Role of Nanoparticle Mechanical Properties inCancer Drug DeliveryYue Hui,† Xin Yi,‡ Fei Hou,† David Wibowo,† Fan Zhang,§ Dongyuan Zhao,*,§ Huajian Gao,*,∥

and Chun-Xia Zhao*,†

†Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, St. Lucia, QLD 4072, Australia‡Department of Mechanics and Engineering Science, Beijing Innovation Center for Engineering Science and Advanced Technology,College of Engineering, Peking University, Beijing 100871, China§Department of Chemistry, Shanghai Key Laboratory of Molecular Catalysis and Innovative Materials, State Key Laboratory ofMolecular Engineering of Polymers, Laboratory of Advanced Materials, Fudan University, Shanghai 200433, China∥School of Engineering, Brown University, Providence, Rhode Island 02912, United States

ABSTRACT: The physicochemical properties of nanoparticlesplay critical roles in regulating nano-bio interactions. Whereas theeffects of the size, shape, and surface charge of nanoparticles ontheir biological performances have been extensively investigated,the roles of nanoparticle mechanical properties in drug delivery,which have only been recognized recently, remain the leastexplored. This review article provides an overview of the impactsof nanoparticle mechanical properties on cancer drug delivery,including (1) basic terminologies of the mechanical properties ofnanoparticles and techniques for characterizing these properties;(2) current methods for fabricating nanoparticles with tunablemechanical properties; (3) in vitro and in vivo studies that highlightkey biological performances of stiff and soft nanoparticles, including blood circulation, tumor or tissue targeting, tumorpenetration, and cancer cell internalization, with a special emphasis on the underlying mechanisms that control thosecomplicated nano-bio interactions at the cellular, tissue, and organ levels. The interesting research and findings discussedin this review article will offer the research community a better understanding of how this research field evolved duringthe past years and provide some general guidance on how to design and explore the effects of nanoparticle mechanicalproperties on nano-bio interactions. These fundamental understandings, will in turn, improve our ability to design betternanoparticles for enhanced drug delivery.KEYWORDS: nanoparticle, nanocapsule, mechanical property, elasticity, stiffness, Young’s modulus, drug delivery, blood circulation,tumor penetration, cellular uptake

Advances in nanotechnology have led to rapid develop-ment of the synthesis, characterization, and applica-tions of nanoparticles (NPs) in cancer treatment.

Various NP-based drug delivery systems have been designed todeliver therapeutic agents specifically to solid tumors toenhance the efficacy of anticancer treatment while minimizingsystemic toxicity.1 The enhanced permeability and retention(EPR) effect has long been considered as the key mechanismto facilitate the preferential accumulation of NPs in tumortissues compared to normal tissues.2,3 However, recent studieshave raised questions about the actual benefits of thisbiological phenomenon. The systemic transport of NPs tosolid tumors following intravenous (i.v.) administration isnever trivial, but extremely complicated, involving several keybiological barriers at all levels from organs and tissues to cells.In general, NPs encounter five key biological barriers from theinjection site to the site of action, including (1) adsorption of

serum opsonin proteins onto the surface of NPs,4 (2)interactions between NPs and the immune system,5 (3)selective extravasation of NPs at tumor sites,2 (4) penetrationof NPs into solid tumor, and (5) internalization of NPs bytumor cells. The eradication of tumor cells requires NPs toovercome the dense tumor extracellular matrix and highinterstitial fluid pressure in the tumor tissue6 as well as toefficiently enter cancer cells.7 Due to the overwhelmingcomplexity of these processes, cancer drug delivery oftenexhibits suboptimal therapeutic benefits, with only 0.7%(median) of the administered NPs reported to reach tumorsites.1,8

Received: May 21, 2019Accepted: July 9, 2019Published: July 9, 2019

Review

www.acsnano.orgCite This: ACS Nano 2019, 13, 7410−7424

© 2019 American Chemical Society 7410 DOI: 10.1021/acsnano.9b03924ACS Nano 2019, 13, 7410−7424

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NP size,9−11 shape,12−14 surface chemistry, and otherphysicochemical properties14,15 have been extensively studied,and their effects on cancer drug delivery have beensystematically investigated. However, the mechanical propertyof NPs has only recently been recognized to play an importantrole in regulating their biological performances. This is largelyinspired by the fact that many cells or even viruses canmodulate their mechanical properties to achieve certainbiological functions.16,17 Despite the growing evidence high-lighting its pivotal role in biology, a consensus about thecorrelation between the mechanical property of NPs and theirbiological fates is still lacking, as manifested by the conflictingviews among different studies. This review focuses on the roleof NP mechanical properties in cancer drug delivery. Basicterminologies of NP mechanical properties, including stiffnessand elasticity, are defined. The commonly used methods forquantifying NP mechanical properties are described, followedby a discussion of the approaches for making NPs with tunablemechanical properties with identical size, shape, and surfaceproperties. Furthermore, the effects of NP mechanicalproperties on several key biological processes involved insystemic drug delivery are highlighted, including bloodcirculation, tumor accumulation/penetration, and cancer cellinternalization, with a particular emphasis on the mechanismsunderlying the distinct biological performances of soft and stiffNPs using experimental and computational approaches. As wefocus on the mechanical properties of NPs and their role inregulating their biological performance, studies of micron-sizedparticles with tunable mechanical properties18−24 will not bediscussed here.

BASIC TERMS AND DEFINITIONS OF MECHANICALPROPERTIES

Mechanical properties describe the behavior of a materialunder loads. Several key mechanical properties includeelasticity, stiffness, rigidity, hardness, and strength, whichhave different definitions and different units. Among thesemechanical terms, elasticity and stiffness have been widely usedin the literature to explore the mechanical properties of NPs(Table 1). In physics, elasticity is defined as the ability of amaterial to resist deformation induced by stress and to returnto its original state when that stress is removed. The elasticityof a material is normally presented as its elastic moduli (N/m2

or Pa), including Young’s modulus and shear modulus forlinear elastic isotropic materials (Figure 1a). Young’s modulus(EY) is defined as the ratio between the uniaxial tensile/compressive stress applied on a material and its correspondingstrain along the direction of the stress. Shear modulus (G), on

the other hand, is the ratio of shear stress to the correspondingshear strain of a material. Another essential mechanicalproperty term, stiffness (N/m), is often used interchangeablywith rigidity to characterize the extent to which a body resistsdeformation upon the application of loadings. Unlike elasticmoduli, which are intrinsic factors determined only by thematerial itself, the stiffness of an object is extensive which isrelated not only to its elastic moduli but also to its dimension,shape, and the directionality of applied forces. In general, thestiffness of an object can be described using Hooke’s law, inwhich the stiffness (also called spring constant) of a spring(Figure 1b) is the ratio of the compressive/tensile forceapplied on it to its compression/extension. Similarly, the

Table 1. Elasticity and Stiffness of a Nanoparticle

mechanicalproperty definition expression unit feature

elasticity the ability of an object to resist deformation caused bystress and to return to original state when the stressis removed

Young’s modulus: The ratio betweenthe uniaxial tensile/compressivestress and strain

Pascal(Pa or N/m2)

•an intrinsic property of thematerial

•independent of geometry•elastic limit existsa

stiffness the extent to which a body resist deformation causedby applied forces

the ratio between applied force and thecorresponding deformation

Newton permeter (N/m)

•determined jointly by theelastic modulus and geometryof an object

•readily derived from theindentation force−displacement curve

aElastic limit is the maximum stress that can arise in a material before the onset of permanent deformation.

