THE NEED FOR NANOMATERIAL EVALUATION IN A PHYSIOLOGICALLY RELEVANT MODEL: CONNECTING ENVIRONMENTAL VARIABLES AND NM BEHAVIOR TO TOXICOLOGICAL RESPONSES

Kristen K. Comfort

Department of Chemical and Materials Engineering

University of Dayton

DEFINING THE NANO-BIO INTERFACE

Nano-Bio interface = dynamic physicochemical interactions, kinetics, and thermodynamic exchanges between nanomaterials (NM) surfaces and biological components

Influenced By: Determines:

MOTIVATION

• Tremendous advances have been made in NM characterization, synthesis, and dosimetry

• Parallel progress in biological models needs to be developed and implemented

• Long term goals:• Generate in vitro models that mimic an in vivo system• Improve predictive modeling of NM-behavior and

bioresponses• Design accurate, high-throughput in vitro systems

IN VITRO VS IN VIVO SYSTEMS

in vitro in vivo

Advantages:• Simplified model

• Lower cost• Rapid assessment/ High-throughput

capabilities• Can explore mechanistic response

• Cell line specificity

Advantages:• Complete physiological response

• Inclusion of immune/inflammatory systems

Disadvantages:• Difficult to extrapolate to human

system• Applicability is dependent on design

Disadvantages:• Ethical concerns – Europe is phasing

out• High cost

• Time requirements• Dosimetry and distribution concerns

• Difficult to puzzle out NM mechanisms

IN VITRO VS IN VIVO SYSTEMS

• in vitro • in vivo

Current Limitation:Poor correlation

Need to improve in vitro models to bridge this gap

LET’S EXAMINE A TISSUE/ORGAN SYSTEM

• What are its unique characteristics?

1) 3-Dimensional

LET’S EXAMINE A TISSUE/ORGAN SYSTEM

• What are its unique characteristics?

1) 3-Dimensional

2) Comprised of multiple cell types• (hepatocytes, endothelial, Kupffer)

LET’S EXAMINE A TISSUE/ORGAN SYSTEM

• What are its unique characteristics?

1) 3-Dimensional

2) Comprised of multiple cell types• (hepatocytes, endothelial, Kupffer)

3) Physiological fluid• Interstitial fluid or secreted bile

LET’S EXAMINE A TISSUE/ORGAN SYSTEM

• What are its unique characteristics?

1) 3-Dimensional

2) Comprised of multiple cell types• (hepatocytes, endothelial, Kupffer)

3) Physiological fluid• Interstitial fluid or secreted bile

4) Dynamic environment• Connected to the CVS

PRIMARY GOALS…To transform this:

Into something that is more representative of:

(1)

(2) Which will lead to

augmented in vitro applicability:

IncreasedCorrelation &

Predictive Modeling

EXPERIMENTAL RESULTS

STUDY APPROACH• Target system: Alveolar region

• Model contains:• Human alveolar epithelial cells• Artificial alveolar fluid (AAF)• Dynamic movement

• 60 nm tannic acid gold nanoparticles (AuNPs)• Characterize• Evaluate nano-bio interface

DYNAMIC FLOW

• Introduced to the cell culture system through use of a peristaltic pump

• Tubing was inserted into lid of 24 well plate• Each well was singularly connected, producing

unilateral flow across the surface

• Target volumetric flow rate was selected:• Velocity in tubing = 0.2 cm/s (capillary rate)• Velocity across cells = 0.003 cm/s (diffusion-based

rate)

ENVIRONMENTAL INFLUENCE ON CELL MORPHOLOGY

A549 cells cultured with:

(A) Media, static

(B) AAF, static

(C) Media, dynamic

(D)AAF, dynamicConclusions:

• Dynamic flow induced

elongation• AAF causes

curvature• BOTH are seen in

vivo

AUNP CHARACTERIZATION

Primary size (nm) 65.1 ± 5.3

Agglomerate size (nm) 74.8 ± 4.6

Zeta potential (mV) -31.8 ± 0.9

Ionic dissolution (%) 0.8 ± 0.5

AUNP CHARACTERIZATION

Conclusions: Exposure to AAF significantly altered

AuNP properties and behavior.

400 500 600 7000.0

0.5

1.0MediaAAF

Water

Wavelength (nm)

Ab

sorb

ance

(a.

u.)

Static Dynamic0

100

200

300MediaAAF

Ag

glo

mer

ate

Siz

e(n

m)

* *

Dis

solu

tio

n (

%)

Static Dynamic0

2

4

6 MediaAAF

**

AUNP DEPOSITION• Deposition = percentage of administered NPs that

are bound to the cell surface or internalized• The deposited dose has been strongly correlated to

cytotoxicity

Dep

osi

tio

n (

%)

Static Dynamic0

25

50

75

100 MediaAAF* *

†

Conclusions: In media: dynamic flow

reduces deposition

In AAF: deposition is unchanged due to

sedimentation of large agglomerates

AUNP INTERNALIZATION

• TEM images of

(A) Media, static

(B) AAF, static

(C) Media, dynamic

(D)AAF, dynamic

Conclusions: • Increased AuNP number with AAF• AAF/dynamic – no internalization

NANO-BIO INTERFACE

Conclusions: • Cells maintained altered morphology

• Increased AuNP number with AAF

TAKE AWAY MESSAGE

• It is possible to modify traditional in vitro systems to more closely mimic in vivo models

• We introduced dynamic flow and biological fluids

• NP characteristics and behavior are strongly dependent upon the surrounding environment

• This has been linked to bioresponses

• Therefore, modified in vitro systems allow for identification of novel responses previously unobtainable.

• Bridging the in vitro – in vivo gap

THANK YOU