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Gianmario Martra,
University of Torino
Department of Chemistry
Molecules Adsorption on Hydroxyapatite:
an Experimental-Theoretical Synergy
dental implant
medullary bone
cortical bone
dental fixture patented by the
University of Torino
(Nanopro project)
drug delivery
From cells toward biomaterials to biomaterials towards cells
nano-HA is used
for both applications
Time = 10-3-10-6 seconds
proteins adsorbs on material
surface displacing
water molecules
conformation, density and
orientation of adsorbed proteins
affect nature of “bioactive sites”in interaction with cell receptors
1. surface + water
2. surface + water + proteins
3. surface + water + proteins + cells
denaturednative proteins
water
different bonding orientations and strengths
water
cells
denaturednative proteins
water
From cells toward biomaterials to biomaterials towards cells
structure of adsorbed biopolymers
Mid-IR Raman
CD-UV PL
biomaterial/biopolymer interface
NMR EPR (labels)
Near-IR
Molecules on biomaterials: the “tool-box” of spectroscopic methods
biomaterial selected:hydroxyapatite,
in nanoform
5Ca(OH)2 + 3H3PO4 Ca5(PO4)3OH + 9H2O
T = 25 °C
T = 40°C
T = 95°C
b
c
L.Bertinetti et al.
Y. Sakhno et al. needles
platelets
HA-am
HA-cry
Nano-hydroxyapatite: tailoring of shape and structure
cnr {01-10}
Chiatti, F.,
Sakhno, Y. et al.
J. Phys. Chem.
C, 117 (2013)
25526-25534
c axis
What about surfaces of nano-HA?
{01-10} Cross-section view of (010)
=bTCP
Chiatti, F.,
Sakhno, Y. et al.
J. Phys. Chem.
C, 117 (2013)
25526-25534
Coexistence of stochiometric,
Ca-rich and P-rich terminations is confirmed by modeling and
UHR- TEM
c axis
Pavlo Ivanchenko - Torino, September, 5-9, 2016
BA
P-rich
Ca-rich
P-rich
P-rich
Ca-rich
{01-10} Cross-section view of (010)
=bTCP
What about surfaces of nano-HA?
Chiatti, F.,
Sakhno, Y. et al.
J. Phys. Chem.
C, 117 (2013)
25526-25534
And in the case of nanosize HA, where a careful inspection and analysis by UHR-TEM can be very difficult?
Cross-section view of (010) {01-10}
c axis
Pavlo Ivanchenko - Torino, September, 5-9, 2016
=bTCP
15/32
What about surfaces of nano-HA?
Chiatti, F.,
Sakhno, Y. et al.
J. Phys. Chem.
C, 117 (2013)
25526-25534
c axis
Ca
O
P
H
{01-10}
Pavlo Ivanchenko - Torino, September, 5-9, 2016
Cross-section view of (010)
16/32
=bTCP
What about surfaces of nano-HA?
The finest tip for surfaces? Molecules!
chemical and physical “resolution”
“seen” by molecular spectroscopies
YES!
21842180
2166
2173
2162
2174
2162
When HR-TEM fails, can CO recognise different HA 01-10 terminations?
Chiatti, F., Sakhno, Y., Corno M., Martra G, Ugliengo P., et al. J. Phys. Chem. C, 117 (2013) 25526-25534
Chiatti, F., Sakhno, Y. et al. J. Phys. Chem. C, 117
(2013) 25526-25534
Revealing HA nanoplatelets surface structure by IR of adsorbed CO
{010}
(2 CO)/ (010)_P-rich
(4 CO)/ (010)_Ca-rich
The only combination reproducing the experimental spectrum indicates that the
(010)_Ca-rich : (010) P-rich ratio should be 1:3
Biomaterial surfaces, water, biomolecules
biomaterial/water biomaterial/biomolecules (small)
too complex
too heavy
too …..
Biomaterial surfaces, water, biomolecules
biomaterial/water biomaterial/biomolecules (small)
too complex
too heavy
too …..
