiowas maintained for prolonged periods of time in the bone marrow[76]. In a phase III trial for secondary AML, patients treated withVyxeos had a median overall survival of 9.56 months compared to5.95 months in patients administered with a combination of freecytarabine and daunorubicin (standard of care) [77]. In addition todelivery of chemotherapy cocktails, it is likely that nanomedicinewill be beneficial for successful implementation of several othertherapeutic combinations. Furthermore, theranostic nanoparticlesthat contain both therapeutic and imaging agents could facili-tate disease staging, treatment selection, response assessment,and recurrence detection [78]. Nanoparticle-mediated combina-tion therapy could also be useful for increasing drug penetrationdepth in tumors. In fact, it is essential that drugs effectively pene-trate tumor tissue to avoid subpopulations of cancer cells survivingand causing tumor recurrence [79]. Nanoparticles that incorpo-rate proteolytic enzymes that break down the dense extracellularmatrix (ECM) of tumors have been developed for improved intra-tumoral drug distribution [80,81]. However, nanoparticles thatcontain permeation enhancers have not yet reached the clinicalstage and are likely to display side effects-induced by proteolyticenzymes.

Targeting

Nanoparticle targeting is based on surface conjugation of molec-ular targeting ligands. This strategy enables the design of drugcarriers that recognize biomolecules that are associated with spe-cific disease conditions. Such nanoparticles are usually designed tobind to molecules that are overexpressed on the surface of tumorvasculature or cancer cells. Targeted nanoparticles have not yetreached the market but several are currently undergoing clinicaltrials. For instance, MM310 is an ephrin type-A receptor 2 (EphA2)-targeted docetaxel-containing liposome that is in phase I clinicaltrials for patients with solid tumors [82]. Notably, EphA2 is presenton the surface of multiple cells in the tumor microenvironment,including cancer cells, stromal cells, and endothelial cells [83]. Thefield of targeted nanodelivery underwent a major setback in 2016,when the promising nanodrug BIND-014 failed in a phase II clin-ical trial for the treatment of cervical and head-and-neck cancers,followed by the bankruptcy of Bind Therapeutics [84]. BIND-014is a docetaxel-containing polymeric nanoparticle with surface lig-ands for prostate-specific membrane antigen (PSMA) [85], whichis expressed on prostate cancer cells and on the vasculature ofmany other solid tumors [86]. Another commonly exploited tar-get on tumor blood vessels is the �V�3 integrin receptor [87],while nanoparticle surface ligands for the transferrin receptor [88]and folate receptors [89] have frequently been utilized for can-cer cell targeting. Notably, molecular recognition of cancer cellsdoes not increase nanoparticle passage from the circulatory sys-tem into the tumor interstitium, but rather promotes retentionafter extravasation has occurred. In addition to enhanced reten-

ari, Clinical cancer nanomedicine, Nano Today (2019),

tion, one of the main benefits of cancer cell targeting is increasedcellular internalization due to ligand-mediated endocytosis. In fact,even untargeted nanoparticles can substantially aid in the cellularuptake of therapeutic agents through various forms of endocyto-

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is [90]. Nanoparticle-mediated internalization can also circumventrug efflux pumps, thereby overcoming drug resistance [91].

There are several potential reasons for disappointing outcomesith targeted nanotherapeutics, including increased immunolog-

cal recognition and clearance due to the presence of surfaceigands. Moreover, targeting molecules enlarge the size of nanopar-icles, which could lead to decreased levels of extravasationnd intratumoral penetration. Another problematic factor is theinding-site-barrier, which occurs when ligands bind with highffinity to target molecules, thereby preventing further diffu-ion throughout the tumor [92]. Therefore, it is important toalance binding affinity and diffusion ability when designing tar-eted nanoparticles. Strategies to achieve this balance include these of fast-penetrating nanoparticles, low-affinity ligands, stimuli-riggered drug release, and ligand unveiling strategies [93,94].

oreover, targeting approaches should be tailored based on tumorypes. For example, the stroma content of tumors is likely to have

major impact on which cells in the tumor microenvironmentould be ideal targets [93]. Accordingly, an understanding of the

issue and cell surface distribution of molecular targets as well ashe effect of ligand-receptor binding on nanoparticle diffusion andellular internalization will be critical for successful clinical trans-ation of targeted nanoparticles. An additional challenge of targetedelivery is the protein corona that forms around the surface ofanoparticles upon exposure to biological fluids [95]. This layer ofiomolecules can mask surface ligands and prevent target recogni-ion [96]. Moreover, protein binding to the nanoparticle surface canead to conformational changes, which could induce an immuno-ogical response [97]. Additionally, the biomolecular layer canffect biodistribution, nanoparticle degradation, and drug release.

