Martin C1-C2

Micro and Nanotechnologies in Health
Pr. Donald MARTIN

(don.martin@univ-grenoble-alpes.fr)
Université Grenoble Alpes

(Faculté Pharmacie)
Parcours: M2-HL_TECH, M2-SMB/BDIV, M2-SMB/TCGIT, M2-SMB/SMB, M2-CHIP

MY TEAM Systèmes Nanobiotechnologiques et Biomimétiques (www-timc.imag.fr/synabi)
LABORATOIRE et
TUTELLES
Prep. Biochem. Biotech., 46:546, 2015 Scientific Reports, 3:1516, 2013

Eur. Phys. J., E39:123, 2016 Chem. Commun., 50:14535, 2014
Langmuir, 33:9988, 2017 Symbiotic ion channel-based systems Symbiotic enzyme-based systems Energy Environ. Science, 8:1017, 2015
Scientific Reports, 7:3399, 2017 Electrochimica Acta, 269:360, 2018
ACS Nano, 12:8867, 2018 Mater. Sci. Eng. C, 108:110359, 2020
Small, 15:1805046, 2019 • ANR – project BioWATTS (Univ. Rennes)
Chem. Commun. 55:13152, 2019 • ANR – project IMABIC (CEA-IRIG-SyMMES-CAMPE, LGP2, Univ. Poitiers, Biopic SAS)
• SATT – project BIOPILE (LRB) (IBFC: CEA-LETI, LGP2, UGA-DCM, Sorin Group)
• SATT – project MICROBIOTA SAMPLER (CHUGA)
• SATT – project SYMBIONT (LRB, CHUGA)
• SATT – project ENDOBIOCRINE (GIN)
Biomaterials, 34:10099, 2013
• Ligue Contre le Cancer (2x projects in nanobiotech & cancer biomarkers) (BCI - IMAC) PlosONE, 9(6):e99416, 2014
• Carnot LSI – project EXSITE (Murata Europe B.V.) Trends Biotechnol., 34:757, 2016
Biotechnol. J., 13:1800463, 2018
• Région Rhône-Alpes Auvergne – project ENDOPROB (Pelican Health SAS) Trends Biotechnol., 35:1035, 2017
• InnovaXN – project Nanostructure of Proteins in Membranes (ILL, Surgical Diagnostics Pty Ltd) FR1654213, 2016
FR1552927, 2015
FR1654215, 2016 • (submitted) ANR Equipex+ – project SHyFlex (CEA-LITEN, CEA-LETI, CHUGA) FR1763088, 2017
FR1655187, 2016 • SATT / UGA Out-of-Labs – project UROLOC FR1902392, 2019
FR1654215, 2017 FR1902393, 2019
Nanomedicine, 2020 (accepted, in press)
Bioinspired diagnostic systems Bioinspired therapeutic systems
équipe inventive et innovante capable d'initier des projets; obtenir de fonds pour la recherche provenant de sources publiques et
Forces Locales
industrielles; créer de startups; (équipements spécialisés: patch-clamp électrophysiologie, électrochimie, biophysique, dispositif médicale)
organ-on-chip; microbiote; smart-hybrid-flexible medical devices; electrochemical biosensors; biofuel cells; biomimetic membrane systems;
Sujets de Collaborations Potentielles
engineering with biology
SyNaBi (Martin) + TIMC + INSERM + UGA-CNRS Institut des Technologies de la Santé Grenobloises
Biomimetic Nanotechnology and Health
Pr. Donald MARTIN



… something to think about …
What is bioinspiration ?
How is bioinspiration turned into medical technology ?
What could the future bring ?

Biomimetic … bioinspiration … nanobiotechnology … health … ?
… inspired by or based on biological structures or processes

(a concept that dates from the Egyptian era … and probably earlier)
An ancient Egyptian false toe found on

a female mummy buried near Luxor
The strapping in the foreground bound it onto
the right foot of the female owner
(Tabaketenmut from around 950–710 BC), who
was the daughter of a priest.
Finch J (2011) Lancet, 377:548-549

Figure 2: Views of right foot and prosthesis:
(A) well-healed amputation area covered by intact layer of skin.
(B) Prosthesis attached.
(C) Intravital usage, indicated by scratches on bottom surface
(D) longstanding intravital amputation of the big toe.
Nerlich AG, Zink A, Szeimes U, Hagedorn HG (2000). Lancet, 356:2176–2179 http://www.orthoanswer.org

