Pharmacokinetics

Nanoparticle Accumulation in Angiogenic Tissues: Towards Predictable Pharmacokinetics

Kristin Yaehne , Amy Tekrony , Aisling Clancy , Yiota Gregoriou , John Walker , Kwin Dean , Trinh Nguyen , Amber Doiron , Kristina Rinker , Xiao Yu Jiang , Sarah Childs , and David Cramb *

Nanoparticles are increasingly used in medical applications such as drug delivery, imaging, and biodiagnostics, particularly for cancer. The design of nanoparticles for tumor delivery has been largely empirical, owing to a lack of quantitative data on angiogenic tissue sequestration. Using fl uorescence correlation spectroscopy, the deposition rate constants of nanoparticles into angiogenic blood vessel tissue are determined. It is shown that deposition is dependent on surface charge. Moreover, the size dependency strongly suggests that nanoparticles are taken up by a passive mechanism that depends largely on geometry. These fi ndings imply that it is possible to tune nanoparticle pharmacokinetics simply by adjusting nanoparticle size.

1. Introduction

Nanoparticles have increasing utility as drug delivery vehi-

cles and diagnostic imaging agents, particularly for treating

cancer. Such nanotherapeutic approaches exploit the

enhanced permeability and retention (EPR) effect, [ 1 ] whereby

© 2013 Wiley-VCH Verlag Gmb

DOI: 10.1002/smll.201201848

K. Yaehne, A. Tekrony, A. Clancy, Dr. Y. Gregoriou, J. Walker, K. Dean, T. Nguyen, Prof. D. CrambDepartment of Chemistry2500 University Dr NWUniversity of CalgaryCalgary, Alberta, Canada, T2N 1N4 E-mail: dcramb@ucalgary.ca

Dr. A. Doiron, Prof. K. RinkerDepartment of Chemical and Petroleum EngineeringSchulich School of Engineering2500 University Dr NWUniversity of CalgaryCalgary, Alberta, Canada, T2N 1N4

X. Y. Jiang, Prof. S. ChildsDepartment of Biochemistry and Molecular BiologyFaculty of MedicineHealth Sciences Centre3330 Hospital Drive, NWUniversity of CalgaryCalgary, Alberta, Canada, T2N 4N1

small 2013, DOI: 10.1002/smll.201201848

nanoparticles become sequestered in angiogenic tissues sur-

rounding growing tumors. Nanoparticle accumulation into

tissues via the EPR effect is partly due to the leakiness of the

newly forming (angiogenic) blood vessels of cancerous tissues

and partly due to poor lymphatic drainage in growing tumors.

For the former, it has been demonstrated that in angiogen-

esis, the endothelial cells comprising the blood vessel walls

do not seal tightly against each other, leaving fenestrations

through which small species can pass. These fenestrations

are approximately 200–800 nm in diameter [ 2 ] and thus nano-

particles smaller than this size are desirable as vehicles for

angiogenesis-related drug delivery. [ 3–10 ] A critical component

to understand and optimize for drug delivery is the physico-

chemical properties of a nanoparticle which can predict their

pharmacokinetics and accumulation in tumors. Predictively

modeling how a given nanocarrier biodistributes relies upon

an accurate understanding of its tissue interactions in various

organs, which can be approximated as compartments in the

organism. Since such predictive pharmacokinetic models for

sequestration of nanoparticles into angiogenic tissues are

unavailable, much of the nanoparticle delivery vehicle devel-

opment has necessarily been empirical. [ 11–19 ] Quantitative

data that relate the size and charge of nanoparticles to rate

constants for bioacummulation in angiogenic tissues would

represent an excellent fi rst step towards holistic, predictive

pharmacokinetics. Such data would allow the refi nement of

nanoparticle deposition mechanisms and thus generate more

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K. Yaehne et al.full papers

predicitive pharmacokinetic models. These models will facili-

tate both rational drug delivery agent design and a predic-

tive understanding of nanotoxicity. It is the aim of the present

study to present quantitative data that connects specifi c nan-

oparticle properties to deposition rates in angiogenic tissues.

To avoid confusion, we propose the following defi nitions.

‘Deposition’ will mean the accumulation of nanoparticles

in angiogenic tissues. This could mean surface deposition or

nanoparticles entering the tissues. ‘Uptake’ will only be used

for denoting when nanoparticles enter individual cells.