potential solution to the corona problem is surface modificationf nanoparticles to promote binding of specific plasma proteinshat dictate preferential transport properties [98]. For instance, onetudy showed that polymeric nanoparticles coated with polysor-ate 80 attracted apolipoprotein binding, which mediated bloodrain barrier crossing [99]. Additionally, carbon nanotube-polymeromplexes have been surface engineered to display binding pock-ts for specific proteins [100]. Similarly, libraries of hydrogelanoparticles with distinct biomolecular absorption properties cane used to dictate the composition of the protein corona [101].lthough studies evaluating interactions between nanoparticlesnd biomolecules following plasma incubation can be informa-ive, studies that in a systematic manner address the impact ofanoparticle characteristics on pharmacokinetics are essential for

mproving tumor-targeting. Coupling in vivo studies with mathe-atical modeling of biomolecule-nanoparticle interactions [102]ill be valuable for establishing a general framework for optimiz-

ng nanoparticle design. Moreover, in situ measurements of theydrodynamic radius of nanoparticles using fluorine-19 diffusion-rdered nuclear magnetic resonance spectroscopy may provideynamic protein absorption measurements in vivo [103]. Addi-ionally, although proteomic studies evaluating the compositionf the protein corona are frequently reported, visualization ofhe biomolecular layer could provide important structural insight.aken together, an increased understanding of interactions thatccur at the bio-nano interface will be critical for the success ofargeted delivery.

riggered activation

Nanomedicine also enables the triggered activation or releasef therapeutic agents in the tumor. There are several stimuli in

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he tumor microenvironment that can be used as triggers, suchs low pH, high levels of glutathione, and elevated amounts ofnzymes. Additionally, exogenous stimuli, such as heat, ultrasound,nd magnetic/electric fields, can be applied to externally acces-

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sible tumors to induce drug release from nanomaterials that areresponsive to specific energy sources. Clinical-stage examples ofstimuli-responsive therapeutics include Probodies, which are anti-bodies that are activated by tumor-associated proteases, such asurokinase-type plasminogen activator, membrane-type serine pro-tease 1, legumain, and various matrix metalloproteinases (MMPs)[104]. The antigen-binding site of Probodies is masked by a peptidethat is cleaved in the tumor microenvironment, thereby reduc-ing antibody binding to normal cells. For instance, CX-072 is aanti-programmed death-ligand 1 (PD-L1) Probody in phase I/II clin-ical trials for patients with solid tumors and lymphoma [105].This Probody has the potential to decrease the side effects ofanti-PD-L1 therapy, including the risk of autoimmune-like con-ditions [104,106]. Another example is CX-2009, an anti-CD166probody-maytansinoid DM4 conjugate that is undergoing phaseI/II clinical trials for solid tumors [107]. CD166 is an antibodythat is upregulated in tumors, while DM4 is a cytotoxic agent.Although Probodies and antibody-drug conjugates are multifunc-tional nanosized therapeutics, they are not generally categorizedas nanomedicine. Nevertheless, similarly to nanodrugs, Probod-ies have the potential to reduce side effects and enable the useof higher doses of therapeutic antibodies. A clinical stage exam-ple of a nanoparticle strategy that utilizes an external trigger fordrug release is ThermoDox, which is a thermosensitive liposomethat releases doxorubicin in response to temperature elevationsabove 39 ◦C [108]. In a Phase III trial of ThermoDox coupled withradiofrequency-induced heating for hepatocellular carcinoma, theprimary endpoint of progression-free survival was not reached[108]. Potential reasons for the failure of this trial include cancer cellresistance to doxorubicin, inadequate dose of doxorubicin, inap-propriate primary endpoints, and unexpected anticancer activityin the control group that received radiofrequency ablation (RFA)without ThermoDox [108]. Additionally, the lack of a standardizedprocedure for RFA is likely to have impacted the results of this study.In fact, retrospective analysis of patients that received RFA for atleast 45 min did show a substantial improvement in progression-free survival [108]. This study highlights the importance of selectingappropriate drug candidates, drug doses, diseases, and treatmentprocedures in order to gain a clear understanding of the benefits ofthe nanoscale component of the treatment strategy.

Exploiting electromagnetic properties

Inorganic nanoparticles can convert energy from externalsources into heat, which can be utilized for therapeutic purposes.A major advantage of these strategies is that the external energysource, such as near-infrared light, radiofrequency waves, and mag-netic fields, can usually be localized to a specific area in the body inorder to reduce damage to healthy tissues. In fact, nanoparticle-based thermal therapy can cause intratumoral temperatures torise above 70 ◦C [109], resulting in cancer cell death. Most of thetherapies that have shown promise in the preclinical and clinicalsetting are based on local injection of nanoparticles. However, it isimportant to develop systemically administered therapies, as themajority of cancer deaths are caused by metastatic lesions thatare impossible to individually target through local interventions.Studies have shown that high concentrations of nanoparticles arenecessary to achieve hyperthermia (> 42 ◦C) or thermal ablation (>50 ◦C) [110]. Therefore, heat-generation capacities and biodistribu-tion profiles should be optimized to potentially achieve therapeuticefficacy through systemic injections.

ari, Clinical cancer nanomedicine, Nano Today (2019),

The only example of a clinically approved treatment strategythat utilizes the unique electromagnetic properties of nanoparti-cles for thermal ablation is NanoTherm. NanoTherm is an iron oxidenanoparticle-based suspension, which is approved in Europe for the

