TUTORIAL Biology for the Physicist, Chemist and Engineer

TUTORIAL

Biology for the Physicist, Chemist and Engineer

Joachim H. Clement

Klinik für Innere Medizin II,

Abt. Hämatologie und Internistische Onkologie,

Universitätsklinikum Jena

E-mail: joachim.clement@med.uni-jena.de

Schedule

• Part 1

– Introduction

• Nanoparticles in biomedicine

– Magnetic Nanoparticles/Nanomaterials

– Biokinetics

– Interaction with Biological Fluids

• General

– Blood

• Part 2

– Interaction with Biological Borders

• Cells

• Tissues/Organs

– Cytotoxicity

• Part 3

– Biodistribution

– Degradation

– Elimination

Tutorial: Biology for the Physicist, Chemist and Eng1neer J.H. Clement

10. International Conference on the Scientific and Clinical Applications of Magnetic Carriers, Dresden 10.-14.06.14

25.06.2014

1

Schedule - Part I

• Part 1

– Introduction

• Nanoparticles in biomedicine

– Magnetic Nanoparticles/Nanomaterials

– Biokinetics

– Interaction with Biological Fluids

• General

– Blood

» Compounds – molecules and cells

» Physico-chemical properties

• Stability

• Part 2

• Part 3



Biomedical applications of nanoparticles

• Drug delivery

• Hyperthermia

• Targeting of cells

• Tracking of cells

• Monitoring of diagnostic parameters



25.06.2014

2

World of nanoparticles

Composition magnetic particle

Physical properties spheres

Targeting ligands face biological systems

Surface chemistry face biological systems

Chou LYT, Ming K, Chan WCW

Chem. Soc. Rev., 2011, 40, 233-245

Natural nanoparticles

Tutorial: Biology for the Physicist, Chemist and Engineer J.H. Clement


Biokinetics of Nanosized Particles

25.06.2014

Oberdörster G et al. Particle and Fibre Toxicology, 2005, 2, 8 doi:10.1186/1743-8977-2-8 (modified)



3

Nanosized Particles in Biofluids

Oberdörster G et al. Particle and Fibre Toxicology, 2005, 2, 8 doi:10.1186/1743-8977-2-8 (modified)

Biological Fluids

Cells/Tissues

25.06.2014



Biological systems

• Biological Fluids

– Blood

– Lymphe

– Liquor

– Urine

– Ascites

– Sweet

– (Breast milk)

• Cells

• Organisms/Organs



4

Blood content



Nanoparticles enter a new world

Ions Ions

Amino acids

Proteins

Lipids

DNA

RNA

Carbohydrates

Hormones

Cytokines

Small molecules

Erythrocytes

Macrophages

Lymphozytes

Granulozytes

Platelets



25.06.2014

5

Blood interferes with Nanoparticles

• protein corona formation

– immediately

– depend on the physico-chemical properties of the nanoparticles

• shell composition

• surface charge

– changes surface charge

• affecting colloidal stability

– agglomeration



Protein Corona – formation protein binding kinetics

25.06.2014

Sahneh FD et al. PLoS ONE, 2013, 8, e64690. doi:10.1371/journal.pone.0064690



6

Protein distribution on nanoparticles – a simple approach

25.06.2014



Protein distribution on nanoparticles – a quantitative approach http://www.biotech.uconn.edu/msf/



7

Protein distribution on nanoparticles – complex protein patterns

25.06.2014

Tenzer S et al., Nature Nanotechnology, 2013, 8, 772-781



Protein corona formation

• is a dynamic process

• and dependent on

– the protein composition

– the time of contact

– the temperature

• proteins are eliminated from medium

• affecting cell interaction



8

Protein Corona leads to a biological identity

25.06.2014 nanowerk.com



Blood interferes with Nanoparticles

• Coaggulation

– prothrombin from tissue

– activated clotting tissue

– activation of coaggulation factor XI

• Clot formation

• Platelet aggregation

• Activation of complement system

• Aggregation of erythrocytes

• Hemolysis (of erythrocytes)