Figure 1. Schematic illustrations of basic mechanical propertyterms and their definitions. (a) Elasticity and elastic moduli. (b)Spring constant and stiffness. (c) Membrane bending rigidity.

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compressive/tensile stiffness of an object measures its ability toresist deformation induced by compressive and tensile forces,whereas the bending stiffness and torsional stiffness (N·m/rad)are the abilities to resist bending or torsional deformationcaused by bending forces or torques (Figure 1b). It should bementioned that, even for the same object, its compressive/tensile stiffness, bending stiffness, and torsional stiffness can bevery distinct. For example, it is normally easier to bend a beamthan to lengthen it. Because the most common type of forceapplied on NPs is compression, Young’s modulus andcompressive stiffness (simply referred to stiffness) are themost relevant characteristics within the context of this reviewarticle. However, as the stiffness of NPs is also affected by theirstructures and geometries, using Young’s modulus allows amore straightforward comparison between the mechanicalproperties of different materials. In this review, the effects ofNP mechanical properties on biological performances will besummarized based on Young’s modulus (elasticity).Atomic force microscopy (AFM) has been the most popular

technique for characterizing the mechanical properties of NPs.AFM uses a nanosized probe to scan a sample surface anddetermine its surface topology. To measure the overallmechanical properties of a NP, a typical way is to exert acompressive force on the NP to cause deformation. Then, boththe applied force and the deformation of the NP are recordedin the form of an indentation force−displacement curve.According to Hooke’s law, the stiffness of a sample is equal tothe slope of the linear part of the curve, whereas the calculationof Young’s modulus involves fitting the force−displacementcurve using certain contact models, such as the Hertz modeland Sneddon model.25−29 The classical Hertz model, which isonly valid at the indentation depth much smaller than theindenter tip radius, is generally not applicable for soft materialssuch as biological tissues and liposomes. To achieve a moreappropriate estimation of the Young’s modulus of softmaterials undergoing finite deformation, the Sneddon theoryis usually adopted.29 This indentation curve method is onlyable to provide one data point for a single indentationexperiment and is very time-consuming to obtain high-resolution Young’s modulus maps. A PeakForce quantitativenanomechanical mapping tapping-mode AFM has beendeveloped recently,30,31 which allows the acquisition ofmechanical information of samples while simultaneouslyimaging the topography at a high resolution and can operateas fast as the regular tapping-mode AFM.31 Due to its highperformance and easy operation, the PeakForce quantitativenanomechanical mapping mode has been used in severalstudies to measure the mechanical properties of NPs.32−34

Another method for quantifying NP mechanical properties isto measure bulk materials at the macroscopic scale rather thanat the nanometer scale.35−37 This approach has only beenapplied to hydrogel NPs because bulk hydrogel materials canbe easily synthesized to have identical chemical composition,thus the same Young’s modulus as the corresponding hydrogelNPs. Measurement of the mechanical properties of bulkhydrogel materials can be conducted by applying macroscopicforces on specimens and analyzing their force−deformationprofiles. For example, a rheometer has been widely used tomeasure the shear modulus of bulk hydrogels,37 which canthen be converted into Young’s modulus using Poisson’s ratios.It is worth noting that the Young’s modulus is a parameter ofhomogeneous and isotropic materials such as the aforemen-tioned hydrogel materials. Therefore, the measured Young’s

moduli of composite NPs (e.g., core−shell NPs) are actuallymore of effective moduli considering the NPs as uniformmaterials.In addition to the mechanical properties of NPs, the bending

rigidity of biological membranes also plays a critical role indictating how cells interact with NPs of different mechanicalproperties. Membrane bending rigidity κ (N·m) measures themechanical resistance of a membrane to loadings (normallybending moments) (Figure 1c). It has become an importantparameter in computational simulations to mechanisticallyunderstand the membrane deformation and wrapping of stiffand soft NPs.38−40 Methods for measuring the bending rigidityof biological membranes include fluctuation spectroscopy41

and micropipette aspiration.42

APPROACHES FOR SYNTHESIZING NANOPARTICLESWITH CONTROLLABLE MECHANICAL PROPERTIESTo investigate the effects of NP mechanical properties oncancer drug delivery, a key prerequisite is the synthesis of NPshaving distinct mechanical properties but identical in otherphysicochemical properties (e.g., size, shape, and surfacechemistry). In this section, current approaches for synthesizingNPs with tunable mechanical properties are introduced,covering hydrogel NPs, hybrid polymer−lipid NPs, and silicananocapsules that have been recently examined in depthtoward the fundamental understanding of the impacts of NPmechanical properties on drug delivery applications (Table 2).

Hydrogel Nanoparticles. A hydrogel is a cross-linkednetwork of hydrophilic polymeric chains surrounded by awater-rich environment.49 Hydrogels can be made intocolloidal NPs, and their mechanical properties can be tunedby adjusting the degree of cross-linking, hence water content,through changing the amount of cross-linking agents duringthe hydrogel NP formation (Figure 2a, left). For example, byincreasing the cross-linker content from 1.7 to 15 mol %, N,N-diethyl acrylamide and 2-hydroxyethyl methacrylate hydrogelNPs were fabricated having Young’s moduli ranging from 18 to211 kPa but similar sizes and zeta-potentials.43 An alternativeway to tune the mechanical properties of hydrogel NPs is bycontrolling the concentration of monomers during thesynthesis (Figure 2a, right). Polyethylene glycol diacrylatehydrogel NPs were synthesized having elastic moduli of 10 and3000 kPa using 10 and 40 vol % of monomers, respectively.37

Other than the above two methods, a lyophilization methodhas also been developed to tune the elasticity of hydrogel NPs.Polymer micelles formed by the self-assembly of diblockcopolymers of poly(ethylene glycol)−poly(lactide) (PEG−PLA) in aqueous solutions exhibited a lower Young’s modulus(165 kPa) owing to the higher water content, whereas anadditional lyophilization step dehydrated the micelle, resultingin an increased Young’s modulus (260 kPa).44 More studiesdescribing the fabrication of hydrogel NPs with tunablemechanical properties are listed in Table 2.It should be pointed out that despite the easy control over

their mechanical properties, hydrogel NPs only exhibit Young’smoduli ranging from dozens of kPa to a few MPa due to theirintrinsic water-rich structures (Figure 3). Therefore, even attheir “stiff” state, hydrogel NPs are softer than othermechanically stiff materials such as polymeric and silica NPs.