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reatment of patients with brain tumors [111]. Iron oxide nanopar-icles are administered through local infusion into the brain, afterhich patients are exposed to an alternating magnetic field that

auses heat-induced tumor ablation. Studies have shown thathis treatment combined with radiotherapy has few side effectsnd extends overall survival compared to conventional treatmentptions [111]. In addition to the clinical utilization of magneticanoparticles, clinical trials are underway to assess the safety andfficacy of laser-heated gold nanoshells (AuroShell) in prostate can-er patients [112,113]. In the preclinical setting, near-infrared light114,115] and radiofrequency irradiation [116] have been appliedo heat gold and carbon nanoparticles for cancer therapy. Besidesumor ablation, less dramatic nanoparticle-induced temperaturelevations have been used to enhance intratumoral permeabilityor improved drug delivery [117]. However, the limited tissue pen-tration depth of near-infrared light brings into question the utilityf this strategy for clinical applications. Although radio wavesan easily penetrate through the body, there is controversy sur-ounding the contribution of gold nanoparticles to radiofrequencyeating. In fact, one study reported that electrolytes in nanoparticleuspensions are responsible for radio wave-induced temperaturelevations [118].

xploiting mass transport characteristics

The circulatory system is responsible for the transport ofutrients, oxygen, waste products, cells, and endocrine factorshroughout the body. The blood circulation is also exploited forhe delivery of intravenous or oral drugs, which account for theargest proportion of therapeutic agents. The characteristics of theascular system differ based on developmental stage, organ type,nd disease condition. Indeed, properties such as vessel diame-er, blood flow velocity, and vascular permeability vary dependingn the physiological function of the tissue. For example, the cap-

llaries in the lungs are narrower than those in other organs tonsure that red blood cells come in close contact with alveolarpaces, enabling efficient gas exchange [119]. Conventional drugsre often smaller than 1 nm and can efficiently diffuse throughoutost tissues in the body regardless of organ-specific vasculature

haracteristics. Although widespread diffusion permits exposuref diseased cells to drugs, dose limitations are necessary due toamage inflicted on healthy cells. Drug doses that are intended tovoid adverse side effects are in many cases insufficient to treatisease. Therefore, there is a need to develop strategies for site-pecific drug delivery. Compared to small molecules, nanoparticleransport in the body is more dependent on vasculature proper-ies. For instance, widespread diffusion of nanoparticles in tissueshroughout the body rarely takes place, as nanoparticles are tooarge to pass through the vascular wall. Moreover, studies havendicated that the cutoff size for renal clearance of rigid particless 5.5 nm [120]. A large portion of nanoparticles in the circula-ion are sequestered by the liver due to vascular fenestrationsnd resident macrophages that are responsible for the clearancef pathogens and cellular debris, which are often in the nanosizeange [121,122]. The dependence of nanoparticle transport on vas-ulature properties can be exploited for tissue-specific delivery tovoid complete liver clearance. Accordingly, the term transportncophysics was coined to denote a different way of viewing can-er, which enables the design of drug delivery strategies that takedvantage of the unique transport properties of tumors [123–125].he physics component indicates that physics is required to under-

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tand the movement of nanoparticles within and between variousiological compartments in the body.

The growth of a tumor beyond 2–3 mm in diameter requires theecruitment of a vasculature network to ensure efficient gas, nutri-

PRESSday xxx (2019) xxx–xxx 7

ent, and waste exchange [126]. The characteristics of tumor bloodvessels differ substantially from healthy vasculature as the neovas-cularization process (tumor-associated angiogenesis) is forced totake place in a short period of time to accommodate rapid tumorgrowth [127]. In particular, tumors often display a disorganizedvasculature network and individual blood vessels exhibit imma-ture properties, such as endothelial fenestrations. Drug deliverysystems can be designed to exploit the unique characteristics ofcancer blood vessels to increase drug accumulation in tumors. Themost well known phenomenon that relates to the utilization ofabnormal tumor vasculature properties for drug delivery purposesis the enhanced permeability and retention (EPR) effect. Anotheremerging drug delivery strategy that exploits differences betweennormal and tumor blood vessels is based on hemodynamics.

EPR effect

The EPR effect, which was first described in 1986 [128,129],entails the increased accumulation of nanosized matter in tumorsprimarily due to vascular fenestrations (up to 5̃00 nm [130]) andpoor lymphatic drainage. Specifically, immature vasculature hasreduced pericyte coverage and less endothelial tight junctions,causing gaps in the vessel wall. Additionally, a lack of functionallymphatic vessels, abnormal blood flow patterns, and a denseECM have been proposed as explanations for enhanced retentionof nanoparticles in the tumor microenvironment after extrava-sation [131,132]. In particular, it is likely that the characteristicintratumoral increase in ECM components [133] leads to adhesiveinteractions with nanoparticles. Fibronectin and collagen have bothbeen referred to as extracellular glue due to the capacity of theseproteins to bind a wide variety of molecules [134,135]. In fact, colla-gen is derived from the Greek word ‘kolla’, which means glue [135].In addition to the biomolecular adhesive properties of ECM compo-nents, it is probable that the structural organization of the proteinfiber network will entrap nanoparticles. Another reason for accu-mulation of nanoparticles in tumors is interactions with cells inthe tumor microenvironment. For instance, it has been shown thattumor-associated macrophages can promote intratumoral uptakeof nanotherapeutics [136]. The beneficial effects of many clinicallyapproved nanoparticles, especially liposomes [137] and polymericnanoparticles [138], have been attributed to the EPR effect. In thepreclinical setting, liposomal delivery has been found to lead toa several fold increase in intratumoral drug accumulation com-pared to administration of free drugs [139–141]. Although thereis evidence of the EPR effect in humans, considerable heterogene-ity exists in the prevalence of this phenomenon among individuals[142]. For example, the accumulation of liposomes in tumors var-ied from 0 to 3.5% of the injected dose in patients with head andneck, breast, and lung cancer [143]. Notably, two out of 17 [143]and two out of 22 [144] cancer patients exhibited undetectablelevels of intratumoral liposomes, suggesting that the EPR effectmay be entirely absent in certain cases. Moreover, there was aninverse correlation between tumor size and liposome accumula-tion/kg tumor tissue [143]. This observation may be due to largetumors having more necrotic areas that contribute to tumor sizebut lack functional vasculature. Indeed, in animal studies, largertumors have been associated with higher levels of necrosis andless liposome uptake normalized by tumor weight [145]. In anotherpatient study, tumor vasculature density, measured by CD31 stain-ing, was found to correlate with liposome accumulation [146]. Inaddition to liposomes, the EPR effect has also been observed inpatient studies with other types of nanoparticles. For instance,