9

The Coagulation Cascade clot



Charge dependent erythrocyte aggregation negative neutral positive

Florian Schlenk, Inst. of Pharmacy, Friedrich-Schiller-University Jena



25.06.2014

10

Nanoparticles can cause hemolysis

25.06.2014 https://ahdc.vet.cornell.edu/sects/ClinPath/



Nanoparticles interact with blood cells

• Erythrocytes

• Macrophages

• Platelets

• Granulocytes

• Immune cells



11

Summary – Part I

• Complex reactions of nanoparticles with blood

• Depending on physico-chemical properties of nanoparticles

• Depending on the biological properties of the blood

• Protein corona formation



25.06.2014

12

TUTORIAL






Schedule – Part II

• Part 1

• Part 2

– Interaction with Biological Borders

• Cells

• Tissues/Organs

– Cytotoxicity

• Part 3

– Biodistribution

– Degradation

– Elimination



25.06.2014

1

Protein Corona leads to a biological identity

25.06.2014 nanowerk.com



Biological systems

• Biological Fluids

• Cells

– Cell surface

– Membrane

– Intracellular “space”

• Cytoplasm

• Organells

– Endosome/lysosome

– Mitochondria

– Nucleus

– Golgi apparatus

• Organisms/Organs

Tutorial: Biology for the Physicist, Chemist and Enieneer J.H. Clement


2

Cells and their abilities to interact with nanoparticles

• Professional phagocytes (Macrophages)

• Proliferating cells

• Cells with transport functions (endothelial cells)

• Docking sites (e.g. growth factor receptors, channels and pores)



A human cell

Compartmentalization

- pH

- enzyme content

- dynamic

 functional areas

Most critical

- mitochondria

- nucleus

TutorVista.com



25.06.2014

3

Cell membrane out Extracellular matrix

25.06.2014 in



Entry into the cell

• Cell membrane of a vital cell is a very restrictive border

• Ion gradients

• Passage through the cell membrane

– Membrane channel

– Receptors/binding site induces change in membrane

– Membrane turn over

– Penetrating peptides

– Forced entry (Transfection, e.g. Magnetofection)



4

Endocytosis

Chou LYT, Ming K, Chan WCW

Chem. Soc. Rev., 2011, 40, 233-245



Intracellular fate of the nanoparticles

25.06.2014



5

Nanoparticles are located in lysosomes nuclear membrane/ nucleus/ vesicle membrane/

Wagner K et al., Appl. Organomet. Chem., 2004, 18, 514




• Nanoparticle shells are degraded in the lysosome

• Liberation of nanoparticles

– Proton sponge effect

• Interaction with components of the cytoplasm

– Nucleic acids (messenger RNA, transfer-RNA)

– Ribosomes

– protein folding machinery



25.06.2014

6


• Penetration into mitochondra or the nucleus

• Impairing essential cellular functions

– Energy production

– Genomic DNA

• Cytotoxicity



Mechanisms of cytotoxicity

• concentration of nanoparticles

– causes oxidative stress by production of reactive oxygen species (ROS)

– activation of transcription factors for proinflammatory mediators

• High surface/size ratio of nanoparticles facilitates generation of free radicals by redox cycling



25.06.2014

7

Reactive oxygen metabolism

Becker LB Cardiovasc Res, 2004, 61, 461-470



Mechanisms of cytotoxicity

• Liberation of iron ions

• Overload of iron ions

– Disturbance of iron homeostasis (balance) causing aberrant cellular processes: cytotoxicity, enzymatic reactions, inflammation, DNA damage  carcinogenesis

• Oxidation status of iron is critical (Fe

2+ ratio)

/Fe

3+

– Fe

3+

is more potent in causing DNA damage than Fe

2+

– Magnetite (Fe

3

O

4

) causes more oxidative DNA lesions than Maghemite (Fe

2

O

3

)



25.06.2014

8

Evaluation of Cytotoxicity

• Using in vitro models:

– fast

– simple

– low costs

– high-throughput

– no ethics

• Methods:

– Metabolic activity

– Membrane integrity

– Proliferation

– Apoptosis



MTS-Assay

Absorption

Formazan

Cytotoxicity assays

Membrane integrity

LDH-Assay

460 nm

ETR

Diaphorase

Ex./Em. [nm]