One of the major challenges in developing predictive

models for bioaccumulation of nanoparticles, particularly with

respect to tumors, is representing the anatomic complexity of

the physiologic environment. This is particularly problematic

when attempting to separate the effects of various angiogenic

characteristics, such as fenestration size, local blood fl ow and

local blood pressure on NP accumulation. [ 20 ] The very prop-

erty of mammalian orthotopic models that makes them pow-

erful mimics of tumors, makes them extremely challenging to

use as quantitative predictors of accumulation. For deconvo-

lution of the nanoparticle deposition kinetics into angiogenic

tissues from tumor distribution and/or sequestration into

the reticuloendothelial system (RES), the chicken embryo

chorioallantoic membrane (CAM) provides an excellent

platform. [ 21 , 22 ] The CAM is an extra-embryonic organ that

functions as the embryo’s lung and provides a large blood

volume connected primarily to angiogenic blood vessels

until day 14 post fertilization (PF). [ 22 ] Moreover, since the

immune system of the chicken embryo is relatively undevel-

oped up to 14 days PF; immune responses to nanoparticles

in the blood stream are minimal. Finally, the CAM expresses

both VEGF-C and the VEGF receptor in the blood vessel

endothelial cells during angiogenesis. [ 22 ] We propose that the

CAM model could be a fi rst screening system for assessing

nanoparticle pharmacokinetics (PK) as this biological system

is more complex than a cell culture model, but is simpler to

use and easier to maintain and grow than a full animal model.

Using the CAM model, we would be able to narrow down

candidate nanoparticle formulations that could undergo a

full evaluation in an orthotopically implanted tumor rodent

model. It has been proposed that it is diffi cult to evaluate

the large amount of different nanoparticle formulations in

animal models in a timely manner. Therefore, novel screening

tools are required to speed the evaluation of potential nano-

particle candidates for in vivo applications. [ 23 ] The CAM is a

powerful tool for understanding, quantitatively and predic-

tively, the behavior of nanoparticles in an angiogenic environ-

ment relevant to tumor drug delivery. Moreover, we suggest

that by using this simple model, one will fi ll a knowledge gap

in the dependence of nanoparticle properties on uptake into

angiogenic tissues.

Presently, nanoparticle bioaccumulation PK models have

only been developed for mature, healthy organisms lacking

angiogenic blood vessels. The most realistic model predicted

quantum dot (QD) distribution in healthy mature rodents

and compared results with literature data. [ 24 ] The blood-

fl ow-limited physiologically based pharmacokinetic (PBPK)

model predicted liver deposition to within 30% of the experi-

mental data but predicted blood and kidney levels with lower

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accuracy (greater than 50% difference from experiment),

suggesting that more specifi c tissue interaction mechanisms

need to be developed. Issues surrounding the modeling of

absorption, distribution, metabolism and excretion (ADME)

of nanoparticle drug carriers have been reviewed by Zolnik

and Sadrieh. [ 25 ] Their review again indicated that more spe-

cifi c mechanism-based guidelines are needed to predict the

effi cacy/risk balance in the development of nanotechnology-

derived therapeutics with particular emphasis on delivery via

angiogenic tissues. As we will see, the work presented in our

study has provided a mechanistic understanding of NP des-

position critical to application of PK models to drug delivery.

Our study provides quantitative relationships between NP

properties and deposition rates not previously available.

Previous nanoparticle deposition studies have exam-

ined nanoparticle size versus circulation half-life in animal

models [ 26 , 27 ] and in the chicken embryo. [ 28 , 29 ] The fi rst nano-

particle size study, performed by Yuan et al. [ 27 ] examined

size-dependent permeability of small nanoparticles (2–5

nm diameter proteins and 90 nm diameter PEGylated lipo-

somes) into xenografted human tumors in mice. There was

a slight decline in permeability to the larger liposomes com-

pared with the protein particles. In another example, Lewis

et al. examined the utility of cowpea mosaic virus (30 nm

diameter, CPMV) for intravital imaging of angiogenic tis-

sues. [ 28 ] They saw direct evidence of nanoparticle uptake into

vascular endothelial cells of the chicken embryo CAM and

compared native CPMV, PEGylated CPMV and 40 nm diam-

eter fl uospheres. The latter two nanoparticles showed lower

absolute uptake. No estimate of CPMV surface charge was

given. In another study, Smith et al. examined a series of QDs

injected into the CAM blood stream and found that there

was longer persistence of aminoPEG dots versus polyacrylic

acid QDs [ 29 ] suggesting greater deposition of aminoPEG QDs

into the endothelium. Dreher et al. examined a size series of

dextran nanoparticles for their propensity to accumulate in

mice tumor models [ 30 ] and found an anticorrelation between

nanoparticle size and angiogenic vascular permeability. More

recently, Bawendi and co-workers [ 26 ] designed a size series

of QD-based nanoparticles (12, 60 and 125 nm diameter) to

image tumors in mice and showed that the main difference in

the particles’ behavior was due to size. They tracked the dis-

tance from the tumor blood vessel wall and found that only

the 12 nm particle traveled any signifi cant distance, 100 mm

in 120 min. Many of the above studies measure the convo-

lution of nanoparticle propensity to extravasate and subse-

quently disperse in surrounding tissue with size and charge.

Therefore, it is challenging to use these results in a quanti-

tatively predictive model without fi rst deconvoluting the two

processes.