ari, Clinical cancer nanomedicine, Nano Today (2019),

polymeric nanoparticles were shown to display increased depo-sition in tumors compared to adjacent non-cancerous tissues innine out of nine cancer patients [147]. Notably, preferential tumoraccumulation of nanoparticles has been observed for both pri-

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ary [143,144,146–148] and metastatic [144,149] cancer patients.nalysis of 17 clinical studies revealed that the EPR effect wasost pronounced in pancreatic, colon, breast, and stomach cancers,hich displayed more than ten times higher intratumoral nanopar-

icle levels compared to those in normal tissue [150]. In contrast,ead and neck cancer and melanoma patients displayed a twofold

ncrease in preferential tumor accumulation [150]. In addition tonterpatient variability in regards to the EPR effect, it is probablehat high levels of intratumoral heterogeneity exist. In fact, in onetudy, nanoparticle levels differed substantially (four-fold) in twoamples taken from different locations of a stomach tumor [151].athematical modeling [152] and experimental studies [79] have

emonstrated that such variations in spatial drug distribution canead to the formation of drug resistance, especially in cases in whichancer cells have limited ability to migrate. The amount of variationbserved in the distribution of nanoparticles in these small cohortsf cancer patients is a strong indicator that the EPR effect in humans

s more heterogeneous than what has been observed in the preclin-cal setting. In fact, the characteristics of tumors in animal modelsnd patients often differ substantially in regards to growth rate,ize relative to body mass, and features of the microenvironment,ll of which are likely to influence the neovascularization process,hich serves as the underlying cause of the EPR effect. In particular,

ancer cell injections in animals result in tumors that lack typicalumor-stroma interactions, which play a major role vascularizationnd nanodelivery. Taken together, the lack of animal models thatccurately recapitulate the EPR effect as well as inter-patient andntra-patient variability is likely to play a major role in the clinicalailure of several nanotherapeutics.

umor hemodynamics

The disorganized structure of tumor vasculature leads to bloodow abnormalities. The flow of fluid through a tube can bexpressed as the shear rate, which is calculated based on the tubeiameter and fluid velocity. In general, the shear rates that occur

n tumor blood vessels are lower (< 100 s−1) than those in normallood vasculature (> 100 s−1) [153,154]. Accordingly, a comple-entary strategy to utilizing the EPR effect for improved tumor

rug delivery is taking advantage of hemodynamics. Specifically,rug carriers can be designed to preferentially attach to the vas-ulature in blood flow conditions with low shear rates (Fig. 2).he two requisites for particle attachment is proximity to the ves-el wall during flow and sufficient adhesive interactions with thendothelium. The size and shape of drug carriers can be tailored toeet both of these criteria. For instance, discoidal microparticles

isplay a tendency to drift laterally against the wall, while spheri-al particles follow the streamline [155]. Additionally, disc-shapedicroparticles have a much larger parallel surface area that can

dhere to the vessel wall [153,156]. Therefore, it is not surpris-ng that platelets display a discoidal morphology as hemostasis isependent on binding of these cell fragments to the endothelium157]. Notably, although the vast majority of drug delivery systemsre spherical, discoidal particles appear to be superior in regards tottaching to the vasculature. Importantly, the diameter and heightf disc-shaped particles can be fine-tuned to obtain different vas-ulature binding patterns in normal and tumor tissues. In fact, inormal vasculature with higher shear rates, strong hydrodynamic

orces cause adhered particles to dislodge from the endothelium153,156]. On the contrary, reduced shear rates in tumor bloodessels enable permanent adhesion of microparticles to the vas-ulature. In essence, strong particle binding to the endothelium is

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romoted by low shear rates, while high shear rates lead to tran-ient particle adhesion. Hemodynamic targeting approaches forancer therapy rely on identifying optimal ratios between particledhesion and hydrodynamic forces. Mathematical modeling and