560/590

Resorufin

Fluorescence

NADH

Lactate

NAD+

LDH

NADH

Resazurin

Pyruvate

MTS ETR reduced

NAD+ mitochondria

LDH mitochondria Franziska Bähring

Dept. Hematology/Oncology

Jena University Hospital

Metabolic activity

NAD+ NADH

ATP ATP mitochondria

Resazurin Resorufin

Fluorescence

Luciferin

Ultra-Glo™

Luciferase

Oxyluciferin

PrestoBlue™-Assay

Ex./Em. [nm] 560/590

Light

CellTiter-Glo ® -Assay

Luminescence

25.06.2014

9

25.06.2014

Cytotoxicity assays necrosis

Ex./Em. [nm]

540/608

Propidiumiodide phosphatidyl serine

130313.002

PS

130313.003

130313.004

AnnexinV-APC

PS

AnnexinV-

APC

Ex./Em. [nm] 633/680

R1 R1 R1

0 0 0 200 400 600 800 1000

FSC-Height

0 0 0 0 0 0 vital

0 Late apoptotic/ necrotic

130313.001

130313.002

130313.003

130313.004

Franziska Bähring

Dept. Hematology/Oncology

Jena University Hospital

100 100 101 102 103 104 100

Propidium Iodide

101 100 102

0

103

1

104

2

102

3

103

10 4

100

0

102

1

100

2

102

Propidium Iodide

104

10 3

102

4

103

Propidium Iodide

0

104

1

100

10 2

101

3

102 101 103 104

Propidium Iodide

104



Sample ID: ungef aerbt

Gate: No Gate

Gated Ev ents: 10000

Total Ev ents: 10000

Sample ID: nur Annexin

Gate: No Gate



Sample ID: nur PI

Gate: No Gate



Sample ID: unbehandelte Zellen

Gate: No Gate



Quad Quad Quad Ev ents Quad Quad Quad Ev ents Quad Quad Quad Ev ents Quad Quad Quad Ev ents % Gated % Total

UL

UR

LL

LR

UL

UR

1

0 0.00

0.01

0.00

LL 99.97

99.97

99.97

LR 2 0.02

UL

UR

151

0 0.00

1.51

0.00

LL 98.47

98.47

98.47

LR 2 0.02

UL

UR

LR

0

1

509

0.01

0.00

0.01

LL 94.90

94.90

94.90

5.09

UL

UR

LL

LR

38

216

95.24

222

95.24

0.38

2.16

95.24

2.22

130313.005

130313.007

130313.008

0 0 0 0 0 0 200 400 600 800 1000 0 0 200 400 600 800 1000 0 0 200 400 600 800 1000

FSC-Height FSC-Height FSC-Height FSC-Height

130313.005

130313.006

130313.007

130313.008

100 100 101 100 101 102 103 104 100 101 100 101 102 103 104 100 100 101 102 103 104 100 100 102 101 102 103 104

Propidium Iodide Propidium Iodide Propidium Iodide Propidium Iodide

File: 130313.005

File: 130313.006

File: 130313.007

File: 130313.008

Gate: G1 Gate: G1

Sample ID: f luidMAG-D 5

Gate: G1

Sample ID: f luidMAG-D 50

Gate: G1 Gate: G1 Gate: G1 Gate: G1 Gate: G1

Sample ID: f luidMAG-CMX 5

Gate: G1 Gate: G1 Gate: G1

Sample ID: f luidMAG-CMX 50

Gate: G1

Gated Ev ents: 9967 Gated Ev ents: 9813 Gated Ev ents: 9967 Gated Ev ents: 9966

Total Ev ents: 10000 Total Ev ents: 10051

Bähring F et al., IEEE Transactions on Magnetics, 2013, 49, 383-388

Total Ev ents: 10008 Total Ev ents: 10006

Quad Quad Quad Ev ents % Gated % Total Quad Ev ents % Gated % Total Quad Ev ents Quad Quad Quad Ev ents % Gated % Total