Hybrid Polymer−Lipid Nanoparticles. Hybrid poly-mer−lipid NPs represent a family of materials having acore−shell structure. In general, polymer−lipid NPs arecomposed of a polymeric NP core and a lipid monolayer or

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Table

2.RepresentativeExamples

ofNanop

articles

withVarying

MechanicalPropertiesandtheEffects

ofMechanicalPropertieson

TheirBiologicalPerform

ances

materials

size

(nm)

elastic

modulus

effectson

biologicalperformances

ref

hydrogelnanoparticles(N

Ps)

N,N-diethylacrylamide;

2-hydroxyethyl

methacrylate

∼160

18−211kPa

•softandstiff

NPs

areinternalized

byRAW264.7macrophagecells

mainlythrough

macropinocytosisandclathrin-m

ediatedroutes,respectively

43

•NPs

with

medium

elasticity

areinternalized

viamultip

lemechanism

s,which

leadsto

ahigher

cellularuptake

rate

comparedto

softandstiff

NPs

poly(ethyleneglycol)diacrylate

∼210

10−3000

kPaa

•softNPs

displaylonger

invivo

bloodcirculationandenhanced

tissuetargetingcompared

tostiff

NPs

37

•softNPs

exhibitlower

invitrouptake

inimmunecells

(J774macrophage),endothelial

cells

(bEn

d.3),and

cancer

cells

(4T1)

comparedto

stiff

NPs

poly(2-hydroxyethylm

ethacrylate)

∼1000

15−156kPa

•softparticlesshow

faster

andhigher

uptake

byHepG2cells

comparedto

stiff

particles

35poly(carboxybetaine)

∼120

180−

1350

kPa

•softerNPs

show

longer

bloodcirculationhalf-lives

than

theirstiffer

counterparts

36•stifferNPs

displayhigher

splenicaccumulationthan

softerNPs

methoxypoly(ethyleneglycol)−

poly(lactid

e)sm

all:17

(soft),1

8(stiff)

soft:

165kPa

•stiffNPs

displayenhanced

melanom

aA375cellularuptake

andtumor

spheroid

penetrationcomparedto

softNPs

44

medium:34

(soft)

51(stiff)

stiff:260kPa

•the

elasticity-dependent

uptake

effect

ismoresignificant

atlarger

NPsizes,indicatin

ga

couplingeffect

betweenNPstiffness

andsize

inregulatin

gcellularuptake

large:

68(soft)

86(stiff)

hybrid

polymer−lipid

NPs

PLGAb−lipid

monolayer

∼100

noquantitativedata;thelipid

bilayer

NPisstiffer

than

thelipid

monolayer

NP

•stiffNPs

exhibitgreaterin

vitrocellularuptake

andmoresignificant

invivo

anticancer

effect

than

softNPs

34PL

GAb−lipid

bilayer

alginate−lipid

bilayer

∼160

45−19000kPa

•softNPs

exhibithigher

cellularuptake

than

stiff

NPs

byboth

neoplasticandnon-

neoplasticcells

45

•softNPs

accumulatemorethan

stiff

NPs

intheorthotopicbreasttumor

modelin

vivo

PLGAb−lipid

bilayer

∼200

5−110MPa

•sem

ielasticNPs

(50MPa)exhibitsuperior

mucosalandtumor-penetratin

gcapability

comparedto

softandstiff

NPs

32

•orally

administeredsemielasticNPs

efficiently

overcomemultip

leintestinalbarriers,

enhancingthebioavailabilityof

doxorubicin

PLGAb−lipid

bilayer

∼40

soft:

0.76

GPa

•stiffNPs

displayhigher

uptake

than

thesoftNPs

byHeLacells

33PL

GAb−water−lipid

bilayer

stiff:1.20

GPa

•bothsoftandstiff

NPs

areinternalized

throughclathrin-m

ediatedendocytosis

tumor-cell-d

erived

lipid

bilayerNPs

∼500

soft:

1kPa

•doxorubicin-lo

aded

softNPs

displaysignificantlyhigher

antitum

oreffect

than

theirstiff

counterpartsin

micebearingH22

tumorsandB16-F10

melanom

acells

46

stiff:3kPa

•com

paredto

stiff

NPs,softNPs

show

enhanced

tumor

accumulation,

bloodvessel

crossing,p

enetratio

ninto

tumor

parenchyma,andpreferentialuptake

bytumor-

repopulatin

gcells

silicananocapsules

(SNCs)

thioether-bridgedsilicaNPs

orNCsc

∼290

stiff:233MPa

•softSN

Csshow

a26-fo

ldhigher

uptake

byMCF-7cells

than

stiff

silicaNPs

47soft:

48MPa

benzene-bridgedsilicaNPs

orNCsc

∼235

stiff:351MPa

soft:

91MPa

•softdrug-lo

aded

SNCsdisplaymoresignificant

killing

effectsforMCF-7cells

than

the

stiff

silicaNPs

ethane-bridged

silicaNPs

orNCsc

∼280

stiff:251MPa

soft:

4MPa

SNCswith

varyingshellthickness,prepared

usingtetraethylorthosilicate

ortriethoxyvinylsilane

∼200

704kPato

9.7GPa

•eith

ernakedor

PEGylated

stiff

SNCsshow

higher

macrophageuptake

than

theirsoft

counterparts

48

•PEG

ylated

stiff

andsoftSN

Csexhibitsimilarcancer

cellularuptake

•folicacid/P

EG-m

odified

stiff

SNCsshow

significantlyhigher

receptor-m

ediatedcellular

uptake

than

thesoftSN

Cs

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bilayer as the shell, and their mechanical properties cannormally be tuned in two ways. One is to change the cross-linking extent of the inner polymeric core (Figure 2b, left), andthe other is to vary the amount of water between the polymericcore and the lipid shell (Figure 2b, right). By controlling thecross-linking degree of alginate hydrogel NPs covered by alipid bilayer, polymer−lipid NPs having Young’s moduliranging from 1.6 to 19 MPa were made.45 Alternatively,increasing the water content between the polymeric core andthe lipid shell decreases the Young’s moduli of the NPs. Twotypes of hybrid NPs with different amounts of water wereprepared using a microfluidic device, that is, poly(lactic-co-glycolic acid) (PLGA)−lipid NPs (less water content) andPLGA−water−lipid NPs (more water content), whichdisplayed Young’s moduli of 1.20 and 0.76 GPa, respectively.33

The same strategy was also adopted32 to synthesize PLGA−water−lipid NPs with variable mechanical properties. As theirT

able

2.continued

materials

size

(nm)

elastic

modulus

effectson

biologicalperformances

ref

silicananocapsules

(SNCs)

•SoftSN

Csshow

superior

tumor

penetratingability

than

thestiff

SNCs;

•StiffSN

Csdisplayhigher

splenicremovalin

vivo,therefore

inferior

tumor

targeting

efficiencythan

softSN

Cs.

aThe

elastic

modulus

istheplateaushearmodulus

(Gp)

measuredby

arheometer.bPo

ly(lactic-co-glycolicacid).c Silica

nanocapsules

areselectivelyetched,h

ollowsilicananoparticles.

Figure 2. Strategies for tuning the mechanical properties ofnanoparticles. (a) Controlling the mechanical properties ofhydrogel nanoparticles by changing the concentration of cross-linking agent (left) or monomer (right), thus the cross-linkingdensity of the nanoparticle. (b) Controlling the mechanicalproperties of hybrid polymer−lipid nanoparticles by changingthe cross-linking degree of the inner polymeric core (left) or byaltering the thickness of the water layer (right) between thepolymeric core and the lipid shell. (c) Controlling the mechanicalproperties of silica nanocapsules by using different types of silicaprecursors (organosilane for soft nanocapsules and silicon alkoxidefor stiff nanocapsules) (left) or by varying the thickness of thesilica shell (right).