PRESSday xxx (2019) xxx–xxx

animal studies have demonstrated that discoidal microparticle-based drug delivery systems can be designed to preferentiallybind to tumor vasculature, resulting in increased tumor accumu-lation and improved therapeutic efficacy compared to sphericaldrug carriers [91,153,158,159]. For instance, in a mouse model ofbreast cancer, tumor accumulation of discoidal silicon micropar-ticles was fivefold higher than that of spherical particles with thesame diameter [158]. Similarly, in a mouse model of melanomalung metastasis, the accumulation of disc-shaped microparticles intumor tissue was ten times higher than that of spherical nanopar-ticles [159]. Although certain dimensions of these particles are inthe micron size range, some dimensions are nano-sized and theparticles have other nanotechnological features, such as nanoscalepores. Other non-spherically shaped particles, such as rods, havealso outperformed spheres in binding to the endothelium of tar-get organs [160]. An important consideration for the developmentof nanotehrapeutics with unconventional shapes is potential man-ufacturing challenges. While lithography-based techniques can beused for large-scale fabrication of silicon [161] and polymeric [162]particles of various shapes and sizes, this may prove less feasiblefor other materials, such as lipids.

Notably, patients have also been shown to display disorga-nized tumor vasculature characteristics [163]. In fact, an intravitalmicroscopy study demonstrated that vasculature shear rates ofhuman tumors are lower than those observed in animal models[163], highlighting the promising potential of hemodynamic tar-geting in the clinical setting. Clinical trials that utilize drug deliverystrategies based on fluid dynamics are currently being planned forthe treatment of breast cancer lung metastasis based on promisingpreclinical results [91,161].

Future directions

The use of nanocarriers has led to major improvements in site-specific drug delivery compared to administration of free drugs.These improvements range from a 10 to 100-fold increase inintratumoral drug accumulation [139,141,164,165]. Despite majoradvancements in the biodistribution of drugs, less than 1% of sys-temically injected nanoparticles typically reach the tumor [57].Over 90% of an injected nanoparticle dose usually ends up in theliver and spleen, as these organs are responsible for the clearanceof nanosized cell debris and pathogens, which are often indistin-guishable from nanotherapeutics [121,122]. Moreover, the reasonsfor failed clinical trials in nanomedicine are largely unknownand are likely to be a combination of multiple complex fac-tors [166]. Accordingly, nanomedicine still faces major challenge(Table 2) that necessitates the development of additional thera-peutic strategies to enhance tumor accumulation. Such approachesinclude engineered extracellular vesicles, drug delivery vehiclesthat undergo spatiotemporal transitions, strategies to prime themicroenvironment, and patient stratification according to tumorproperties.

Engineered extracellular vesicles

Extracellular vesicles are released by all cells and play a cen-tral role in cell communication by serving as endogenous carriersfor biomolecules over both short and long distances [167]. Geneticor chemical engineering approaches can transform extracellu-lar vesicles to drug delivery vehicles [168,169]. The complexityof natural vesicle membranes is challenging to replicate in the

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synthetic setting and provides unique functional properties. Forinstance, it was recently shown that extracellular vesicles carry-ing gene silencing agents against oncogenic KRAS display improvedtherapeutic efficacy in pancreatic cancer models compared to lipo-

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Fig. 2. Hemodynamic-based tumor targeting. Spherical nanoparticles follow the streamline (upper panel), while discoidal microparticles flow close to the vessel wall (lowerpanel). Proximity to the endothelium and a large parallel surface area that can attach to the vessel wall make disc-shaped particles ideal for vascular adhesion. Uniquehemodynamics in normal and tumor blood vessels lead to different patterns of particle adhesion. Specifically, the disorganized vasculature network of tumors leads to lowershear rates and permanent attachment of discoidal microparticles.

Table 2Limitations of nanotherapeutics.

Type Limitations

Clinically approvedNon-pegylated nanoparticles • Rapid clearance by the immune system

• Low tumor accumulation

Pegylated liposomes • Polyethylene glycol (PEG) dilemma (reduced interactions with cancer cells)• Transient protection from immunological clearance• Potential activation of the complement system• Accelerated blood clearance (ABC) phenomenon (antibody-mediated

clearance following repeated injections)

Iron oxide nanoparticles • Require local injection to achieve sufficient quantities for thermal ablation• Unknown effects of long-term accumulation in the body

Preclinical/Clinical trialsRNA interference nanoparticles • Complete protection required due to susceptibility of RNA to degradation

• Potential side effects• Endosomal escape strategies necessary• siRNA-based therapeutics are unlikely to be effective as monotherapy• Toxicity of cationic nanoparticles that result in higher loading efficiency

DNA, mRNA nanoparticles • Problematic nanoparticle encapsulation due to large size• Immune responses in intracellular compartments• Endosomal escape strategies necessary

Immunotherapeutic nanoparticles • Unspecific immune activation• Costly personalized approaches

Nanoparticles with permeation enhancers • Side effects• Risk of increased metastasis

Targeted nanoparticles • Masking of targeting ligands by the protein corona• Binding-site-barrier (ligands bind with high affinity to target molecules,

thereby preventing further diffusion throughout the tumor)• Decreased extravasation and intratumoral penetration due to increased size

(targeting ligands on the surface)• Potentially increased immunoactivation (targeting ligands on the surface)