0 2 4 6
40
80
120
nads (H2O molecules/Ca2+
)
-a
dsH
(kJ/m
ol)
-LIQ
H(H2O)
Water molecules on hydroxyapatite: energetic & structural features
0 100 200
0
5
10
Int (H
2O
)
Int (OH)
R2=0.994
CO= 42 cm-1; -ads H ~ 45 kJ/mol
strong H2O – Ca2+cus interaction
strong H2O – H2O interaction
Ca2+cus: moderate Lewis acid strength
IR of adsorbed CO~ 300 K ~ 100 K
H2O on HA:
enthalpy of adsorption
ads. I run
ads. II run
(from IR data)
0 2 4 6
40
80
120
nads (H2O molecules/Ca2+
)
-a
dsH
(kJ/m
ol)
-LIQ
H(H2O)
Water molecules on hydroxyapatite: energetic & structural features
I
II
III
IV
I
II
III
IV
0 100 200
0
5
10
Int (H
2O
)
Int (OH)
R2=0.994
CO= 42 cm-1; -ads H ~ 45 kJ/mol
strong H2O – Ca2+cus interaction
strong H2O – H2O interaction
Ca2+cus: moderate Lewis acid strength
IR of adsorbed CO~ 300 K ~ 100 K
H2O on HA:
enthalpy of adsorption
ads. I run
ads. II run
H2O on HA: IR
(from IR data)
IR H2O bulk
Chiatti, F., Sakhno, Y. et al. J. Phys. Chem. C, 117
(2013) 25526-25534
Revealing HA nanoplatelets surface structure by IR of adsorbed CO
{01-10}
(2 CO)/ (010)_P-rich
(4 CO)/ (010)_Ca-rich
The only combination reproducing the experimental spectrum indicates that the
(010)_Ca-rich : (010) P-rich ratio should be 1:3
Chiatti, F., Sakhno, Y. et al. J. Phys. Chem. C, 117
(2013) 25526-25534
Revealing HA nanoplatelets surface structure by IR of adsorbed CO
{01-10}
(2 CO)/ (010)_P-rich
(4 CO)/ (010)_Ca-rich
The only combination reproducing the experimental spectrum indicates that the
(010)_Ca-rich : (010) P-rich ratio should be 1:3
Source of Calcium: from Ca(OH)2 to CaAc2:→ effect on particle lengthBUT→ {01-10} facets remain the most abundant
Shape and surface structure of nano-HA: effect of preparation conditions
HA-2HA-3
Source of Calcium: from Ca(OH)2 to CaAc2:→ effect on particle lengthBUT→ {01-10} facets remain the most abundant
Shape and surface structure of nano-HA: effect of preparation conditions
Ca_rich/
P_rich
1/1.4
Ca_rich/P_rich
1/5
HA-2HA-3
measure of the amount of adsorbed water microgravimetry
structural arrangement of adsorbed water molecules at different coverage
Mid- and Near -IR spectroscopy
Ca-rich:P-rich = 1:1.4 (HA2: Ca(OH)2, T=25oC)
Ca-rich:P-rich = 1:3 (HA1: Ca(OH)2, T=95oC)
Ca-rich:P-rich = 1:5 (HA3: CaAc2, T=25oC)
Water on nano-HA: meterials and methods
water/HA interaction Conditions considered:
Water on nano-HA: combined quantitative microgravimetric and Mid-IR study
3500 3000 1700 1600
3400
Wavenumber, cm
-1
16453500 3000 1700 1600
1645
3150
36
80
3320
Wavenumber (cm-1
)
0.1
15-22 H2O moleculesca. 4 H2O molecules
Limitation of Mid-IR: complexity of OH signal and low sensitivity of H2O signal to intermolecular structure
H2O H2O
HA-2
HA-3
HA-1
H2O:- anharmonicity;- coupling- H-bond
HOH:interaction through H: up-shiftinteraction through O: down-shift
Pavlo Ivanchenko - Torino, September, 5-9, 2016
HA-2
HA-3
HA-1
Ca_rich/
P_rich
1/1.4
Ca_rich/
P_rich
1/3
5200 – 5000
H-bonded
OH-groups
5263 – O-H
vibrations of
free OH-
groups
surface
A. Burneau, et.al, The surface properties of silicas (1998) 199
Surface coverage: ca. 4 molecules/nm2
=
1 molecule per surface Ca2+ site
Spectral patterns divided in two main parts:
Assignment on the basis of literature data on other materials
first principle assigment for H2O on HA
Water on nano-HA: combined quantitative microgravimetric and Near-IR study
5500 5250 5000 4750 4500
5200-49005260
Wavenumber, cm-1
Ca_rich/
P_rich
1/5
HA-1
HA-2
HA-3
asymHOH+HOH
5500 5250 5000 4750 4500
5200-4900
5380
5310
Wavenumber, cm-1
Ca_rich/
P_rich
1/1.4
Ca_rich/
P_rich
1/3
Ca_rich/
P_rich
1/5
Water on nano-HA: Combined theoretical and IR study
Thus, is the Ca-rich/P-rich ratio effective or ineffective
for the interaction of HA with water?
Ca-rich P-rich
Pictures and simulated spectra are contribution of Dr. Chiatti
Near-IR asym+ mode of adsorbed water:- possibility to obtain insights on the orientation of H2O molecules on nano-HA surfaces- limited differences in the spectral pattern in dependence on the (010)_Ca-rich/P-rich ratio
HA-1
HA-2
HA-3
asymHOH+HOH
Water on nano-HA: buiding-up interfacial water multilayers (by Near-IR)
15-22 molecules/nm2
+water
Wavenumber, cm-1
HA-2
HA-3
HA-1
Ca_rich/
P_rich
1/1.4
Ca_rich/
P_rich
1/3
Ca_rich/
P_rich
1/5
Differences in the atomic structure of {010} surfaces
Differences in the relative location of water molecules
in contact with the surface with similar orientation,
which act as ‘seeds’ for the adsorption of further
water molecules
Origin of structural differences when building up
water overlayers
Water on nano-HA: buiding-up interfacial water multilayers (by Near-IR)
15-22 molecules/nm2
+water
Wavenumber, cm-1
HA-2
HA-3
HA-1
Ca_rich/
P_rich
1/1.4
Ca_rich/
P_rich
1/3
Ca_rich/
P_rich
1/5
Differences in the atomic structure of {010} surfaces
Differences in the relative location of water molecules
in contact with the surface with similar orientation,
which act as ‘seeds’ for the adsorption of further
water molecules
Origin of structural differences when building up
water overlayers
…also water (multylayers) has (have) a surface…..