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water content decreased, the Young’s moduli of the NPsincreased from 5 to 110 MPa. Recently, a biosynthesis methodhas also been developed to make two types of NPs (around500 nm) having different mechanical properties. These lipid-shell (cell membrane) cytosol-core NPs with Young’s moduliof around 1 and 3 kPa were produced using cells cultured insoft gels and on rigid plastics, respectively, as a result of thedifferent expression levels of a cytoskeleton-related protein:cytospin-A.46

Compared to the lipid shell, the polymeric core mainlycontributes to the overall mechanical properties of a hybridpolymer−lipid NP. For example, by excluding the polymericcore from the hybrid NPs to form hollow lipid shells(liposomes), the Young’s modulus decreased significantlyfrom 19 MPa to 45 kPa.45 Polymer−lipid NPs can be verysoft with Young’s moduli as low as 1.6 MPa when hydrogelNPs are encapsulated,45 whereas they can also be very stiff(e.g., 1.2 GPa) when containing dense polymeric NPs.33

However, it remains challenging to produce polymer−lipidNPs with identical size, shape, and surface chemistry but variedmechanical properties covering a wide range of Young’s modulifrom kPa to GPa (Figure 3).Silica Nanocapsules. Silica nanocapsules (SNCs) have

recently emerged as an attractive NP system having control-lable mechanical properties. A SNC is a nanosized core−shellstructure with a core region surrounded by a silica shell.50

When silicon alkoxides (e.g., tetraethyl orthosilicate (TEOS)and tetramethyl orthosilicate (TMOS)) are used as silicaprecursors, the resultant silica shells are very rigid. However, byusing organosilanes containing organic groups (e.g., vinyl,thioether, benzene, ethane, and methyl), soft and deformablesilica shells can be prepared as a result of the low cross-linkingdensity (Figure 2c, left).51−53

Thioether-bridged, benzene-bridged, and ethane-bridgedmesoporous organosilica nanocapsules were synthesizedusing bis[3-(triethoxysilyl)propyl]tetrasulfide, 1,4-bis-

(triethoxysilyl)benzene, and 1,2-bis(triethoxysilyl)ethane assilica precursors, respectively, in combination with apreferential etching approach.47 These etched SNCs displayedgood deformability and much lower Young’s moduli (48, 91,and 3 MPa, respectively) compared to those of the non-etchedSNCs prepared using the same precursors (233, 351, and 251MPa, respectively). The mechanical properties of SNCs canalso be tuned by varying the silica shell thickness (Figure 2c,right). We recently reported the fabrication of nanoemulsion-templated stiff and soft SNCs by using TEOS and anorganosilane, triethoxyvinylsilane (TEVS), as precursors.48 Byincreasing the reaction time from 30 to 50 h, the silica shellthickness can be increased from ∼10 to ∼25 nm, resulting inan increase in stiffness from 0.04 to 0.4 N/m for the soft TEVSSNCs and from 8.7 to 20.1 N/m for the stiff TEOS SNCs. Thederived Young’s moduli of the softest and the stiffest SNCswere 704 kPa and 9.7 GPa, respectively.It is worth noting that, due to the different silica precursors

used for making soft and stiff SNCs, different surface groupscan be introduced onto the surfaces of the SNCs.50 To takeadvantage of the facile functionalization of the silica shells,surface modification, such as PEGylation, is normally adoptedto render the surface of soft and stiff SNCs identical andfunctional.47,48 PEGylation does not significantly change themechanical properties of SNCs as it is the silica shell (thicknessand chemistry) that mainly determines the Young’s modulus ofSNCs.48 As reflected in the above examples, the mechanicalproperties of SNCs can be adjusted in a very broad range, fromkPa to GPa (Figure 3), which makes it possible tosystematically study the effect of NP mechanical propertieson nano-bio interactions.

EFFECTS OF NANOPARTICLE MECHANICALPROPERTIES ON CANCER DRUG DELIVERY

Generally, NP-based drug delivery systems administered viaintravenous injection go through a series of biological

Figure 3. Spectrum of the mechanical properties of different nanoparticles. Hydrogel nanoparticles normally have elastic moduli rangingfrom dozens of kPa to a few MPa. The elastic moduli of polymer−lipid nanoparticles can be down to kPa or up to GPa ranges. However, fora certain core material, it remains challenging to adjust its mechanical properties over a broad range. Silica nanocapsules display a very broadmechanical property range, with elastic moduli ranging from kPa to GPa. Refer to Table 2 for the biological effects due to the variedmechanical properties of these nanoparticles.

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processes from the injection site to the side of action, includingthe formation of a protein corona, the clearance by theimmune system, the selective accumulation and penetration insolid tumors, and the internalization by cancer cells. Thissection reviews the latest studies on the crucial role of themechanical properties of NPs in their blood circulation, tumortargeting and biodistribution, tumor penetration, and tumorcell internalization (Table 2).Blood Circulation. The blood circulation time of NPs has

long been recognized to be positively correlated with theiraccumulation in the tumor tissue.54 Therefore, great effortshave been attributed to design NPs to have prolongedcirculation time by overcoming biological barriers, includingimmune cell uptake and blood filtration. This is commonlyachieved by rationally engineering the physicochemicalproperties of NPs such as size, shape, and surface charge.54,55

Recently, the mechanical properties of NPs have also proven tobe an important parameter that can be leveraged to regulatetheir blood circulation time.The circulation time of hydrogel NPs (∼200 nm) having

Young’s moduli of 10 and 3000 kPa in mice was examined.37

The soft NPs displayed a persistence in the vasculaturesignificantly higher than that of their stiff counterparts,especially in the first 2 h, and this difference became muchless prominent at 4 h (Figure 4a). The different bloodcirculation performances of the soft and stiff NPs wereattributable to their distinct phagocytosis profiles. The stiffNPs displayed an uptake by J774 macrophages 3.5-fold higherthan that of the soft NPs over a 12 h period in vitro, and thehigher macrophage capture of stiff NPs likely led to more rapidelimination.37 Similar preferential macrophage uptake of stifferNPs compared to the softer NPs was also reported in ourrecent study, wherein RAW264.7 murine macrophagesexhibited uptake of both unmodified and PEGylated stiffSNCs (9.7 GPa) significantly greater than that of the soft ones(700 kPa).48 Macrophage sequestration is a major reasonaccounting for the removal of NPs during circulation, and thelower macrophage uptake of soft NPs is likely due to theirability to deform upon the forces exerted by macrophagecells.37 It has been reported that the extent of phagocytosis canpotentially be decreased when the NP becomes elongated orstretched.56

The sieving effect of the biological filtration systems (e.g.,lungs and spleen) represents another important mechanismresponsible for NP removal. The blood circulation half-lives ofhydrogel NPs (∼120 nm) in mice decreased from 19.6 to 9.1 has their Young’s moduli increased from 18 to 1350 kPa (Figure4b).36 The longer circulation time of the softer NPs wasattributed to its superior deformability, allowing them to passthrough filters with a pore size smaller than its diameterwithout losing structural integrity (Figure 4c). In comparison,the stiffer NPs failed to pass through the filter. This sievingeffect, as will be elaborated in next section, also applies tospleen which acts as a filter for purifying blood. Due to theircompromised deformability, stiffer NPs normally exhibit moresignificant spleen accumulation than softer NPs, which in turnshortens their blood circulation time.Overall, soft NPs exhibit blood circulation longer than that

of their stiff counterparts due to their lower macrophagecapture and the ability to avoid the removal by biologicalfiltration systems. This characteristic to some extent reflectsthe defending mechanisms against foreign substances andpathological tissues, considering that most endogenoussubstances in the bloodstream (e.g., healthy red blood cells)possess very low stiffness, whereas pathological cells (e.g.,diseased and aging red blood cells) and foreign matter (e.g.,viruses) are relatively stiffer.