Triggered-release strategies • Tumors need to be accessible if using external energy triggers• Rarely completely specific to tumor tissue if using triggers in the

microenvironment

Gold nanoparticles • Limited tissue penetration depth of infrared light• Unknown effects of long-term accumulation in the body

Non-spherical nanoparticles • Manufacturing challenges• Material limitations

Transitional/multistage nanoparticles • Complex manufacturing• Complex characterization• Regulatory challenges

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omes [170]. Notably, it was postulated that the presence of theransmembrane protein CD47 was a major contributing factor forncreased anticancer activity, as this protein reduces immuno-ogical clearance [170]. The use of extracellular vesicles as drugelivery vehicles is promising due to the abundance of surfaceroteins that can be exploited for tissue-specific targeting. How-ver, successful implementation of these biological nanoparticlesor drug delivery will be dependent on the identification andsolation of vesicles that display favorable biomolecular profiles.nother approach that also exploits biological membranes for drugelivery is the use of particles coated with cell membranes, includ-

ng those derived from leukocytes [51] and erythrocytes [52].n the contrary to engineered extracellular vesicles, membrane-oated particles are not confined to spherical shapes [51]. Theajor challenge for clinical translation of extracellular vesicles and

ther biological membrane-based therapeutics is the developmentf scalable cost-effective production methods [171,172]. Indeed,he most common method for the isolation of vesicles is ultra-entrifugation, which is expensive, labor-intensive, and difficulto scale-up for clinical-grade manufacturing [173]. Additionally,atch-to-batch consistency, purity, integrity, and recovery are lowith this isolation method, necessitating the use of alternative pro-

uction techniques for engineered extracellular vesicles. Despitehese challenges, extracellular vesicles for immunotherapy enteredlinical trials more then ten years ago. Specifically, in 2005, theesults from metastatic melanoma and non-small cell lung can-er phase I clinical trials were published [174,175]. These trialsevealed that tumor antigen-loaded exosomes derived from autol-gous dendritic cells were safe and in a few cases lead to a minor orartial therapeutic response. Accordingly, further research is nec-ssary to identify the most effective ways of utilizing extracellularesicles for immunotherapy. There are also several other promisinganotechnology-based strategies for inducing anticancer immuneesponses. These approaches are more than a decade away fromlinical translation and have been extensively reviewed elsewhere176–178].

patiotemporal nanoparticle transitions

In addition to the incorporation of multiple functional elements,rug delivery systems can be designed to display transitional prop-rties in a spatiotemporal manner. Namely, the characteristics ofanoparticles change with time as they move from one compart-ent in the body to another. In fact, nanoparticle properties that

re beneficial in some biological settings are unfavorable in others.or example, while pegylation provides an immunoevasive func-ion in the circulation, this surface modification reduces cellularptake in the tumor microenvironment. Multistage or transitionalrug delivery systems that display adjustable stability, size, andurface properties can be developed to overcome such challenges179]. The multistage vector is a drug delivery system that consistsf several components that are active at various stages in the drugelivery process [161,180]. The first stage is a silicon micropar-icle that is designed to navigate in the circulatory system andind to inflamed vasculature. The microparticle then releases aecond stage, which consists of nanoparticles that display optimalroperties for transport within the tumor interstitium. The finaltage comprises a therapeutic molecule that binds to an intracel-ular target. The multistage vector is a versatile platform that hasreviously been used for the delivery of chemotherapeutic agents91,181], anti-inflammatory drugs [182], gene-silencing agents183–185], and therapeutic proteins [186] encapsulated in vari-

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us nanocarriers. Recently, a new multistage platform based on annjectable nanoparticle generator was developed [91]. This genera-or is an intravenously administered discoidal silicon microparticlehat spontaneously produces and releases polymeric doxoru-

PRESSday xxx (2019) xxx–xxx

bicin nanoparticles into the tumor microenvironment. Specifically,nanoparticles are formed by self-assembly of polymeric doxoru-bicin strands loaded inside the microparticle. The nanoparticlesare then internalized by cancer cells through endocytosis, therebyovercoming drug efflux pumps [91]. Doxorubicin is released insideacidic endosomes following the cleavage of a pH-sensitive linker. Ina breast cancer lung metastasis model, the survival time doubledin mice administered with the nanoparticle generator comparedto those treated with a liposomal formulation containing the samedose of doxorubicin [91]. Other examples of multistage drug deliv-ery system are 80–100 nm nanoparticles that in response to tumorenzymes or acidity release smaller 5–10 nm nanoparticles that canmore easily diffuse throughout the tumor [187–189]. An alternativeapproach to the sequential release of nanoparticles is the designof multilayer nanodrugs. For instance, PEG layers can be shed inresponse to enzymes in the tumor microenvironment [190]. Addi-tionally, the acidic environment of the tumor can be exploited toinduce surface modifications that expose cell-penetrating [191]and nucleus-targeting peptides [192]. In conclusion, nanopropertychanges are necessary to ensure beneficial bio-nano interactions indifferent biological compartments. Although spatiotemporal tran-sitions substantially improve drug delivery, it is important to notethat complex nanotherapeutics face greater manufacturing andregulatory challenges. Nevertheless, large-scale production of theinjectable nanoparticle generator based on photolithography andelectrochemical etching according to the Current Good Manufac-turing Practices (CGMPs) enforced by the United States FDA hasbeen established [161]. Toxicity studies based on CGMP qualityparticles have been performed and an investigational new drugapplication for breast cancer lung metastases will be submittedwithin the next 12 months. Clinical approval of a wide range of mul-tifunctional and transitional nanoparticles will require retraining ofthe pharmaceutical industry to perform cost-effective manufactur-ing of nanoparticles.