… by applying nano-micro-biotechnology … example: snake-skin

laser-created textures inspired by the scales on snake-skin ... reduces sliding friction (dry)
(a) Scanning electron

micrograph of a single (a) Friction coefficient for lubricated
snake-skin scale. sliding
(b) images of scales

overlapping in x- and y
direction created on
100Cr6 steel pins, (b) Friction coefficient for dry sliding
(c) images of the scales

arranged in rows created
on 100Cr6 steel pins
Greiner & Schäfer (2015) Bioinspir. Biomim. 10:044001

… by applying nano-micro-biotechnology … example: artificial insect eye
Production of artificial
dragonfly eye insect eye
Kuo et al (2015) Bioinspir. Biomim. 10:056010

… by applying nano-micro-biotechnology … example: artificial insect eye
fast-moving targets could be

detected, in a manner similar to
that of the compound eyes of
insects
Technology applications: mobile camera lenses, credit card fingerprint recognition systems,
medical diagnostic system, surveillance imaging systems, and light-field photography
Kuo et al (2015) Bioinspir. Biomim. 10:056010

… non-medical devices inspired by biological structures
(a) floating fern Salvinia molesta has two types of leaves

(b) water droplet on eggbeater-shaped superhydrophobic trichomes
covering the green floating leaves
(c) SEM images green leaf surface
(d) fast and selective oil absorption by a green leaf
Oil absorption capacity of leaves (Salvinia molesta, Nelumbo nucifera and Pistia
stratiotes) is better than existing commercial (Nanofur, Deurex Pure, Öl-Ex)
Zeiger et al (2016) Bioinspir. Biomim. 11:056003


curiosity
what is the question to ask What keeps the colour in church windows ?
how to find the answer to the question
a dilute Ag salt
does the answer mean anything solution applied to the
surface of glass
heat (500° - 650° C)

causes Ag to penetrate
into the glass
Ag forms a colloidal
distribution of nanoparticles
(10 - 200 nm)
the Ag nanoparticles selectively

scatter light, producing bright from
pale to bright yellow or orange
Nanoparticles create colours in colloidal suspension
curiosity
what is the question to ask
does the answer mean anything
1857: Michael Faraday published method for making gold

colloidal suspensions (”red gold hydrosols”)
1903: Richard Zsigmondy made uniform-sized gold nanoparticles
of 10 nm and an instrument to make the measurements of those
nanoparticles (ultramicroscope)
- solution changed in colour from red to blue when gold
nanoparticles coagulate by removing their electrical charge
Richard Zsigmondy
with salts … this is the method used by modern biochemical
tests (e.g. a pregnancy test)
 first person to use the term "nanometer" explicitly for characterizing particle size
 won the Nobel prize for Chemistry in 1925
curiosity
Schematic representations of:
(A) a thrombin sensor that operates based on fibrin-

mediated aggregation of Au NP,
(B)(C) fibrinogen and thrombin sensors that operate

based on fibrinogen modulation of the formation of
Thr–Au NP aggregates.
Chen YY et al (2011). Gold nanoparticle-based colorimetric assays for coagulation-related

proteins and their inhibition reactions. Biosensors and Bioelectronics, 26:3160–3166
curiosity

 3D bioelectrodes designed for sustainable function when
implanted
 compatible with the internal environment, not causing

inflammation and able to use biomolecules inside the body
genepin-crosslinked
chitosan
+
MWCNT
+
laccase
First 14 days: slight inflammatory reaction (fat necrosis, fibrin,

After 167 days
neutrophils) implanted
After 90 days (C): new tissues, thin layer mesenchymal tissue
After 167 days (E,F): more mesenchymal growth and collagen.
Blood vessels between tissues (vascularised matrix) and thin
connective tissue membrane (D).
Bioelectrode nanotechnology for symbiotic implantable devices

 after the 18th day of implantation, the tele-transmission system wirelessly
charged and discharged the operational GBFC in vivo through a 100 k
load for 30 min each day.
 for 16 days: the GBFC delivered 16 mW/ml continuously during each 30
minute discharge
 reduced long-term performance probably due to inflammatory reaction
compared to non-functional implanted control GBFC (identified at
autopsy)
Enzymatic biofuel cell implanted inside the body

www.ibfc.fr
 main challenge is biocompatibility  eliminate biofouling to ensure stable supply of substrates
 stable supply of substrates  stable level of sufficient power
 choice of appropriate biological components for implanted device
Improve the bioengineering of symbiotic implantable devices