In the current study, we use a fl uorescence-based assay

to measure the effects of NP properties on deposition into

the angiogenic tissues of the CAM. It is possible to quanti-

tiatively determine nanoparticle deposition kinetics if the

concentration of nanoparticles can be measured in the blood

stream. Fluorescence-based measurement of nanoparticle

concentration in the blood stream is not trivial. Using direct

fl uorescence intensities may give misleading information, as

aggregation of nanoparticles may not change the average

Verlag GmbH & Co. KGaA, Weinheim small 2013, DOI: 10.1002/smll.201201848

Nanoparticle Accumulation in Angiogenic Tissues

Figure 1 . a) Microscope set-up for micro-injection into and fl uorescence correlation spectroscopy measurements of chicken embryo blood vessels. (See text for details of experiment). b) Example of nanoparticle solution injection into a CAM blood vessel. Note the laminar fl ow of the clear injectate solution. c) Confocal fl uorescence image of a fi xed blood vessel that was injected with QDs. The sample shows a venule of approximately 70 μ m inner diameter that was extracted from an embryo following systemic injection of 300 μ L of 40 nM amino605 QD solution. Fluorescence is evident on the one side of the blood vessel but less apparent on the opposite side. The sample was fi xed 10 minutes after injection.

fl uorescence rate. However, fl uorescence

correlation spectroscopy (FCS) reports

on aggregation of nanoparticles in solu-

tion [ 31 , 32 ] and therefore provides a method

to measure accurately nanoparticle con-

centration in the blood, as we have pre-

viously demonstrated. [ 31 ] Briefl y, FCS

measures and correlates fl uctuations in

fl uorescence intensity from a well-defi ned

volume of excited molecules, where the

fl uctuations result from a changing number

of fl uorescence emitters (fl uorophores)

within the volume. Thus, any phenomenon

that alters the number or diffusion behav-

iour of fl uorophores within the volume

will induce a change in the time depend-

ence of fl uorescence intensity fl uctuations

around the average. These effects arise

from simple diffusion, fl ow, chemical reac-

tivity, aggregation, or photochemistry. The

time scales of the fl uctuations are related

to the kinetic rates of the phenomena.

The goal of the current study is to

quantitatively determine the contribution

of nanoparticle size and charge to bioac-

cumulation in the CAM angiogenic tissues,

as this can then inform the development

of nanoparticle-based drug delivery and

diagnostic imaging vehicles. To our knowl-

edge, there is no study that covers the

range of nanoparticles investigated here

and that separates NPs exiting the blood

vessels from local tumor distribution. It

is challenging to develop a single type of

nanoparticle with broadly tunable size

and charge. Therefore, we chose to study

a series of different types of nanoparticles

(polymer coated quantum dots, QDs, polystyrene nanospheres

and liposomes) that provide a range in diameter from 24–500

nm and in surface zeta potential from ∼ 40 mV to − 66 mV. We

have employed the chicken embryo CAM as our primary

angiogenic tissue model organism and also have examined

nanoparticle charge effects in zebrafi sh embryos and cultured

human umbilical vein endothelial cells (HUVEC). We have

measured nanoparticle deposition kinetics and distributions

and related the quantitiative data to nanoparticle proper-

ties in an attempt to provide broad ranging insight into the

mechanism of deposition into angiogenic tissues. Our results

suggest that one can tune the angiogenesis-related NP phar-

macokinetics by size, regardless of the nanoparticle chemistry.

2. Results and Discussion

2.1. Nanoparticle Charge and Accumulation in Angiogenic Tissues

Imaging of microinjection of nanoparticle solutions into the

CAM blood vessels provided preliminary insight into charge

© 2013 Wiley-VCH Verlag Gmbsmall 2013, DOI: 10.1002/smll.201201848

effects. Figure 1 shows the microscope set-up (Figure 1 a) and

results of a typical injection (Figure 1 b,c) into a moderate

sized blood vessel ( ∼ 150 μ m diameter, Figure 1 b). Near the

injection points, fl ow was generally laminar as indicated by

the streamlines of clear fl uid. Mixing occurs further down-

stream where blood vessels are more serpentine. Fluores-

cence imaging of the CAM (Figure 1 c) reveals that amino605

CdSe/ZnS QDs (amino terminated polyethylene glycol, emis-

sion maximum 605 nm, ζ = − 10 mV) are localized to the wall

of a CAM venule, preferentially on one side of the vessel.

This non-uniform vessel wall deposition could be attributed

to the distribution of injectate solution immediately down-

stream of the injection site where the injectate encountered

only one side of the venule. This qualitative, rapid redistri-

bution behavior occurred only for particles that had small

negative to positive zeta potential charge ( − 10 to + 41 mV,

Table 1 ).

It is important to note the slight negative potential of the

amine-terminated particles. This range of zeta potential meas-

urements for amine-functionalized polystyrene NPs is not

uncommon. [ 33 ] It was suggested by Roebben et al. that soni-

cation, while necessary to maintain a monodisperse sample,

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K. Yaehne et al.

4

full papers Table 1. Blood deposition rate constants, hydrodynamic radii and zeta potentials of nanoparticles.

