Nanomedicine Lab | Centre for Tissue Injury and Repair | Institute of Inflammation and Repair | Faculty of Medical and Human Sciences | & National Graphene Institute | The University of Manchester | UK

@Nanomedicinelab www.nanomedicinelab.com

Cyrill Bussy, PhD

Graphene: Biomedical applications & safety profile

Nanomedicine Lab | Engineering & testing nano-carriers for diagnosis & therapy

Nano-carrier platforms

Aptamers

Antibodies

Nucleic Acids

Gadolinium

Indocyanine Green

Doxorubicin

Iron Oxide

Protein based Nano-objects

Fullerene Graphene

(Nano-hydrogels)

Artificially enveloped adenovirus

Liposomes

C nanotubes C nanohorns Gold nanoparticles Nanoswimmers

Graphene | A biomaterial ?

The idea: using graphene as a biomaterial

Solution 1 : using graphene as it is Solution 2 : modifying graphene to make it a biomaterial performing better

than existing ones

what is interesting about graphene (intrinsically) ?

Is graphene a good platform ?

Bitounis et al., Adv Mat, 2013

Graphene as Biomaterial | Current Landscape – Non Covalent Modifications

Graphene Graphene Oxide

Graphene as Biomaterial | Current Landscape – Covalent Modifications

Bitounis et al., Adv Mat, 2013

Long lasting blood circulation

Targeting

Imaging

Biocompatibility

Therapy

Capture

Graphene in Biomedicine | Current Landscape – Most mature bio-applications

The high chemical versatility of graphene, GO and rGO allows the conjugation of a vast variety of small molecules, macromolecules, and bioactive agents => promising biomedical opportunities

Expected (forecasted) applications

Servant et al.,Bioorg. Med. Chem. Lett., 2014 | Bitounis et al., Adv Mat, 2013 | Shen et al, Theranostics 2012

Most Mature applications

Biosensing & Diagnostic Devices 64%

Toxicity 12%

Drug Delivery & Imaging carriers 8%

Antibacterial Agents 4% Tissue engineering & Scaffolds 4%

Photothermal Therapy 3%

Gene Delivery 2%

Biochemistry General 2%

Enzymatic Interations 1%

Drug & Gene Delivery 13%

Graphene in Biomedicine | Current Landscape (2013)

Orecchioni et al, Theranostics, 2015

Status of Graphene publications in the last 7 years for Cancer Fight.

D. Jasim, 1st transfer report, 2014

73%

32%

10%

31% 27%

• Which graphene materials to use?

• How is the interaction with cells ?

• What happens with graphene in the body ?

• Limitations: Toxicity - Biodegradation - Biopersistence?

• Use specific intrinsic properties (conductivity, optical, …) to identify uniqueness of graphene in biomedical application

Novoselov, K. et al., Nature, 2012

Graphene in Biomedicine | the Roadmap View

Kostarelos, K. and Novoselov K., Nat Nanotechnologies, 2014

Kostarelos, K. and Novoselov K., Science, 2014

What do we need to know/study to transform hope into real opportunities ?

STEP 1: Understand the material

Graphene Material engineering and characterization

Understand your material | Specific Physicochemical Features / Proper name

Bussy et al., Acc Chem Res, 2013 Bianco et al, Editorial of Carbon, 2013

It is essential to know and name properly the materials used to prevent confusion, misunderstanding, and generalisation

Ali-Boucetta et al., Adv Health Mat, 2013 + Lozano et al., in preparation

Modified Hummers’ Method for Biologically-relevant GO prepared under endotoxin free conditions

K. Chen et al, J. Mater. Chem. A, 2015,3, 2441-2453

Understand your material | Engineer the Required Materials

Graphene

Graphene Oxide

In ionic (physiological) fluids [e.g. Graphene Oxide ]

Understand your material | Characterisation/Behaviour in Relevant Fluids

The theory The Reality

In water In culture medium In saline solution

Smal

l La

rge

Understand your material | Characterisation/Behaviour in Relevant Fluids

STEP 2: Understand the interactions with cells

Graphene Cell biology

Graphene cell biology | Colloidal Suspension vs Substrate

Bussy et al., Acc Chem Res, 2013

Colloidal suspensions = developing tools for imaging, diagnosis and therapy in nanomedicine

Substrate = platform for cell growth, differentiation in regenerative medicine (and tissue engineering)

Shah, S. et al., Advanced Materials, 2014

Ph

ase

con

tras

t B

righ

t fi

eld

Naïve Exposed to small flakes

Graphene cell biology | Interactions of Cells with Colloidal GNM Suspension

2. UPTAKE

24hrs after exposure

Ph

ase

con

tras

t B

righ

t fi

eld

Naïve Exposed to small flakes Exposed to small flakes

Graphene cell biology | Interactions of Cells with Colloidal GNM Suspension

2. UPTAKE

30 days after exposure

STEP 3: Understand the interactions with tissues

Graphene Pharmacology

Graphene Pharmacology | Biodistribution, Elimination and Accumulation

111InCl3

=

Jasim D et al., Chem Sci, 2015.

Radioactive marker

Required parameters: • Good dispersibility => GO • Small size (blood) => small GO • Able to carry tracer => functionalised

Metabolic Profile

Jasim D et al., Chem Sci, 2015.

1h

4

h

24

h

Lung

Liver

Kidney

Bladder

Spleen

Lung

Liver

Kidney

Bladder

Spleen

Lung

Liver

Kidney

Bladder

Spleen

SPECT/CT Imaging

111InCl3

=

Gamma Scintigraphy

0

2

4

6

8

10

DOTA-111In EDTA-111In GO-DOTA-111In

%ID

de

tect

ed

aft

er

24

hr

Urine

Feaces

GO – DOTA[111In] EDTA[111In] DOTA[111In]

* *

* *

Graphene Pharmacology | Biodistribution, elimination and accumulation

Consequences, long term fate ?