Biodistribution and Tumor Targeting. Biodistributiondescribes the spatiotemporal localization of NPs in the tissuesand organs (e.g., lungs, kidney, spleen, etc.) of animals orhumans. Broadly speaking, the accumulation of NPs in solidtumors is within the scope of biodistribution. In fact, NPs’biodistribution, blood circulation, and tumor accumulation arethree closely associated biological processes. Understandingthe distribution profiles of NPs in different organs and tissueswill shed light on the mechanisms involved in their clearancefrom the bloodstream by the mononuclear phagocyte system,which, in turn, determine their tumor-targeting ability. As such,the effect of the physicochemical properties of NPs on theirbiodistribution has been extensively explored to promote thetumor-targeted delivery of NPs while reducing their off-targetdeposition, which are considered the grand challenges for NP-based drug delivery.1,57

As demonstrated by Figure 4b in the last section, the in vivocirculation time of hydrogel NPs decreased from 19.6 to 9.1 h

Figure 4. Effect of the mechanical properties of nanoparticles on their blood circulation. (a) Circulation data of soft (10 kPa) and stiff (3000kPa) hydrogel nanoparticles over 12 h. %ID represents the percentage of injected nanoparticle dose remaining in blood circulation. Insethighlights the data from 0 to 2 h.37 (b) Circulation profiles of poly(carboxybetaine) (pCB) hydrogel nanoparticles with varying mechanicalproperties over 72 h, with the pCB concentration from 2 to 15% corresponding to Young’s moduli from 180 to 1350 kPa.36 (c) Stiff (1350kPa) and soft (260 kPa) hydrogel nanoparticles with a mean hydrodynamic size of 250 nm to pass filters with a pore size of 220 nm.Schemes show that the stiff nanoparticles cannot pass the filter slits, whereas the soft nanoparticles can deform and pass through the slitswithout losing their structural integrity.36 (a) Reprinted from ref 37. Copyright 2015 American Chemical Society. (b,c) Reprinted from ref36. Copyright 2012 American Chemical Society.

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as their Young’s moduli increased from 18 to 1350 kPa. Toelucidate how these NPs were cleared from the bloodstream,their biodistribution was examined (Figure 5a). At 48 h afterintravenous injection, both the stiff and soft NPs were mainlyfound in livers and spleens, which are the major mononuclearphagocyte system organs responsible for the clearance offoreign substances.36 Moreover, the accumulation of softer andstiffer NPs in the liver did not show a significant difference,whereas the splenic accumulation of stiffer NPs wassignificantly higher than that of the softer NPs, exhibiting a

negative correlation with their blood circulation times (Figure5b). This observation indicates the critical role of the spleen inthe filtration of NPs in the bloodstream, which can be betterunderstood by taking a closer look into the microanatomy ofthe splenic venous sinuses (Figure 5c). Briefly, splenic venoussinuses are lined with a layer of discontinuous endothelial cellsorganized in parallel and connected by stress fibers, and thisspecial arrangement delimits narrow slits serving as a filtrationsystem.17,58 When blood from the splenic red-pulp cordscollects in the sinuses by passing through the narrow slits

Figure 5. Effect of the mechanical properties of NPs on their biodistribution and tumor targeting. (a) Biodistribution of pCB hydrogelnanoparticles with varying mechanical properties, with the pCB concentration from 2 to 15% corresponding to Young’s moduli from 180 to1350 kPa.36 (b) Circulation half-life and splenic accumulation of the pCB hydrogel nanoparticles.36 (c) Schema of a venous sinus located inthe cords of the splenic red pulp.17 Semiquantitative analysis displaying the accumulation of stiff (9.7 GPa) and soft (704 kPa) silicananocapsules between (d) tumor and liver and (e) tumor and spleen. Values represent the ratios of integral fluorescence per unit mass (IF/g).48 (f) Distribution of ICAM-conjugated stiff and soft hydrogel nanoparticles in various organs.37 (a,b) Reprinted from ref 36. Copyright2012 American Chemical Society. (c) Reprinted with permission from ref 17. Copyright 2005 Nature Publishing Group. (d,e) Reprintedfrom ref 48. Copyright 2018 American Chemical Society. (f) Reprinted with permission from ref 37. Copyright 2015 American ChemicalSociety.

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(shown by arrows), healthy red blood cells that aremechanically flexible are able to deform and squeeze throughthe slits to remain in the bloodstream, whereas aging orpathological red blood cells are stiffened so that they aretrapped in the macrophage-rich red pulp and ultimatelydestroyed.17 This mechanism has been proven to also apply tothe splenic removal of intravenously injected NPs.17 Werecently demonstrated how decreasing NP elasticity can reducetheir splenic clearance and thus enhance their tumortargeting.48 SNCs (∼200 nm) having Young’s moduli of 704kPa and 9.7 GPa were modified with poly(ethylene glycol)(PEG) and folic acid (FA)−PEG and were injected into micebearing a SKOV3 tumor xenograft that overexpresses folatereceptors. The ratios of SNC accumulation in tumor−liver(Figure 5d) and tumor−spleen (Figure 5e) were examined for72 h. The tumor−liver ratios of the stiff and soft SNCs did notshow an evident difference, whereas the tumor−spleen ratiosof the stiff SNCs were much lower than that of their softcounterparts, implying a higher splenic clearance of the stiffSNCs and a lower tumor-targeting efficiency. It is worth notingthat, at 24 h postinjection, the tumor−liver and tumor−spleenratios of FA-PEG-modified stiff SNCs were significantly higherthan those of its PEG-modified counterpart, indicating anenhancing effect of FA functionalization on the tumor-targeting ability of stiff SNCs, which was, however, notobserved for soft SNCs. This is likely because FA conjugationpromotes the folate-receptor-mediated cellular uptake of the

stiff SNCs to a higher extent than the soft ones. At all othertime points, FA-PEG-modified SNCs did not show tumoraccumulation higher than that of their PEG-modified counter-parts, which reflects a key hindrance to the clinical success ofactive tumor targeting: ligands on the surface of NPs can onlyenhance cell−NP interactions at a very close proximity (<0.5nm) but cannot substantially alter the overall in vivo NPbiodistribution.3