Nanoparticle k fast [s − 1 ]

k slow [s − 1 ]

r H [nm]

ζ ± std dev [mV]

QD-AminoPEG 605 2.42 0.02 12 − 10 ± 5

QD-Methoxy 605 0.01 0.01 13 − 15 ± 5

QD-Carboxy 605 0.04 0.01 14 − 25 ± 7

DOTAP Lipsosomes > 3 – 55 41 ± 11

DOPC Liposomes 0.003/0.002 – 50/82 − 40 ± 10

Amino Polystyrene nanosphere > 3 100 − 10 ± 5

Carboxy Polystyrene nanosphere 0.02 – 20 − 66 ± 12

Carboxy Polystyrene nanosphere 0.003 – 50 − 60 ± 11

Carboxy Polystyrene nanosphere 0.002 – 100 − 53 ± 7

Carboxy Polystyrene nanosphere no loss No loss 250 − 50 ± 5

could degrade the surface chemistry and remove surface

species. It is also possible that for amino-PEG coated nano-

particles, the amine termination does not completely com-

pensate for the negative charge of the PEG. Zeta potential

Figure 2 . Distribution pattern of Amino- and Carboxy-terminated quantum dots in Zebrafi sh Caudal Tail Plexus. In all panels green represents GFP expressed in endothelial cells, while red represents Quantum dots (A,B). The ventral-most vessel of the caudal tail plexus in a 53 hpf zebrafi sh embryo 0.5 hours post-injection of quantum dots is portrayed in uninjected animals. C) Injected amino-terminated quantum dots (Amino-QD) form a visible punctate pattern along the vessel walls 30 minutes after injection (white arrows). D) At the same timepoint, a large portion of injected Carboxy-QDs remain circulating in the lumen, often in large aggregates of the Carboxy-QD (blue arrows). Circulating aggregates are rarely observed in Amino-QD injected embryos. E) and F) are overlays for the amino-QD and carboxy-QD and GFP images, respectively.

measurements are not an absolute indica-

tion of surface charge but do give a rela-

tive idea of particle charge in relation to

other particles, which we can use to pre-

dict the behaviour of the NPs.

To determine whether or not this charge

dependent, fast accumulation is specifi c to

the CAM blood vessels, analogous experi-

ments were performed using the zebrafi sh

embryo as an alternate model for angio-

genesis. Zebrafi sh embryos were injected

with equivalent concentrations of nano-

particles (quantum dots) that had either

amino-PEG or carboxy-poly acrylic acid

terminated surfaces ( ζ = − 10 and − 22 mV,

respectively) to assess qualitatively, charge

dependent accumulation behavior. The

distribution and dynamics of the quantum

dots were examined using confocal micro-

scopy imaging of live embryos. Figure 2

displays the fl uorescence confocal micro-

scopy images of zebrafi sh embryo blood

vessels. The amino-terminated quantum

dots were observed to adhere more quickly

to the endothelial layer of the blood ves-

sels. This was evidenced by the immediate

and lasting small punctate nature of the

red channel images, which has been shown

to indicate cellular internalization of fl uo-

rescent particles. [ 34–39 ] By contrast, similar

punctate patterns were not observed for

the carboxy-terminated quantum dots

in the short term. Larger clusters of red

signal may be evidence of quantum dots

in the lumen of the blood vessels, since

the fl ow is quite slow in the tortuous small

vessels.

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HUVEC were used as a system to examine the inter-

action of nanoparticles of differing charge directly with

endothelial cells. We compared amino- ( ζ = − 10 mV) and car-

boxy- ( ζ = − 53 mV) terminated 200 nm diameter polystyrene

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Nanoparticle Accumulation in Angiogenic Tissues

Figure 3 . HUVEC grown on a thin coating of collagen and exposed to 200 nm polystyrene nanoparticles. A,B) Confocal microscopy images of cells exposed for one hour to amino-modifi ed particles (A) and carboxy-modifi ed particles (B), from left to right of cell membrane stained with CellMask, nuclei stained with Hoechst, red nanoparticles, and the overlay of the three. Scale bar applies to all images and denotes 50 μ m. C,D) Number of adherent and internalized particles over time per cell surface area (n = 3). Cells were incubated at 37 ˚C with either positive (C) or negative (D) particles, and fl uorescence intensity was measured. The goodness of fi t (R 2 ) for each fi tted curve is 0.935 (C) and 0.911 (D).

nanoparticle (at equivalent concentrations) uptake behavior,

as shown in confocal microscopy images in panels A and

B of Figure 3 . It can be seen that the particles are brighter

in panel A than they are in panel B, indicating increased

adsorption of the amino-terminated polystyrene spheres over

the carboxy-terminated ones. In addition, from panels C and

D in Figure 3 , we found that the adsorption rate for amino-

terminated spheres was 1.3 × 10 7 M − 1 s − 1 , whereas the car-

boxy-terminated spheres was 1.8 × 10 6 M − 1 s − 1 (see Section

S3, Supporting Information for deposition rate measurement

details). Intuitively, this difference in rates can be observed in

the y-axis by the larger number of amino particles per surface

area in comparison to the carboxy-terminated particles. Thus

the less negative nanoparticles show a 10x faster adherence to

the endothelial cells, supporting our hypothesis that a similar

charge selection for fast adhesion to the endothelium occurs

in the chicken embryo and zebrafi sh embryo. Moreover, the

punctate nature of internalization is evident in this fi gure.