UL

UR

LL

LR

UL

UR

LR

14 0.14

30 0.30

UL 19 0.19

13


LL 96.95

97.27

96.95

164 1.64

LL 92.01

94.24

92.01

LR 243 2.42

LL 95.72

96.12

95.72

LR 150 1.50

0.13

3.01

LL 94.26

94.64

94.26

LR 220 2.20

130313.009

130313.010

File: 130313.009

Sample ID: f luidMAG-PEI/750/O 5

Gate: G1 Gate: G1 Gate: G1

0 0 0 200 400 600 800 1000

FSC-Height

Gated Ev ents: 9875


0 0 0

Gate: G1

FSC-Height

Gate: G1

200 400 600 800 1000

File: 130313.010

Sample ID: f luidMAG-PEI/750/O 50

Gate: G1

Gated Ev ents: 8545


130313.009

Quad Quad Ev ents % Gated % Total

UL

UR

LL

LR

UL

UR

LL

LR

22

222

63.60

776

89.67

0.16

1.59

63.60

5.57

130313.010

Quad Quad Ev ents % Gated % Total

UL

UR

LL

LR

UL

UR

LL

LR

11

482

5627

2425

0.02

0.66

7.68

3.31

100 100 101 100 101 102 103 104

Propidium Iodide

100 100 100 101 102 103 104

Propidium Iodide

10

Evaluation of Cytotoxicity

• Using in vitro models

• Caution!

– Interference with assay components

– Little/no correlation to in vivo situation

• because of

– no perfect cell-cell contact

– no perfect cell-matrix contact

– mostly 2-dimensional cell culture models instead of 3dimensional cell culture models



Cytotoxicity assays – assay components affect measurements absorption fluorescence

25.06.2014 luminescence

Bähring F et al., IEEE Transactions on Magnetics, 2013, 49, 383-388 fluorescence



11

Nanoparticle derived cytotoxicity

• Cytotoxicity correlates with nanoparticle

– Size

– Shape

– Surface conditions

• Charge

e.g. rods show lower endocytosis rates than spheres



Summary – Part II

• Nanoparticles can enter cells via different routes

• The endosomal/lysosomal pathway is the most common

• Nanoparticles can affect mitochondrial and nuclear processes and can cause cytotoxicity

• Comprehensive analysis of potential cytotoxicity of nanoparticles is crucial for their future biomedical application



25.06.2014

12

TUTORIAL






Schedule – Part III

• Part 1

• Part 2

• Part 3

– Biodistribution

– Degradation

• Shell components

• Core materials

– Elimination



25.06.2014

1

Biokinetics of Nanosized Particles

25.06.2014

Oberdörster G et al. Particle and Fibre Toxicology,2005, 2, 8 doi:10.1186/1743-8977-2-8 (modified)



Fate of nanoparticles

• depends on physico-chemical characteristics

– Size

– Size distribution

– Surface structure

– Charge

– Shell material

• depends on composition of the protein corona

– Biological identity

• depends on interaction tissue/cells



2

Applying nanoparticles into organisms

• Open questions

– Pharmacokinetics

• Organ specific distribution

• unspecific/artificial interactions with tissues and cells

• Degradation, elimination, clearance

• Effect of increased iron content

• repeated application (MRT, drug delivery)



Biodistribution

Applying nanoparticles into organisms :

• Rapid clearance from circulation

• Distribution

– >80% liver, >5% spleen, 1-2% bone marrow

• Nanoparticles affect homeostasis of organs, especially liver and kidney

• Liver

– Metabolic capacity is very high; 10% of the liver is able to fulfil all essential functions

– high regenerative potential

• But: sensitive to inflammation



25.06.2014

3

Biodistribution

• Directed distribution (to cells; to overcome barriers)

– Binding proteins on cell surface

• Receptors, e.g. HER2 receptor

– Ligand

– Antibody

• Glycoproteins, e.g. Mucin1

– Tight junctions

– Transcellular transport (Transcytosis)

• Endothelia

• Airway-epithelia

• Blood-brain-barrier

• Gastrointestinal duct

– … using a magnet

– Blocking cellular interaction/uptake



Biodistribution

• Receptor-mediated

distribution

25.06.2014

Mai WX and Meng H Integr. Biol., 2013, 5, 19



4

Biodistribution - barriers http://www.neuro24.de/show_glossar.php?id=288



Biodistribution - barriers

• Tight junctions http://php.med.unsw.edu.au/cellbiology/images/c/c3/Tight_junction3.jpg