The above studies demonstrate the crucial role of biologicalfiltration systems (e.g., splenic sinusoid) in the clearance ofintravenously injected NPs. Soft NPs are able to squeezethrough these barriers and remain in the blood circulation,whereas stiff NPs are prone to be trapped due to their poordeformability. Normally, this higher accumulation in themononuclear phagocyte system organs, including spleen andliver, leads to shortened blood circulation time and inferiortumor accumulation of stiff NPs. However, different resultshave also been observed. For instance, the accumulation of softhydrogel NPs (∼200 nm, 10 kPa) in the spleen and lung wassignificantly higher than that of the stiff NPs (3000 kPa)(Figure 5f), which was attributed to the longer bloodcirculation of the soft NPs.37 The hydrogel NPs werefunctionalized with anti-intercellular adhesion molecule(ICAM) as the ICAM protein is overexpressed in the spleenand lungs. In this case, the spleen cannot be simply consideredas a biological filter but a target organ. Given the complicatedresults discussed above, more explorations and better under-

Figure 6. Effect of the mechanical properties of nanoparticles on tumor penetration. (a) Fluorescence intensity distribution of stiff (9.7 GPa)and soft (704 kPa) silica nanocapsules across tumor spheroids at a scanning depth of 150 μm and (b) schematic illustration showing thepenetration of the stiff and soft silica nanocapsules in the tumor spheroids.48 (c) Distribution of soft and stiff hydrogel micelles influorescently labeled tumor spheroids: blue, DAPI (nuclei); green, micelles; purple, DiD (cell membrane).44 (d) Soft (5 MPa), semielastic(50 MPa), and hard (110 MPa) polymer−lipid nanoparticle penetration into tumor spheroids. Z-stack images were obtained starting fromthe top to the center of the spheroid at an interval of 20 μm. Scale bars = 50 μm.32 (a,b) Reprinted from ref 48. Copyright 2018 AmericanChemical Society. (c) Reprinted with permission from ref 44. Copyright 2017 Elsevier. (d) Reprinted with permission from ref 32.Copyright 2018 Springer Nature.

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standing on the effect of the mechanical property of NPs ontheir in vivo biodistribution and tumor targeting are needed.Tumor Penetration. After reaching the tumor tissue, most

NPs accumulate at the periphery of the tumor mass. Thisinefficient tumor penetration of therapeutic NPs often leads to

the difficulty in eradicating all tumor cells, thus resulting insuboptimal treatment outcomes and consequent cancerrecurrence.59,60 Penetrating into the deep interior of a solidtumor is not trivial for NPs owing to a number of physiologicalbarriers. Compared to normal tissues, tumor tissues are

Figure 7. Effect of the mechanical properties of nanoparticles on their interactions with cancer cells. (a) Confocal fluorescent imagesshowing the uptake of stiff (1.2 GPa) and soft (0.7 GPa) polymer−lipid nanoparticles by HeLa cells. Colocalization of the PLGA core (red)and the lipid layer (green) shows a yellow color. Scale bar = 5 μm. (b) MD simulations showing the slower cell membrane wrapping of thesoft nanoparticles compared to the stiff nanoparticles.33 Cellular uptake of (c) bare and (d) ICAM-conjugated soft (10 kPa) and stiff (3000kPa) hydrogel nanoparticles by 4T1 cells at various time points.37 (e) Uptake of PEG- and FA-PEG-modified stiff (9.7 GPa) and soft (704kPa) silica nanocapsules by SKOV3 cells.48 (f) Adhesion of soft (160 kPa) and stiff (260 kPa) hydrogel micelles with different sizes to A375cells.44 (g) Transmission electron microscopy images (top 1−4) and schematic illustration (bottom 1−4) of the uptake process of soft silicananocapsules (47 MPa) by MCF-7 cells, with arrows indicating the cell membrane. Scale bars = 100 nm.47 (h) Schematic illustrationshowing that soft (45 kPa) liposomes enter cells via membrane fusion (predominant) and endocytosis (inferior), whereas hard polymer−lipid nanoparticles (19 MPa) enter cells via only clathrin-mediated endocytosis.45 (a,b) Adapted with permission from ref 33. Copyright2015 John Wiley & Sons Inc. (c,d) Reprinted from ref 37. Copyright 2015 American Chemical Society. (e) Reprinted from ref 48. Copyright2018 American Chemical Society. (f) Reprinted with permission from ref 44. Copyright 2017 Elsevier. (g) Reprinted from ref 47. Copyright2018 American Chemical Society. (h) Reprinted with permission from ref 45. Copyright 2018 Springer Nature.

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normally featured by a dense extracellular matrix due to theirhigh content of collagen and lysyl oxidase.6,59 The interstitialfluid pressure inside tumors are also increased because of therapid cell proliferation and impaired lymphatic drainage.59

These barriers prevent the diffusion of NPs into tumorinterstitium. Researchers have attempted to improve NPpenetration by optimizing their physiochemical propertiessuch as size, shape, and surface charge. Recently, themechanical properties of NPs have also been demonstratedto play a role in regulating their tumor penetration.The tumor penetration of NPs is often investigated using

tumor spheroids owing to their simple and easy preparation,their 3D architecture and spatial heterogeneity, and theirsimilar chemical and biological characteristics to tumortissues.61 By studying the interactions between FA-function-alized stiff/soft SNCs (∼200 nm) and folate-receptor-positiveSKOV3 tumor spheroids, we demonstrated the superiortumor-penetrating ability of the soft SNCs (704 kPa), whichexhibited a penetration depth of around 80 μm in the tumorspheroids (Figure 6a).48 In comparison, the stiff SNCs (9.7GPa) were mainly located at the surface of the spheroids,although they exhibited overall cellular uptake higher than thatof their soft counterparts. The different performances of thesoft and stiff SNCs were attributed to their distinct flexibilityand deformability. The soft SNCs were able to squeezethrough the intercellular spaces and transport deeper into thetumor spheroids, whereas the stiff SNCs were unable to passthrough the narrow interstitial space and were thereforetrapped at the peripheral area of the spheroids (Figure 6b).48

Additionally, the poorer tumor penetration of the stiff SNCsmight also be a result of their stronger cell interaction, leadingto their immobilization at the exterior of the tumor spheroids.The penetration study of polymer micelles (∼80 nm) with

Young’s moduli of 165 and 260 kPa in BxPC3 pancreatic celltumor spheroids revealed that the stiff micelles penetrated intoall layers of the spheroid (the diameter of tumor spheroids was∼350 μm) including the center site (Figure 6c), whereas notmuch softer micelles were found in the inner layer of thespheroid.44 In another case, polymer−lipid NPs (∼ 200 nm)with a moderate elasticity (50 MPa) exhibited a greater tumorpermeability in BxPC3 and human pancreatic star cellmulticellular spheroids when compared to that of their soft(5 MPa) and rigid (110 MPa) counterparts.32 Thefluorescence signal of the semielastic NPs could be detectedinside the tumor spheroids at a scanning depth of 70 μm,whereas the fluorescence signals of the soft and rigid NPs onlyoccurred at the marginal area of the tumor spheroids at ascanning depth of 30 μm (Figure 6d). The stronger tumorpenetration of the semielastic NPs was attributed to theirenhanced diffusivity in biological hydrogels (e.g., mucus) bothin vitro and in vivo. As elucidated by super-resolutionmicroscopy and molecular dynamics (MD) simulation results,the semielastic NPs deformed into ellipsoids and performedrotational motion in biological hydrogels.32 This alteration inshape led to a higher aspect ratio, which boosted the diffusivityof the NPs. The very soft and stiff NPs, however, eitherdeformed excessively or retained their original shape inbiological hydrogels, thus exhibiting weaker diffusivity.The inconsistent results discussed above reflect the complex

effect of NP mechanical properties on tumor penetration,which might stem from the distinct experimental settings incurrent studies. For example, big gaps exist between themechanical properties of NPs fabricated in individual studies