Symbiotic implantable devices ?
Why do we need to do engineering with biological components ?
 To construct devices with better biocompatibility with the body,

 To construct devices that have nano- or micro-scaled dimensions,
 To construct devices that can be manufactured with self-assembly processes,
 To construct devices that can be easily scalable,
 To construct devices that need less energy to operate (thus produce less heat during operation)
Bioengineering of biomimetic membrane systems … with biological components

SyNaBi takes an original approach to bioinspiration
Instead of trying to find artificial materials to mimic biology,

we utilise biological components to engineer symbiotic devices
BIOINSPIRATION
‟utilising the self-assembly of biological molecules to develop
nanostructured biomimetic systems”
‟biotechnology systems are hybrids of synthetic materials that support and

integrate self-assembled biological components”

… argh, too complex ! …
… yes, perhaps too complex !
No, there is a simple and common feature …

… is it simply the technology of biology
But, where is the science … ?

BIOINSPIRED ENGINEERING OF NANOSTRUCTURED SYSTEMS
utilising properties of natural proteins/lipids
membrane ion channels

(engineering cells to modify function)
change in biological function
… bioengineering of
natural systems
extracellular space
GLUT2 glucokinase
glucose glucose Glucose-6-
phosphate
1 = HEPG2ins/g (add insulin gene & GLUT2)
2 = MIN6 (+ve control)
3 = HEPG2ins (add insulin gene) 35 kDa METABOLIC
SIGNALS

4 = HEPG2 (untransfected liver cells) KATP
+
depolarisation K
Blocker: glibenclamide, (glucose)

Activator: diazoxide
Insulin secretion
CLSM
glucose
glucose
Liu GJ, Simpson AM, Swan AM, Tuch BE, Martin DK (2003). ATP-sensitive potassium +
channels induced in liver cells after transfection with insulin receptor and GLUT2
transporter regulate glucose-stimulated insulin secretion. FASEB Journal. 17:1682-1684
diazoxide
utilising properties of natural proteins/lipids
membrane ion channels

(engineering cells to modify function)
… bioengineering of
artificial systems
(external stimulus)
utilises biological components,

makes naturally biocompatible “synaptic” connections with body tissues or
organs,
easily assembled into 3-D array architectures (with nanoscaled dimensions),
easily scalable,
lower operational energy requirements (fJ) compared to state-of-the-art
ENODe systems (pJ) that are designed to mimic biological synapses (1000-fold
less power requirements)

control
ion channel
+ inhibiting signals
drug
The maximum aposteriori
(MAP) state estimates of the
"smart" underlying Markov chain are
ion-transporting computed via the forward
vesicles backward smoothing
algorithm. Also shown are
the observations (Yk)
IEEE Sensors Journal
(2007) 7:1281-1288
+ + stochastic (Markov) algorithm to

detect changes in ion channel signals
lipid bilayer
polyelectrolyte
microcapsules engineered  understand the nanostructure
ion channel  change the type of ion channel
Adv. Func. Mater. (2009) 19:201-208

Bioengineering of sensing systems … with biological components
Bioengineering of sensing systems … with biological components
utilising properties of natural proteins/lipids + utilising properties of synthetic materials
from 2D
Martin DK et al (2016). Ion-transporting Supported and
Tethered Lipid Bilayers that Incorporate Biological Membrane
with protein
Transport Proteins. In Pabst G et al (eds.) “Liposomes, Lipid
Bilayers and Model Membranes: From Basic Research to
Application”, CRC Press, ISBN 9781138198753 NaCl
no protein
to 3D
[US 9,577,280]
The power output from such a 3D system with a volume of 72 ml is

simulated to be 300 mW with a density of transport proteins of 350 per mm2.
… for example, 4 mW for a device with a volume of 1 ml.
… approximately 4 W/kg … Toyota Prius 2016 produces 64 W/kg (total 90.2 kW, 1414 kg)
Bioinspired (biological) engineering of nanostructured systems

utilising properties of natural proteins/lipids utilising properties of synthetic materials
nanobiotechnology
change in biological function physical, chemical or electrical output
Biomimetic output
So what … are nanotechnology systems useful for health ?
How does the system interact with the body ?