We next set out to measure quantitatively nanoparticle

deposition kinetics in the CAM and determine how charge

and size infl uence rate constants. Thus, a time series of auto-

correlation decays (ACDs) were measured shortly after

systemic injections of nanoparticles into large vessels signifi -

cantly upstream of the region of interrogation. Systemic injec-

tions with subsequent 3-minute circulation time were found

© 2013 Wiley-VCH Verlag Gmbsmall 2013, DOI: 10.1002/smll.201201848

to be optimal for FCS measurements. [ 24 ] Figure 4 a shows a

sample of a partial data set used to measure the nanoparticle

blood concentration versus time, where each point in the

graph is derived from each corresponding color in the inset.

Figure 4 b gives an example of a full data set of the number of

nanoparticles in the CAM blood stream focal volume versus

time post injection. From this we are able to obtain a decay

rate constant from the decay fi t shown in red. The concen-

tration of emitters is derived from the ACD amplitude, G(0),

of each of the graphs in the Figure 4 a inset (see Section S1,

Supporting Information). Since FCS differentiates between

loss due to deposition and apparent loss due to aggrega-

tion, [ 31 ] only kinetics data (rate constants and number of

particles taken up) that were not infl uenced by aggregation

were used. The aggregation could be induced by interactions

with platelets, although for PEGylated nanoparticles, this

is rare. [ 40 ] Aggregation was observed in no more than 10%

of the data collection runs. Interestingly, a slight decrease

in the fractional number of nanoparticles leaving the blood

stream was observed for increasing size and/or charge, but

this trend is within experimental error (see Figure S8, Sec-

tion S1, Supporting Information). Estimates of nanoparticle

mass balance through fl uorescence imaging suggest that the

loss of nanoparticles from the chicken embryo blood stream

is largely due to accumulation into the endothelial layer of

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K. Yaehne et al.full papers

Figure 4 . a) Simplifi ed example of nanoparticle loss from the bloodstream versus time after injection. The concentration data are taken from a series of autocorrelation decays collected (ACDs) using TPE-FCS within a 200 μ m venule following systemic injection of 100 μ L of 20 nM methoxy565 solution. ACDs are shown in the inset of panel a). The dotted line is a single exponential decay fi t to the data. b) Complete data set from one CAM injection of number of 20 nm polystyrene nanopsheres in the focal volume versus time post injection. The red line is a single exponential decay fi t to the data. c) High resolution fl uorescence deconvolution microscopy image of an arteriole in the CAM following systematic injection of 200 μ L of 40 nM carboxy655 QD (red channel). The blue channel is a nucleic acid stain. Scale bar is 5 μ m. d) High resolution fl uorescence deconvolution image of an intact arteriole from the CAM after injection of amino-PEG605 QD (yellow channel). Blue and red channels are nuclear and membrane stains, respectively. Scale bar is 15 μ m.

the CAM blood vessels (Figure 4 c and d). Analysis of the

chicken embryo organs revealed almost no accumulation of

nanoparticles (data not shown). For less negatively charged

nanoparticles ( ζ > − 10 mV), the kinetics were best modelled

using a bi-exponential decay (see Figure S5c), suggesting two

mechanisms for nanoparticle loss; rapid adherence to the vas-

cular endothelium or slower deposition into nanofenestra-

tions (see Section S1, Supporting Information for details on

all kinetics data used). For 110 nm diameter DOTAP lipo-

somes ( ζ = + 41 mV) adherence to the vessel wall was so rapid

Figure 5 . Plot of rate constant for loss of nanoparticles from the CAM blood stream versus zeta potential. In the left panel a cut off is apparent near − 10 mV, where nanoparticles larger zeta potentials display rapid loss. The right panel is an expansion of the more negative region of the plot. No clear trend between zeta potential and rate constant is found. Data were taken from Table 1 .

that the deposition kinetics could not be

measured accurately. In addition, the non-

uniform amino-PEG quantum dot distri-

bution shown in Figure 1 c suggests there is

very rapid nanoparticle deposition into the

endothelial layer. This is further evidenced

by the relatively higher fl uorescence that

was observed for DOTAP liposome accu-

mulation in the blood vessel walls very

shortly after nanoparticle injection (see

Figure S9). Signifi cantly, more negatively

charged species displayed a lower rate

constant for exiting the blood stream

(Table 1 ) and tended to follow a single

exponential decay (Section S1, Figure S5).