Priming the microenvironment

A complementary approach to developing innovative nanopar-ticles for treatment of disease is the design of strategies aimed attargeting the microenvironment. Such approaches have generallyfocused on modifying tumor characteristics, such as the vascula-ture and ECM, for improved nanodelivery [193]. Indeed, there areseveral barriers in the tumor microenvironment that prevent effec-tive nanomedicine delivery. These obstacles include uneven bloodflow distribution, interstitial fluid pressure, a dense ECM, and alarge population of stromal cells [93]. In animal models, antiangio-genic and angiogenic agents have been administered to normalizecancer vasculature for improved nanoparticle accumulation [194].Other studies have utilized hyperthermia to enhance nanoparti-cle permeability across the tumor endothelium [117]. Furthermore,degradation of modification of the ECM can decompress cancer vas-culature and improve intratumoral diffusion of nanoparticles [93].The tumor microenvironment can also be restructured through theinactivation or destruction of cancer-associated fibroblasts [93]. Inthe clinical setting, angiotensin inhibitors, which alter the vascu-lature and ECM of tumors [195], have been explored for improveddelivery of chemotherapeutic agents. The findings from such clin-ical trials are inconclusive as retrospective studies have indicatedpromising results [196,197], while other trials have failed to showany improvement [198]. It is worth noting that the effect of tumormicroenvironment alterations on nanoparticle permeation maydiffer substantially from that of small molecules, as nanodrugs

ari, Clinical cancer nanomedicine, Nano Today (2019),

display unique transport characteristics. Moreover, a potentialdrawback of increased vascular permeability and reduced ECMthickness is the promotion of cancer cell invasiveness. In addition tomodifying the tumor microenvironment for improved drug deliv-

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ry, healthy organs such as the liver and spleen can be targetedo alter nanoparticle biodistribution [199]. For example, pretreat-

ent of mice with gadolinium chloride was found to deactivateupffer cells in the liver, leading to increased accumulation ofuantum dots in tumors [200]. Saturation of the innate immuneystem with decoy liposomes prior to injection of therapeuticron oxide nanoparticles also demonstrated promise for improvingumor homing [201]. Additionally, preconditioning of the mononu-lear phagocyte system with the clinically approved antimalarialgent chloroquine was shown to reduce nanoparticle uptake in theiver and improve intratumoral accumulation in a mouse model202]. This pretreatment strategy is a simple, broadly applicable,nd easily implementable approach to potentially increase site-pecific nanodelivery in the clinical setting [203]. Based on commoniodsitribution trends [121,122], a 1% redirection of nanoparti-les from the mononuclear phagocyte system to the tumor woulde sufficient to double intratumoral drug levels. Assuming thathere is a correlation between tumor drug concentration and anti-ancer efficacy, a minor redistribution of nanoparticles could be aetermining factor between life and death. Namely, small changes

n liver and spleen accumulation could have major implicationsor therapeutic efficacy. The clinical translation of strategies to

odify the mononuclear phagocyte system for improved tumorelivery of nanoparticles should be easily implementable in cer-ain cases, as agents such as chloroquine that deactivate the innatemmune system are already on the market and have shown accept-ble safety profiles when combined with chemotherapeutic agents204]. However, such strategies could also impact tumor-associated

acrophages, which could have favorable or unfavorable effectsepending on the tumor type [202]. Notably, the translation ofherapeutic strategies to modify the tumor microenvironment hasimited relevance unless animal models resemble clinical patho-iology. Accordingly, the suitability of tumor models should bessessed by comparing gene expression, histological features, andeterogeneity to biobanks of patient tumors [166]. In general,enetically engineered and patient-derived xenograft models tendo most accurately recapitulate the tumor microenvironment ofatients [166].

ompanion diagnostics

Another approach to improve the performance of nanothera-eutics is to stratify patients according to tumor characteristics,uch as vasculature density, vessel permeability, blood flow veloc-ty, pressure gradients, and ECM density. Methods for patienttratification can be based on biomarker profiles or imaging. Fornstance, in animal models, it was shown that the ratio of MMP-

to tissue inhibitor of metalloproteinase 1 (TIMP-1) can serve as serum biomarker of nanoparticle accumulation in tumors [205].oreover, the content of capillary-wall collagen has been shown to

e a biophysical marker of nanoparticle extravasation from tumorasculature [206]. Additionally, several angiogenesis biomarkersave been reported, including circulating cells, proteins, genexpression profiles, and functional parameters [207]. For instance,igher levels of circulating endothelial cells and endothelial cellrogenitors are indicative of increased angiogenesis [208]. In thelinical setting, it has been demonstrated that imaging techniquesan be used to predict intratumoral nanoparticle accumulation andherapeutic efficacy. For example, the uptake of iron nanoparticlesn patients with advanced solid tumors was quantified by magneticesonance imaging prior to treatment with Onivyde (irinotecaniposomes) [209]. Notably, intratumoral levels of iron nanopar-