 to sense something
 to deliver something
 to replace a body function
 to augment a body function
What is needed for good interaction ?

 biocompatibility
 appropriate interface with body tissues
What is biomimetic nanotechnology ?
 bioinspired engineering
nanobiotechnology
Biomimetic output
nanobiotechnology
Biomimetic output
… building blocks …
“UroLOC” system to mimic exocrine glands: therapies and diagnostics
Chip to grow Collect mL ‟acini” Include real-time sensors for

‟acini” secretions secreted molecules
Proof-of-Concept
WPE1-int cells cultured for 5 days. Pseudo-colouration of
measure‟acini” secretions
depth in hemispherical “acini" (red = 0 μm, blue = 50 μm). (A) WPE1-int – DHT (unstimulated)
(B) WPE1 + DHT (stimulated)
A B C D (C) RPTEC (-ve control)
(D) 3 ng of pure PSA (+ve control)
Martin DK, Picollet-d’hahan N (2016)

Trends in Biotechnology, 34:757-769, 2016 “Cell co-culture chip and process for the production thereof”
WO 2017/194894 A1
Martin DK, Picollet-d’hahan N (2016)

“Method for diagnosing genitourinary cancersf”
WO 2017/194895 A1
Biomimetic membrane nanotechnology for symbiotic devices

Relevance of technology to health ?
 to replace a body function (augment the function ?)
Flexible and Stretchable Physical Sensor Integrated Platforms for Wearable
Human‐Activity Monitoring and Personal Healthcare
Advanced Materials Volume 28, Issue 22, pages 4338-4372, 3 FEB 2016 DOI: 10.1002/adma.201504244
Flexible and Stretchable Physical Sensor Integrated Platforms for Wearable
Human‐Activity Monitoring and Personal Healthcare
(a) Optical image of a multifunctional EES (epidermal electronic

system) that includes an EP (electrophysiological) sensor, a
temperature sensor, and a mechanical strain sensor.
(b) EES mounted on the forearm
(c) The optical images of multifunctional wearable patches laminated
on the wrist of the human body under tension and compression
(d) Top: the time-dependent resistance change of a Si strain sensor
generated by simulated hand tremors at various frequencies.
Bottom: the multilevel-cell operation of memory cells.
(e) Images of the transparent stretchable iHMI (interactive human
machine interface) laminated on human skin
(f) Schematics of the closed-loop system of the iHMI
(g) Representative image showing human motion controlling the robot
arm.
Advanced Materials Volume 28, Issue 22, pages 4338-4372, 3 FEB 2016 DOI: 10.1002/adma.201504244
Practical needs for nanoparticles in systemic drug delivery
Nanoparticles must evade the reticulendothelial system (RES):

 RES includes cells that can do phagocytosis (mononuclear phagocytes, neutrophils,
eosinophils)
 mononuclear phagocytes –
 circulating peripheral blood monocytes
 non-circulating tissue macrophages (Kupffer cells of liver, histiocytes, Littoral cells of spleen)
Nanoparticles must exhibit tissue-specific targeting:
Nanoparticles must overcome in vivo barriers including the vascular endothelium:

Nanotechnology to replace a body function
• Most obvious use  nanostructured materials for tissue engineering (e.g. cell scaffolds):
• Skin
• Cartilage
• Bone
• Nerve
• Cardiovascular
• Goals of nanostructured tissue engineering:
• “space-filler” until damaged tissue is regenerated by the body,

• mimic characteristics of natural tissue,
• replace tissue functions (temporary or permanent),
• scaffold for tissue ingrowth
Adult mesenchymal stem cells (MSCs) have wide application in
musculoskeletal tissue engineering:
 immunoregulatory,
 anti-inflammatory,
 modulate endothelial cells,
 multi-lineage differentiation potential
In order to have those activities, the MSCs must first home to the target tissue
and then differentiate properly
 “home” = be directed towards
(Tuan RS. (2012). Nat. Rev. Rheum., 9:74-76)

Improving the scaffold to grow MSCs:
 highly stretchable hydrogels developed …

 alginate-polyacrylamide hybrid hydrogel (the 2 types of polymer intertwined and further
cross-linked via covalent bonds between amine groups on polyacrylamide chains and
carboxyl groups on alginate chains)
(Sun JY et al. (2012). Nature, 489:133-136)

Alveolar Defect and Reconstruction with Tissue Engineering
Patient (male, 20 years) has lost his upper left incisor during an accident. The alveolar defect was reconstructed with tissue engineering techniques.
(A) Radiograph of the significant alveolar defect as a result of the accident.