This trend is similar to that observed for

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HUVEC although the rate constants differ by two orders

of magnitude in the CAM (for similar sized QDs). It may

be that the endothelial cells of the angiogenic tissues in the

CAM carry a greater negative charge than cultured HUVEC

(31). This would enhance the difference in the rate constants

between carboxy- and amino- terminated nanoparticles in

CAM versus HUVEC.

The dependence of rate constant for loss of nanoparti-

cles from the blood stream of the CAM is plotted versus zeta

potential in Figure 5 . This plot reveals two points. The fi rst

rlag GmbH & Co. KGaA, Weinheim small 2013, DOI: 10.1002/smll.201201848

Nanoparticle Accumulation in Angiogenic Tissues

Figure 6 . Plots of the rate constants for nanoparticle loss from the blood stream of chicken embryos versus a) the particle radius, b) the inverse of the particle radius, and c) the inverse squared of the particle radius. The plots include three different types of nanoparticles with negative zeta potentials in the range − 20 to − 66 mV. d) Fluorescence confocal image of an intact CAM blood vessel that was injected with 100 μ L of a 160 nM carboxyPAA 655 QD solution. Blue regions represent signal detected from a nucleic acid stain (Hoescht 33258) to indicate location of endothelial cells and red-pink regions indicate signal from the injected QDs. Scale bar is 10 μ m.

is that there is an apparent cutoff around − 15 mV. The loss

is much more rapid for particles whose zeta potentials are

more positive than this cutoff. The second is that for nano-

particles with zeta potential more negative than − 15 mV, the

rate constant is small, with no apparent dependence on the

zeta potential value.

The effect of charge on deposition into angiogenic tissues

is challenging to assess quantitatively. Elegant work by Del-

lian on charged serum proteins [ 41 ] and later by Krasnici et al.

on liposomes [ 42 ] suggests a rapid adherence of positive nano-

particles to the angiogenic endothelium surrounding tumors.

However, adherence rates and the apparent charge cutoff for

this effect were not presented. Lewis et al. [ 28 ] observed nan-

oparticle uptake into CAM vascular endothelial cells with

enhanced uptake for positive particles, without accounting

for size differences in the nanoparticles. The longer persist-

ence of aminoPEG dots versus polyacrylic acid QDs in the

CAM observed by Smith et al. [ 29 ] could be rationalized by

greater adherence and possibly uptake of aminoPEG QDs

into the endothelium. In their work, Smith et al. did not dif-

ferentiate between nanoparticles in the lumen and those in

or on the endothelium.

Our current study also evaulates the different interactions

of nanoparticles with angiogenic tissues, based on charge.

We demonstrate that there is much more rapid adherence

of more positive nanoparticles to endothelial cells across

three species (cultured HUVEC, zebrafi sh and chicken).

© 2013 Wiley-VCH Verlag Gmsmall 2013, DOI: 10.1002/smll.201201848

Moreover, from the CAM study we can suggest a cutoff for

this interaction of approximately − 15 mV (Figure 5 ). Nano-

particles more negative than –15 mV display a stronger pro-

pensity to exit the blood vessels slowly (possibly through

fenestrations), whereas nanoparticles with a potential greater

than this tend to adhere to the endothelium (Figure 3 and

Supporting Information, section S3). We used this cutoff to

help separate size and charge effects of apparent deposition

into the angiogenic tissues.

2.2. Effects of Size on Nanoparticle Accumulation

We propose that studying more negative nanoparticles ( ζ

< − 15 mV), with smaller rate constants, will permit the

measurement of size dependencies and thus allow the sep-

aration of charge and size effects. Indeed, for the CAM

system, using the more negative, carboxy-terminated PAA

quantum dots, carboxy-terminated polystyrene nanospheres,

and 1,2-dileoyl-sn-3-glycero-3-phospocholine (DOPC) lipo-

somes, there appears to be a trend in rate constant versus

size (Table 1 , Figure 6 a-c). That the rate constants are much

smaller than those for the amino-terminated nanoparticles

may result from electrostatic repulsion with the endothelial

cells, which have a net negative membrane charge. [ 25 ] This

suggests that direct endothelial membrane adherence may

not be the mechanism of deposition for nanoparticles with

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K. Yaehne et al.full papers

Figure 7 . Schematic representations depicting the relative sizes of the nanoparticles compared to the angiogenic fenestrations in the vessel wall. It is proposed that the linearity of the k vs. 1/r 2 plot is derived from a loss mechanism that depends primarily on the relative sizes (i.e. footprints) of the particles.

a zeta potential less than − 15 mV. Indeed more negative

nanoparticles are distributed within the endothelial layer

after systemic injection into the CAM blood vessel system

(Figure 6 d, for polyacrylic acid coated, carboxy605 QDs). It

is notable that no loss was observed for negative nanoparti-

cles whose diameters were 500 nm or greater (see Figure S6).