25.06.2014

5

Biodistribution - barriers

• Transcytosis

Caveolae-mediated

Endocytosis http://www.iitk.ac.in/infocell/



Biodistribution

• Blocking cellular interaction/uptake

25.06.2014

Image credit: Mary Leonard, Biomedical Art & Design, University of Pennsylvania

Rights information: University of Pennsylvania/Biomedical Art & Design | http://www.med.upenn.edu/art/info.html



6

Biodistribution

• Methods to study distribution

– Tissue sections/Histology (e.g. Prussian blue)

– Magnetic resonance imaging (MRI)

– Magnetic particle spectroscopy (MPS)

– Magnetic Particle Imaging (MPI)

– Magnetrelaxometry

– Fluorescence based methods

– Multi-spectral Optoacustic Imaging

– …



Degradation



25.06.2014

7

Degradation

• Polysaccharide shells are rapidly degraded

• Magnetite/Maghemite particles

– Degradation needs several weeks

• PEG and other compounds can hinder degradation and clearance of iron oxide based cores (stealth particles)



Degradation

• Magnetite/Maghemite particles

– Degradation needs several weeks

– Change of iron homeostasis

– Oxidative stress

– ROS production

– High intracellular iron content leads to increased risk of cancer (e.g. liver cancer: spindle cell carcinoma, pleomorphic carcinoma)



25.06.2014

8

Reactive oxygen metabolism

Becker LB Cardiovasc Res, 2004, 61, 461-470



Clearance



25.06.2014

9

Clearance - liver

Kupffer cells

Sinusoidal endothelial cells

Parenchyma

Wang et al., Accounts of Chemical

Research, 2013, 46,761-769



Clearance - spleen

25.06.2014


Research, 2013, 46,761-769



10


Research, 2013, 46,761-769

Clearance - kidney mesangial cells tube podocytes



Clearance - lung


Research, 2013, 46,761-769

Interstitial space macrophages

25.06.2014



11

Clearance


Research, 2013, 46,761-769



Summary - Part III

• Vertebrates are complex

• Little is known about long term effects of nanomaterials in organisms

• In vivo application of nanomaterials need global and in-depth understanding of biological consequences

• A Gold standard for cytotoxicity determination is needed



25.06.2014

12

Perspectives

• High content approaches

• Systems biology

• Collecting data – extend the nanoparticle databases

• Bridge the gap between in vitro and in vivo



Take home message

- Nanomaterials are amazing tools -

- Biology offers great opportunites -

Lets bring them together!



25.06.2014

13

Selected references

Bähring F et al., IEEE T. Magn., 2013, 49, 383-388

Becker LB, Cardiovasc. Res., 2004, 61, 461-470

Chou LYT et al., Chem. Soc. Rev., 2011, 40, 233-245

Da Rocha EL et al., Mat. Sci. Eng. C, 2014, 34, 270-279

Kim J-E et al., Arch. Toxicol., 2012, 86, 685-700

Lartigue L et al., ACS Nano, 2012, 6, 2665-2678

Lei L et al., Chin. Phys. B, 2013, 22, doi 10.1088/1674-1056/22/127503

Mahmoudi M et al., ACS Nano, 2013, 7, 6555-6562

Mahmoudi M et al., Chem. Rev., 2012, 112, 2323-2338

Mai WX & Meng H, Integr. Biol., 2013, 5, 19

Oberdörster G et al. Part. Fibre Toxicol., 2005, 2, 8 doi:10.1186/1743-8977-2-8

Prakash YS & Matalon S, Am. J. Physiol. Lung Cell. Mol. Physiol., 2014, 306, L393-L396

Sahneh FD et al., PLoS ONE, 2013, 8, e64690. doi:10.1371/journal.pone.0064690

Singh N et al., Nano Rev., 2010, 1, 5358

Tenzer S et al., Nature Nanotechnol., 2013, 8, 772-781

Wagner K et al., Appl. Organomet. Chem., 2004, 18, 514

Walkey CD & Chan WCW, Chem. Soc. Rev., 2012, 41, 2780-2799

Wang B et al., Accounts Chem. Res., 2013, 46, 761-769



25.06.2014

14

TUTORIAL Biology for the Physicist, Chemist and Engineer

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