(B) Radiograph of the defect reconstructed with ceramic scaffold (HA) covered with cultured MSCs (white arrow).
(C) Radiograph; four months later a dental implant was installed.
(D) Intra-oral view; arrow points to the crown, which was fixated on the implant.
Samples and Histology of the Patient Shown above
(A) HA particles stained with methylene blue immediately after seeding of the MSCs, showing cell distribution.
(B) Idem stained with trypan blue after one week of culturing, showing cell vitality.
(C) Histology six weeks after subcutaneous implantation in mice, showing in vivo bone formation (white arrow) in contact with HA particle (black arrow).
(D) Histology after four months of implantation in the upper left tooth region, showing bone formation (white arrow) induced by the implanted cells in
contact with the HA (black arrow).
KEY POINTS:
… and the “blade runner” ?
Schematic representation of “Flex-Foot®” and prosthetic
components (socket and liner) with representation of a residual limb. The schema is based
on transtibial (below-knee) amputees.
Hobara H (2014). バイオメカニズム学会誌, 38(2):105-110
T43 class = double below-knee amputees and other athletes with impairments that are comparable to a double-
below knee amputation
T44 class = any athlete with a lower limb impairment/s that meets minimum disability criteria for lower limb
deficiency, impaired lower limb passive range of motion, impaired lower limb muscle power, or leg length difference
3 “mechanical” variables that help us run faster ?
how quickly the limbs can be repositioned for successive steps (shorter swing
time)
the forward distance the body travels while the foot is in contact with the ground
(longer contact length)
how much force the limbs can apply to the ground in relation to the body’s weight
(greater body-mass-specific ground reaction force)

Does the amputee have an unfair advantage ?
Advantages: shorter swing time, longer contact length
Disadvantages: lower vertical-ground-reaction-force, leg stiffness in lower-leg

amputees

So what … are nanotechnology systems useful for health ?


 Intention for the implanted medical
device to have communication of
materials with the body
 ‟Duplex devices”
 Extends the definition of a

biocompatible system  requires
stable exchange of materials between
the implanted device and the body





Alcaraz et al (2018). Biotech J, 13:1800102

Symbiotic implantable medical devices should
benefit from a porous encapsulating membrane
 to allow transport of beneficial molecules
 to avoid immune reactions

SUMMARY… are nanotechnology systems useful for health ?


Role of Biosensors in Disease Detection
Pr. Donald MARTIN



Components of a biosensor
A biological sensing system:
is a self-contained integrated device
is capable of providing specific quantitative or semi-quantitative analytical

information using a biological recognition element
biological recognition element is retained in spatial contact with an

electrochemical transduction element
Bioinspired (biological) sensing systems for cancer biomarkers
Components of a Biosensor
Detector
http://www.dddmag.com/images/0409/HTS1_lrg.jpg
Principles of detection
Biosensors can be divided according to the type transducer into four basic groups:
1) Optical biosensors - can be further divided into
• colorimetric,
• fluorescent,
• luminescent
• interferometer;
2) Gravimetric biosensors - can be based on the piezoelectric effect or operate on the principle of
acoustic waves;
3) Calorimeters (thermometric) biosensors;
4) Electrochemical biosensors:
• amperometric,
• potentiometric
• conductometric,
• impedimetric.
Electrochemical biosensor
• Measure electrical signal, which is generated during the biochemical interaction between the
biologically active part and the substrate.
• Potentiometric biosensors have ion selective electrodes that detect a change in voltage
depending on the analyte concentration.
• Amperometric biosensors measure current generated by applying constant potential between two
electrodes.
• Working electrode is formed either by noble metal or printed layer covered with biologically active
comound.
• Conductometric and impedimetric biosensors