STEP 4: Prove efficacious/valuable function (intrinsic)

Graphene Therapeutics / Diagnostics

HYDROGELS

3D water swollen polymer network

Formed by chemical or physical cross-linking

Equilibrium swelling/shrinking behaviour

High water content and resemblance with natural tissues

Biocompatible

• Pre-programmed drug delivery systems – Multi-layered polymeric matrix: layer loaded with drug and a

‘spacer’ layer

– Release controlled by the degradation of the polymer matrix

• ‘Smart’ materials: – Glucose or enzyme responsive

– pH responsive

– Temperature sensitive

– Electro-sensitive

– Light sensitive

Graphene & drug delivery | Polymeric implants for pulsatile drug release

How to make them responsive to stimuli ?

MAA, MBAM, PPS

70°C, 20 hours

Electrical

stimulation

I)

II)

III)

14C-sucrose loading

into the gel matrix

Servant et al., Adv. Health. Mater., 2012

Servant et al., J. Mat. Chem., 2013

Graphene & drug delivery | Electro-responsive hydrogels for pulsatile drug release

The idea: using the electrical conductivity of C-NMs to release drug from a gel. First, using CNTs

Servant et al., Adv. Healthcare Mater. 2014

In situ polymerisation

Graphene & drug delivery | Electroresponsive hydrogels for pulsatile drug release

The idea: using the electrical conductivity of C-NMs to release drug from a gel, this time using graphene

Ball mill graphene

• Graphene gels outperform MWNT gel hybrid in vivo: higher amounts of 14C-sucrose are released

• Reproducibility between cycles implying less damage upon electrical stimulation

0

10

20

30

40

50

60

70

80

90

100

0 50 100 150

% 1

4C

-su

cro

se r

elea

sed

in

vit

ro

Time (min)

graphene hybrid (0.2 mg/ml)

MWNT hybrid (0.2mg/ml)

Blank gel

0

1

2

3

4

5

6

7

0 50 100 150 200 250

14C

-su

cro

se r

elea

se

in b

loo

d (

%)

Time (min)

MWNT 0.2 mg/ml

Blank gel

graphene 0.2 mg/ml

Graphene & drug delivery | Electroresponsive hydrogels for pulsatile drug release

Servant et al., Adv. Healthcare Mater. 2014

Graphene

Nanotubes

Blank gel

Graphene

Nanotubes

Blank gel

Graphene-based electro-responsive hydrogels perform (Release Drugs) better

Blank gel Graphene gel MWNT gel

Graphene & drug delivery | Electroresponsive hydrogels for pulsatile drug release

Servant et al., Adv. Healthcare Mater. 2014

Graphene-based electro-responsive hydrogels are SAFER

• Gels were implanted subcutaneously and electrically stimulated for 5 mins

• Significant inflammation for MWNT hybrid gels due to gel heating during stimulation

• under normal conditions, GO are non-toxic materials

• under irradiation (light), materials starts to produce oxidative stress related products that induce cell death

The idea: using the intrinsic optical (photothermal) properties of graphene related materials to induce toxicity

Graphene & therapy | Therapies based on induced toxicity

Li et al, Int J Nanomedicine, 2015

Wu et al, ACS nano, 2015

Cancer cells

Bacteria

The idea: using both the chemical functionalization potential (to attract and collect) and the optical absorption (photothermal) properties to kill bacteria

STEP 5: Understand the limitations

Graphene Safety profile: Toxicology / Biocompatibility

Bussy et al., Acc Chem Res, 2013

Graphene safety profile | Potential Limitations (environmental health)

Bussy et al., Acc Chem Res, 2013

Graphene safety profile | Analogy with Carbon Nanotubes (environmental health)?

Diaphragm tissue Inflammatory response

Evaluation of:

Intra-peritoneal injection

(50 µg/animal) n=8-10

After 24 hr & 7 days

Ali-Boucetta et al., Adv Health Mat, 2013

C57 BL/6 mice (6weeks old)

Graphene safety profile | Analogy with Carbon Nanotubes – Mesothelium model

Graphene safety profile | Potential Limitations (beyond environmental health)

Bussy et al., Nanoscale, 2015

Main outcome: Pulmonary system is tissue of highest risk, regardless of administration route. Lungs are organs: • with highest accumulation of GBMs (>100 nm) & • site of reported adverse effects

STEP 6: Put everything into perspective

Graphene For medicine (drug delivery)

Bussy et al., Acc Chem Res, 2013

SAFETY RULES 1. to use small, individual CNMs that macrophages in the body can efficiently

internalize and remove from the site of deposition; 2. to use hydrophilic, stable, colloidal dispersions of CNMs to minimize

aggregation in vivo; 3. to use excretable CNMs or chemically-modified CNMs that can be degraded

effectively.

Graphene in Biomedicine| put everything into perspective

Kostarelos, K. and Novoselov K., Science, 2014

Wick et al., Angew Chem Int Ed Engl., 2014 Bianco, Angew Chem Int Ed Engl., 2013

Graphene in Biomedicine| Proposed Classification

3 essential features for bio-interactions:

• Lateral size • Thickness • Functionalization

Increase in Thickness

Increase in Lateral Dimensions Increase in

Functionalisation

Connect with us:

www.nanomedicinelab.com

@Nanomedicinelab

WP 2 – Health and Environment

Faculty of Medical and Human Sciences | The University of Manchester | UK

Graphene: Biomedical applications & safety profile

Connect with us:

www.nanomedicinelab.com @Nanomedicinelab