This would likely be a consequence of the fenestrations

between the endothelial cells having an effective diameter of

500 nm or less, and is consistent with those found in rodent

tumor models [ 27 ] and in the angiogenic tissues surrounding

human tumors. [ 43] ]

When the rate constants are compared with nanoparticle

size (nanoparticles with ζ < − 15 mV), a nonlinear anticorrela-

tion between rate constant and size (r) can be seen (Figure

6 a) suggesting an inverse relationship. If the rate constants

depend on the diffusion coeffi cients, then a plot of k vs. 1/r

should be linear. Such a plot was also non-linear (Figure 6 b),

suggesting that the deposition rate constant is not directly

proportional to diffusion rate. When the rate constant was

plotted versus 1/r 2 for the nanoparticles (including carboxy-

polyacrylic acid coated QDs, carboxy-terminated polystyrene

nanospheres and DOPC unilamellar vesicles) a linear rela-

tionship is observed (Figure 6 c). It follows that either the sur-

face area or the footprint of the nanoparticles is related to

the mechanism of deposition into the endothelial layer. Since

there appears to be no charge dependent relationship that

agrees with 1/r 2 (see zeta potentials in Table 1 and Figure 5 ),

8 www.small-journal.com © 2013 Wiley-VCH

this therefore suggests that the footprints of nanoparticles

are related to their deposition rate constants ( Figure 7 ).

In a random nanoparticle-fenestration collision regime,

this 1/r 2 geometric dependence would arise because there is

a greater probability of a smaller area object passing through

an opening of constant area. The rate of passage is propor-

tional to the number of objects that can occupy the opening.

For spherical nanoparticles, the occupation number in the

opening of a fenestration is proportional to 1/r 2 (as shown

in Figure 6 c). Therefore, for nanoparticles where adherence

to the endothelium does not dominate loss from the blood

stream (Table 1 ), we suggest the rate of deposition into the

endothelium of angiogenic blood vessels is predominantly

based on the nanoparticle footprint area and is independent

of NP surface chemistry.

There have been no previous studies that assessed, quan-

titatively, nanoparticle deposition rates versus nanoparticle

size while separating effects due to surface charge. However,

we can still compare our results with prior, qualitative work.

Our results are consistent with the size study by Yuan et al.

(2–5 nm proteins and 45 nm PEGylated liposomes), [ 27 ] where

there was a slight decline in angiogenic vascular permeability

to the larger liposomes compared with the protein particles,

similar to the trends observed presently. The size-related,

QD-tumor study by Bawendi and co-workers [ 26 ] indicated

that only small nanoparticles ( ∼ 10 nm diameter or less) pen-

etrate signifi cantly into the tumor mass. Since their study was

directed towards optimizing nanoparticle design for tumor

imaging, they did not measure extravasation rates, but rather

circulation half-lives. It is challenging to directly compare

half-lives with deposition rates across species, because of the

vastly different angiogenic tissue volumes for xenografted

tumors in mice and in the CAM, but the trends are similar.

Dreher et al. [ 30 ] examined rhodamine labeled dextrans of

various molecular weights for apparent permeability, P app , (a measure of the fl ux, i.e. rate) across the endothelial walls

surrounding a xenografted tumor in a mouse model. They

plotted P app versus hydrodynamic radius ( Figure 8 a) and

observed an anticorrelation between the two parameters,

similar to that from Figure 6 a. Signifi cantly, we have replotted

the data as P app vs. 1/r and 1/r 2 (Figure 8 b and c, respectively).

The plot of P app vs. 1/r 2 is linear (R 2 = 0.98), suggesting agree-

ment with our current study and further indicating that this

size relationship may indeed be universal for endothelial per-

meability to nanoparticles.

3. Conclusion

We have demonstrated the successful use of the CAM to

study the transport of nanoparticles. In this work, the effects

of nanoparticle zeta potential and size on deposition rate con-

stants into angiogenic tissues have been effectively separated.

Also, our results help to deconvolute the data from these pre-

vious studies by quantitating interactions specifi cally with the

endothelial layer of new blood vessels, whilst eliminating the

convolution of dependences on local blood fl ow and blood

pressure in the complex tumor environment. Finally, our

results suggest that the radius squared is the most important

Verlag GmbH & Co. KGaA, Weinheim small 2013, DOI: 10.1002/smll.201201848

Nanoparticle Accumulation in Angiogenic Tissues

Figure 8 . Apparent permeability, P app , of the vascular endothelial wall surrounding a mouse model tumor to a dextran nanoparticle size series (26). a) P app as a function of increasing nanoparticle radius. b) P app as a function of inverse radius. c) P app as a function of inverse radius squared. Linear regression fi t (R 2 = 0.98) also shown.

parameter in predicting deposition rates into angiogenic tis-

sues when nanoparticles carry moderate negative zeta poten-

tial (less than − 15 mV). The deposition rate dependence on

the footprint area of the nanoparticles holds for fi ve different

general surface chemistries spanning solids (polystyrene, dex-

tran), lipid liquid phase (DOPC) and semi solid (poly acrylic

acid, polyethylene glycol) and two species (chicken and

mouse). We identifi ed specifi c nanoparticle parameters that

dictate the deposition rates in the CAM from a drug delivery

perspective. Consequently, the CAM model may be useful

in assessing nanoparticle toxicity. The study presented here

has added to our understanding of the mechanism of nano-

particle deposition into angiogenic tissues but also presents a

new biological model that can accelerate our understanding

of nanoparticle toxicity and tumor targeting.