measure the change in electrical conductivity or
resistance of the solution depending on the number of
ions or electrons produced during biochemical
reactions between biologically active component and
the analyte.
An electrochemical biosensor:
is a self-contained integrated device
is capable of providing specific quantitative or semi-quantitative analytical

information using a biological recognition element
biological recognition element is retained in spatial contact with an electrochemical

transduction element
1ST Component: Biological Element
The component used to bind the target molecule.
Must be highly specific, stable under storage conditions, and immobilized.
Microorganism
Tissue
Cell
Organelle
Nucleic Acid
Enzyme
Enzyme Component
Receptor
Antibody http://www.chemistry.wustl.edu/~edudev/LabTutorials/HIV/DrugStrategies.html
2ND Component: Physiochemical
Transducer
Acts as an interface, measuring the physical change that occurs
with the reaction at the bioreceptor then transforming that
energy into measurable electrical output.
3RD Component: Detector
Signals from the transducer are

passed to a microprocessor where
they are amplified and analyzed.
The data is then converted to

concentration units and transferred to
a display or/and data storage device.
www.modernmike.com
Principles of Detection
measures change in mass
measures change in electric distribution
measures change in light intensity
measures change in heat

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Micro and Nanotechnologies in Health

Pr. Donald MARTIN

Université Grenoble Alpes

Parcours: M2-HL_TECH, M2-SMB/BDIV, M2-SMB/TCGIT, M2-SMB/SMB, M2-CHIP

Prep. Biochem. Biotech., 46:546, 2015 Scientific Reports, 3:1516, 2013

Pr. Donald MARTIN

Université Grenoble Alpes

Parcours: M2-HL_TECH, M2-SMB/BDIV, M2-SMB/TCGIT, M2-SMB/SMB, M2-CHIP

How is bioinspiration turned into medical technology ?

What could the future bring ?

… inspired by or based on biological structures or processes

An ancient Egyptian false toe found on

Finch J (2011) Lancet, 377:548-549

Nerlich AG, Zink A, Szeimes U, Hagedorn HG (2000). Lancet, 356:2176–2179 http://www.orthoanswer.org

… by applying nano-micro-biotechnology … example: snake-skin

(a) Scanning electron

(b) images of scales

(c) images of the scales

Greiner & Schäfer (2015) Bioinspir. Biomim. 10:044001

… by applying nano-micro-biotechnology … example: artificial insect eye

Kuo et al (2015) Bioinspir. Biomim. 10:056010

… by applying nano-micro-biotechnology … example: artificial insect eye

fast-moving targets could be

Kuo et al (2015) Bioinspir. Biomim. 10:056010

(a) floating fern Salvinia molesta has two types of leaves

Zeiger et al (2016) Bioinspir. Biomim. 11:056003

How is bioinspiration turned into medical technology ?

What could the future bring ?

heat (500° - 650° C)

the Ag nanoparticles selectively

1857: Michael Faraday published method for making gold

Schematic representations of:

(A) a thrombin sensor that operates based on fibrin-

(B)(C) fibrinogen and thrombin sensors that operate

Chen YY et al (2011). Gold nanoparticle-based colorimetric assays for coagulation-related

How is bioinspiration turned into medical technology ?

What could the future bring ?

 compatible with the internal environment, not causing

First 14 days: slight inflammatory reaction (fat necrosis, fibrin,

Bioelectrode nanotechnology for symbiotic implantable devices

Enzymatic biofuel cell implanted inside the body

 stable supply of substrates  stable level of sufficient power

 choice of appropriate biological components for implanted device

Improve the bioengineering of symbiotic implantable devices

Why do we need to do engineering with biological components ?

 To construct devices with better biocompatibility with the body,

Bioengineering of biomimetic membrane systems … with biological components

Instead of trying to find artificial materials to mimic biology,

‟biotechnology systems are hybrids of synthetic materials that support and

Improve the bioengineering of symbiotic implantable devices

No, there is a simple and common feature …

But, where is the science … ?

utilising properties of natural proteins/lipids

membrane ion channels

change in biological function

Blocker: glibenclamide, (glucose)

utilising properties of natural proteins/lipids

membrane ion channels

change in biological function

utilises biological components,

Bioengineering of biomimetic membrane systems … with biological components

+ + stochastic (Markov) algorithm to

Bioengineering of biomimetic membrane systems … with biological components

The power output from such a 3D system with a volume of 72 ml is