(Figure 3). By comparing the tumor penetration behavior ofNPs having Young’s moduli of 165 and 260 kPa, one maydevelop a conclusion that a higher elasticity is favorable for thetumor penetration of NPs. However, 260 kPa is still far softerthan real stiff materials. As the elasticity increases, NPs with amoderate elasticity may turn out to have better tumor-penetrating ability than the softer and the stiffer ones. Anothermajor limitation is that many key in vivo physiochemicalsettings are oversimplified in tumor spheroid models, such asthe interstitial fluid flow and pressure, the stiffness of the tumortissue, and the component of extracellular matrix, etc. Theabsence of precise control of these parameters can lead to poorrepresentation of what really happens in vivo. Designingmicrofluidic devices that better mimic the in vivo conditions byintegrating tumor spheroids with necessary tumor micro-environments (e.g., flow, pressure, tumor stiffness, andpermeability) may be a way to address this issue.62,63

Interaction with Cancer Cells. The efficient internal-ization of drug-loaded NPs by tumor cells is a key prerequisitefor successful drug delivery, hence great efforts have beendedicated to explore the design principles of NPs withenhanced cancer cell uptake. Although the majority of relevantstudies report that stiff NPs display macrophage internalizationgreater than that of their soft counterparts, there remains agreat discrepancy in the literature regarding the effect of NPelasticity on cancer cell uptake.Many studies claim that NPs with higher elasticity are more

favorable for internalization by cancer cells. By usingPEGylated polymer−lipid NPs (∼40 nm) with Young’s moduliof 0.76 and 1.2 GPa, the stiff NPs showed higher nonspecificuptake by HeLa cells compared to the soft NPs (Figure 7a).33

As a result, doxorubicin and combretastatin A4 dual-drug-loaded stiff NPs displayed a cytotoxic effect greater than that ofboth free drug and drug-loaded soft NPs. MD simulationsreveal that the decreased cellular uptake of the soft NPsstemmed from their deformation during internalization, whichraised the energy level required for their complete cellmembrane wrapping (Figure 7b).33 Similar MD simulationresults have also been reported elsewhere.38,39 Interestingly,there is increasing evidence that the cellular uptake differencebetween the stiff and soft NPs can be magnified by modifyingother characteristics such as surface chemistry. Hydrogel NPs(∼200 nm) with Young’s moduli of 10 and 3000 kPa werestudied for their uptake by 4T1 epithelial tumor cells.37 Whenthe surface of NPs was not modified, no statistical differencebetween the uptake of stiff and soft NPs was observed at shorttime points (≤4 h), and the stiff NPs were only bound to orinternalized by 4T1 cells greater than their soft counterparts at8 and 12 h (Figure 7c). Surface modification with anti-ICAMantibody enhanced the uptake of both stiff and soft NPs by4T1 cells that express ICAM receptors on cell surfaces.However, this enhanced uptake was more significant for thestiff NPs, with their uptake becoming statistically higher thanthat of the soft NPs at time points of 2, 4, 8, and 12 h (Figure7d).37 These results indicate an interdependent effect betweenthe mechanical property and surface property of NPs ondetermining their cancer cell uptake, which is in accordancewith our recent studies. By studying the uptake of SNCs (∼200nm) having Young’s moduli of 704 kPa and 9.7 GPa by folate-receptor-positive SKOV3 cancer cells, we found that the PEG-modified stiff and soft SNCs displayed similar nonspecificcellular uptake, whereas the FA-PEG-modified stiff SNCsexhibited enhanced receptor-mediated uptake by SKOV3 cells

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that overexpressed folate receptors on their surfaces to asignificantly higher extent than the soft ones (Figure 7e).48

Mechanical properties have also been demonstrated to work intandem with the size of NPs to affect their cancer cell uptake.Stiff (260 kPa) and soft (165 kPa) PEG−PLA micelles havinga diameter of ∼17 nm showed no significant difference in thenumber of NPs adhered to A375 cancer cells (Figure 7f).44

However, increasing the diameters of the micelles to ∼40 and∼75 nm increased the cellular binding capacity of the stiffmicelles much more significantly than that of the soft micelles,which consequently influenced their cellular internalizationefficiency.There are also studies wherein soft NPs display tumor cell

uptake higher than that of stiff NPs. For example, PEG-modified solid and hollow silica NPs (∼300 nm) with Young’smoduli of 233 and 47 MPa, respectively, were studied for theiruptake in MCF-7 breast cancer cells, and their morphologicaltransition during cellular internalization was observed usingtransmission electron microscopy.47 The soft SNCs couldeasily enter MCF-7 cells via a spherical-to-oval morphologicalchange (Figure 7g), resulting in a cellular uptake 26-fold higherthan that of their stiff counterparts which remained sphericalduring the internalization. As a result, MCF-7 cells co-incubated with the doxorubicin-loaded soft silica NPs showedcytotoxicity higher than that of the stiff NPs. In another case,the uptake of hydrogel NPs (∼800 nm) by HepG2 cancer cellsdecreased as their Young’s moduli increased from 15 to 156kPa, which was attributed to the different uptake mechanismsinvolved in the endocytosis of stiff and soft NPs.35 The softestNPs were mainly internalized via macropinocytosis, whereasthe stiffest NPs largely adopted clathrin- and caveolae-mediated pathways as well as macropinocytosis to enter thecells. The different endocytosis mechanisms involved in thecellular uptake of stiff and soft NPs have also been reported forliposome-based NPs. The uptake of soft liposomes (∼160 nm,45 kPa) by MDA-MB-231 and MCF-7 cancer cells was viaboth endocytosis and membrane fusion (Figure 7h), whereasthe stiff hydrogel lipid NPs (∼160 nm, 19 MPa) could onlyenter the cells through endocytosis.45 When membrane fusion,which requires less energy than endocytosis, was adopted byliposomes as a predominant way of entering the cells, softerliposomes displayed cancer cell uptake significantly higher thanthat of their stiff counterparts.Although it is clear that the cancer cell uptake of NPs can be

modulated by modifying their mechanical properties, itremains challenging to paint a complete picture about howthe elasticity of NPs affects their interactions with cancer cells.A main reason for this is the heterogeneity in different cancercells and the associated tumor microenvironments. Theinconsistent experiment findings may also stem from thecoupling effect of NP elasticity with other parameters such assize and surface chemistry. As discussed above, cellularinternalization behaviors of stiff and soft NPs can bedramatically affected by altering their sizes or by functionaliz-ing with targeting ligands. Another important reason is that theNPs used in most current studies possess a relatively narrowrange of Young’s modulus with considerable gaps betweendifferent systems (Figure 3), leading to inconsistent results. Toaddress this issue, NP systems with a broad elasticity rangefrom very soft (Pa) to very stiff (GPa) should be used toobtain a full spectrum result.Computational simulation remains a powerful tool to