4. Experimental Section

All commercial nanoparticles (QDs and fl uospheres) were obtained from Invitrogen (Burlington, ON) and diluted into phosphate buffered saline to a concentration of 10 nM prior to injection. Fluorescence (1,2-dioleoyl- sn -glycero-3-phosphoethanolamine- N -(lissamine rhodamine B sulfonyl) (ammonium salt)) labeled 1,2-dileoyl-sn-3-glycero-3-phosphocholine (DOPC) and 1,2-dileoyl-sn-3-glycero-3-trimethylammonium-propane (DOTAP) liposomes were prepared via extrusion as described in detail in section S2 of the Supporting Information. Nanoparticles’ hydrodynamic radii in buffer solution were characterized via dynamic light scattering (Malvern Nano ZS) and FCS. Zeta potentials were also measured (Malvern Nano ZS, details in Section S2, Supporting Information). All nanoparticles were tested for stability in chicken blood sera as

© 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheimsmall 2013, DOI: 10.1002/smll.201201848

described previously. [ 31 ] They were all found to be stable for the timeframe over which they were used (1–3 days).

The chicken embryo CAM was prepared according to Samkoe et al. [ 44 ] and detailed in Section S1 of the Supporting Infomation. Systemic injections and measurements were performed on day 9 PF. A photograph of an intravenous injection into a blood vessel of the CAM with a glass micro-needle ( ∼ 0.02 mm tip diameter) is shown in Figure 1 B. Injections were typically 50–100 μ L. The blood is cleared by the injectate, which then remixes with blood as the bolus circulates, typically 2-3 round trips ( ∼ 1 min). [ 31 ] FCS measurements were taken at least 100 μ m downstream to avoid artifacts due do occasional leakage of blood at the injection site.

Two-photon excitation-FCS (TPE-FCS) data were collected and autocorrelation decays calculated in real time using an ALV 3000 cor-relator board. The sample was excited with a 780 nm (100 fsec) laser and signal collected in an epifl uorescence confi guration through a 1 cm working distance, 0.4 NA objective lens (Zeiss, Canada). This arrangement delivered a TPE volume of approximately 33 fL (an ovoid

with axes, 3 and 10 μ m). [ 31 ] Autocorrelation decays were analyzed as described in the Section S1 of the Supporting Information (Figure S4).

In addition to TPE-FCS measurements, confocal fl uorescence imaging of nanoparticle distributions in the CAM, zebrafi sh and HUVECs was performed. For the CAM, nanoparticles and/or dye (1 mg/mL of Hoescht 33258; ∼ 40 nM QD) were injected into blood vessels and allowed to circulate for 2−3 hours. Vessels were then fi xed with formaldehyde and laid fl at on slides or cross-sectioned for imaging. Sectioning was performed using a Leica CM1850 cryostat microtome (kindly made available in the laboratory of Dr. William Stell, University of Calgary) at − 20 ° C with the sectioning thickness set between 8 and 30 μ m. Tissue was embedded in Tissue-Tek embedding medium (Sakura, CA) prior to sectioning. Fixed blood vessel samples were imaged using a Leica DM RXA2 upright confocal microscope equipped with a 63X DIC-D oil immer-sion lens (with a numerical aperture of 1.32). Further details can be found in the Supporting Information.

Zebrafi sh embryos were immobilized with 5% tricaine in E3 solution prior to injection of approximately 5–10 nL of amino or carboxy conjugated QD 605 solution (Invitrogen, Q21501MP and Q21301MP respectively). Injected embryos were embedded in 1% low-melting-point agarose after 10 min of recovery. Images were taken, on a Zeiss LSM510 Meta confocal microscope at 1 μ m intervals using a 40x objective lens, of 3 independently injected embryos at each time point and injection type. Images were taken at 30 min post-injection (30 min p.i.), 1.5 hpi, and 3.5 hpi. The images presented are a maximum intensity projection of 5 con-secutive sagittal confocal slices of the zebrafi sh caudal tail plexus, processed with a KalmanStack Filter using ImageJ.

HUVEC (Lonza, Walkersville, MD, USA) were cultured according to manufacturer’s directions in endothelial growth medium (EGM,

9www.small-journal.com

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Supporting Information

Supporting Information is available from the Wiley Online Library or from the author.

Acknowledgements

The authors are grateful to the Natural Sciences and Engineering Council of Canada and the Canadian Institutes of Health for a Col-laborative Health Research Project Grant. We thank Warren Chan (University of Toronto) for insightful discussions.

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Received: July 31, 2012 Revised: October 11, 2012Published online:

erlag GmbH & Co. KGaA, Weinheim small 2013, DOI: 10.1002/smll.201201848