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icles were predictive of therapeutic responses to the liposomes209]. Additionally, in metastatic breast cancer patients treatedith radiolabeled doxorubicin-containing liposomes, tumor accu-ulation quantified by positron emission tomography was shown

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to correlate with treatment outcome [210]. This example highlightsthe value of theranostic nanoparticles that provide informationabout the success of drug delivery in addition to having thera-peutic effects. Taken together, companion diagnostics present apromising tool for identifying patients that are likely to respondto nanotherapeutics. Future applications also include further pre-selection of patients according to the most suitable nanoparticleproperties, such as size, shape, and charge. In order for compan-ion diagnostics to achieve clinical relevance in nanomedicine, adecision-making framework needs to be created. The generation ofsuch a framework requires improved tools and databases to assesstumor heterogeneity, especially in regards to parameters that dic-tate nanoparticle delivery to tumors. Moreover, the focus should beshifted from formulation-driven development of nanotherapeuticsto design criteria that are based on treating specific tumor types[166].

Conclusions

The reasons for the failures of clinical trials to reach proposedobjectives are invariably multi-fold, complex, and interwoven witheach other. Unfortunately, it is the norm rather than the exceptionthat therapeutic agents (chemo-, biological, or nanotechnological)prove to be very effective and cell-selective in targeting certain can-cer cell populations in vitro, and even in suitable animal models, yetfail dramatically in clinical trials. It stands to reason that the biodis-tribution of the agent may be a fundamental factor for these failures,with insufficient concentrations being realized at target sites, andunwanted concentrations elsewhere causing dose-limiting toxici-ties. The biodistribution of therapeutic agents is largely controlledby the ability of the drug to penetrate across biological barriers,especially in the evolving forms they present in the course ofcarcinogenesis. The strategy of adding targeting moieties to thera-peutic nanoparticles to increase their localization specificity hasnot yielded clinically approved drugs, to date, despite 30 yearsof attempts by many laboratories and pharmaceutical companies.This pitfall is related to that the fact that the addition of molec-ular targeting agents increases recognition specificity, but at thecost of much greater difficulties in addressing biological barriers.This review highlights critical obstacles in nanomedicine and thenecessity to develop strategies for addressing them in a sequentialfashion for improved outcomes in clinical trials. The various bene-fits and characteristics of nanoparticles summarized in this reviewform the knowledge basis from which further strategies can bedeployed to improve cancer therapeutics. Specifically, the majoradvantages of nanotherapeutics are multifunctionality, uniqueelectromagnetic characteristics, and distinct transport properties.Although many of these benefits have been individually imple-mented in the clinical setting, successful future nanotherapeuticswill likely integrate a wider range of valuable nanoscale character-istics combined with strategies to modify the microenvironment.In regards to clinical oncology, nanomedicine has the potential tosubstantially improve the treatment of aggressive diseases, suchas triple negative breast cancer and pancreatic cancer. Specifically,the microenvironment of pancreatic tumors poses unique chal-lenges due to poor vascularization and dense stroma, requiringa multipronged approach to treatment. In the case of metastaticbreast cancer, nanoparticle-based treatments show promise forovercoming drug resistance and achieving site-specific deliverythrough approaches such as hemodynamic targeting. Althoughnanomedicine offers new and improved solutions for the treatment

ari, Clinical cancer nanomedicine, Nano Today (2019),

of cancer, there is a need for an improved understanding of bio-nano interactions in the body, as this interface is a determiningfactor of the success of nanotherapeutics. Accordingly, systematicstudies that evaluate the impact of nanoparticle characteristics

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n biomolecular interactions and pharmacokinetics will be criticalor the design of superior treatments. Furthermore, the commer-ialization process for nanotherapeutics is challenging due to aack of industry experience in large-scale clinical-grade manufac-uring. Indeed, pharmaceutical production facilities are primarilypecialized in small molecule and antibody production. In conclu-ion, an increase in industry knowledge of scalable nanoparticleynthesis coupled with the design of transitional multifunctionalanoparticles, microenvironmental priming strategies, and com-anion diagnostics is likely to radically transform cancer treatment.

eclaration of interest

None.

cknowledgements

Mauro Ferrari is the Ernest Cockrell Jr. Presidential Distin-uished Chair at the Houston Methodist Research Institute. Thisork was supported by the National Cancer Institute Physical

ciences-Oncology Network of the National Institutes of Health,nder award number U54CA210181. The content is solely theesponsibility of the authors and does not necessarily representhe official views of the National Institutes of Health. This work waslso supported by the Office of the Assistant Secretary of Defenseor Health Affairs, through the Breast Cancer Research Program,nder award number W81XWH-17-1-0389. Opinions, interpreta-ions, conclusions, and recommendations are those of the authorsnd are not necessarily endorsed by the Department of Defense.dditionally, this work was supported by intramural funding fromayo Clinic. The authors thank Dr. Piotr Godzinski, Director of theffice of Cancer Nanotechnology Research at the National Can-er Institute, for reviewing the manuscript and providing valuableomments.

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