explore the kinetics of cell adhesion and endocytosis of elastic

NPs. Cell adhesion of NPs is a result of the binding betweenligands on a NP surface and receptors on a cell membrane, andit is the driving force for membrane wrapping of NPs.64 Severalsimulation studies showed that softer NPs display strongermultivalent binding to target cells compared to that of stifferNPs because the former is able to deform into a flattenedconfiguration.64,65 For example, in a coarse-grained MDsimulation study, softer NPs deforms into a hemisphere tomaximize their contact area with the cell membrane toestablish more ligand−receptor binding in the early stage ofcell uptake.65 However, this enhanced binding between softerNPs and cells does not necessarily lead to more efficient NPinternalization, mainly because the softer NPs are energeticallyless prone to be fully wrapped than the stiffer NPs. Thewrapping of NPs by the cell membrane during endocytosis hasalso been widely studied by simulation. For clathrin- andcaveolin-independent endocytosis, the cellular uptake of NPscan be modeled by simple membrane wrapping based on theHelfrich−Canham membrane theory,66 on which continuumscale theoretical analysis indicates that stiff NPs are moreenergetically favorable to full membrane wrapping than theirsoft counterparts, and this effect gradually becomes lesssignificant as the NP stiffness increases.39 Similarly, aspreviously discussed, MD simulation also suggests a fullwrapping of stiff NPs that is faster than that of the soft ones bya simple membrane (Figure 7b).33 In real situations, however,the cellular uptake of NPs may not only involve the lipidmembrane. As another membrane deformation processadopted normally by receptor-mediated cellular uptake,clathrin-mediated endocytosis not only requires the substantialmembrane deformation as in the simple membrane wrappingbut also involves the adaptor protein-induced spontaneouscurvature and actin-mediated force on budding.67,68 Recently,theoretical models at the continuum scale have been developedto describe clathrin-mediated endocytosis incorporatingprotein-induced heterogeneities in the deformed mem-brane.67,68 However, as the presence of NPs and the featureof NP−membrane interactions are not considered in thesemodels, they are only valid for the clathrin-mediatedendocytosis of NPs with radii smaller than that of the clathrinscaffold. For relatively larger NPs, depending on the stiffnessratio between the engulfed NPs and the self-assembled clathrinscaffold, the mechanically compressive interplay between theNPs and clathrin-coated membrane either squeezes the softNPs to induce substantial NP deformation or expands theclathrin scaffold to accommodate the NPs, thus influencing theentire endocytic process. Other challenges remaining to betackled include modeling the scission of clathrin-coatedvesicles and endocytic kinetics such as the deterministicreceptor diffusion and stochastic receptor−ligand binding andcoat assembly. As the remodeling of the actin-based networkand explicit consideration of its interaction with the lipidmembrane are not fully established in MD simulations and areextremely computationally expensive and time-consuming,existing MD modelings in literature on the cellular uptake ofelastic NPs are restricted to the cases of simple membranewrapping.40,69,70 More sophisticated MD approaches integrat-ing the lipid membrane and associated component remodelingare yet to be developed.

SUMMARY AND PERSPECTIVESNP-based cancer drug delivery depends on a well-orchestratedseries of biological events that requires synergistic effects of the

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physicochemical properties of NPs to overcome key biologicalbarriers to drug delivery in cancer. This review highlights theimpacts of the mechanical properties of NPs on cancer drugdelivery through regulating their blood circulation, biodis-tribution, tumor penetration, and tumor cell uptake. Comparedto stiff NPs, soft NPs normally display less macrophage uptakeand removal by biological filtration systems (e.g., splenicvenous sinuses) owing to their superior deformability, leadingto longer blood circulation times and higher tumoraccumulation. This may to some extent explain why mostcurrently commercialized anticancer drug delivery systems arebased on soft materials (e.g., liposome and albumin NP).71

Despite these major findings, definitive conclusions on howNP elasticity affects tumor penetration and interactions withcancer cells are still lacking, which is attributable to theoverwhelming heterogeneity between different cancer cells andcomplex tumor microenvironments. The large gaps betweenthe mechanical properties of NPs in different studies, as well asthe inconsistency in other physicochemical parameters (e.g.,size, shape, and surface modification), also contribute to thecontradictory results of different studies. Therefore, thecoupling effect between the mechanical properties and otherparameters of NPs on modulating nano-bio interactions needsto be more carefully elucidated in future studies. NP systemshaving a wide spectrum of mechanical properties should beused, and their mechanical properties should be quantifiedusing standardized methods to acquire comparable results.Furthermore, other important factors warrant more attention,such as the role of NP mechanical properties in regulating theirextravasation from tumor vasculature and retention at tumortissues by taking advantage of the EPR effect as well as theirinteractions with tumor microenvironments including theextracellular matrix, tumor-associated cells, and tumor stromalcells. Tumor-on-a-chip models offer valuable tools forexploring these topics.62 Until now, studying cellular uptakepathways (using internalization inhibitors) and the energycosts involved in cellular internalization (using computationalsimulation) of stiff and soft NPs remains the main method forexploring mechanisms governing mechanically regulated NP−cell interactions. Other important aspects, such as the effects ofNP mechanical properties on the formation of protein coronaand on cell signaling pathways (using the emerging omicstechnique),72 which affect NP cellular uptake, should also besystematically studied. This would require the efforts ofmultidisciplinary communities including nanotechnology,biophysics, cell biology and mechanical engineering. A morecomplete understanding of these topics would prompt thedevelopment of mechanically engineered NPs for superiorcancer drug delivery: for example, NPs that are very soft duringcirculation for improving blood circulation time andconsequently tumor accumulation but then alter their elasticityby responding to tumor microenvironment or externalstimulation for enhanced cancer cell internalization.

AUTHOR INFORMATION

Corresponding Authors*E-mail: dyzhao@fudan.edu.cn.*E-mail: huajian_gao@brown.edu.*E-mail: z.chunxia@uq.edu.au.

ORCIDYue Hui: 0000-0002-1057-5671Xin Yi: 0000-0002-4726-5765

David Wibowo: 0000-0001-6919-3355Fan Zhang: 0000-0001-7886-6144Dongyuan Zhao: 0000-0001-8440-6902Chun-Xia Zhao: 0000-0002-3365-3759NotesThe authors declare no competing financial interest.

ACKNOWLEDGMENTSThis work was supported by the Australian Research Councilunder Discovery Project (DP150100798) and Future Fellow-ship project (FT140100726). Y.H. acknowledges Ph.D.scholarships from the University of Queensland and theChinese Scholarship Council. H.G. acknowledges financialsupport from the National Science Foundation (Grant CMMI-1562904). X.Y. acknowledges support from the NationalNatural Science Foundation of China (Grant No. 11872005).

VOCABULARYmechanical properties, physical properties that a materialexhibits upon the application of forces; indentation, acommon method of testing the mechanical properties ofmaterials by pressing the materials using an indentor tip;force−displacement curve, a curve specifying the displace-ment of an object that results from applied forces;biodistribution, a method of tracking where nanoparticles ofinterest travel in an experimental animal or human subject;endocytosis, the cellular process in which substances aresurrounded by plasma membrane and brought into the cell

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