Biomaterial surfaces, water, biomolecules
biomaterial/water biomaterial/biomolecules (small)
complex
heavy (QM)
…..
step-wise adsorption
procedure
control of the
adsorbed amount
cmore than 1 component
Glycine adsoprtion in controlled and model conditions
no identification of
experimental surfaces
MM, DM:
- FFs accuracy
- occurrence of chemical
reactions?
2) adsoprtion of biomolecules in
highly controlled conditions
4) first principle QM
3) in-situ spectroscopy (IR)
simple biomolecule:
glycine
1) nano HA with defined surfaces
state-of-the-art our targets
Gly on HA: a step toward a surface science model for biology
for each adduct:
optimum structure
IR spectrum
reacted with water
(-OH formation)
+ co-adsorbed H2O (W)
B3LYP/6-31G(d,p)
CRYSTAL09
Chemisorption of Gly on HA: QM calculations
+ minor contributions
from other species on
less abundant
different terminations
A. Rimola, Y. Sakhno et al.
J. Phys. Chem. Lett., 2 (2011) 1390-1394
Recognition of components in the Exp spectrum
Biomaterial surfaces, water, biomolecules
biomaterial/water biomaterial/biomolecules (small)
complex
heavy (QM)
…..
biomaterial/water biomaterial/biomolecules (small)
complex
heavy (QM)
…..
Biomaterial surfaces, water, biomolecules
biomaterial/water biomaterial/biomolecules (small)
complex
heavy (QM)
…..
Biomaterial surfaces, water, biomolecules
MATERIALS
HA LThexagonal hydroxyapatite
Ca10(PO4)6(OH)2 T = 25 °C
…and the protein
Bovine Serum Albumin (BSA) secondary structure ≈ 67% a-helix
MW = 66 KDa
583 residues
3 homologous domains
pI = 4.7
negatively charged at pH 7.4
• the most aboundant in blood (60%);
• relevant physiological importance;
• high structural homology with Human
Serum Albumin (75%);
• relative ease of isolation/purification;
• intensively studied and characterized.
HA HThexagonal hydroxyapatite
Ca10(PO4)6(OH)2 T = 95 °C
the biomaterials…
SSABET = 76 m2/g SSABET = 73 m2/g
XRD
20 25 30 35 40 45 50 55 60
2
LT
(104)
(213)
(222)
(310)
(300)
(202)
(211)
(210)
(102)
Inte
nsit
y (
a.u
.)
(002)HT
BSA ADSORPTION: PROCEDURE
Amount of adsorbed proteins: adsorption isotherms in HEPES (pH 7.4)
Primary isotherm
HA suspension
(25 mg) in HEPES
BSA solution of different concentrations
(0.1-7.5 mg/mL)
incubation (15 min)
centrifugation Secondary isotherm
resuspension/centrifugation
cycles of precipitates in HEPES
total amount of BSA
adsorbed
irreversibly
adsorbed fraction
Measure of adsorption
intensity of supernatant
(λ= 280 nm)
Measure of adsorption intensity
of supernatants (λ= 280 nm)
precipitate
recovery: BSA
irreversibly
adsorbed on HA
NP agglomeration: from theoretical to actual surface coverage (1)
ADSORPTION ISOTHERMS
decrease of the surface actually
accessible to BSA for adsorption
HEPES
(pH 7.4)
0 1 2 3 4 5 6 7 80
2
4
6
8
10
12
14
16
18
[BSA] incubation (mg/mL)
ABOVE
BELOW
theoretical monolayer
overstimated!
0 1 2 3 4 5 6 7 80.0
0.2
0.4
0.6
0.8
1.0
[BSA] incubation (mg/mL)
(theoretical monolayer)
HA HT BSA
HA LT BSA
HEPES, BSA
(pH 7.4)
MULTILAYERS OF IRREVERSIBLY
ADSORBED PROTEINS:
PROTEIN HARD CORONA
NP agglomeration: from theoretical to actual surface coverage (1)
ADSORPTION ISOTHERMS
decrease of the surface actually
accessible to BSA for adsorption
HEPES
(pH 7.4)
0 1 2 3 4 5 6 7 80
2
4
6
8
10
12
14
16
18
[BSA] incubation (mg/mL)
ABOVE
BELOW
theoretical monolayer
overstimated!
0 1 2 3 4 5 6 7 80.0
0.2
0.4
0.6
0.8
1.0
[BSA] incubation (mg/mL)
(theoretical monolayer)
HA HT BSA
HA LT BSA
HEPES, BSA
(pH 7.4)
MULTILAYERS OF IRREVERSIBLY
ADSORBED PROTEINS:
PROTEIN HARD CORONA
PROTEIN CONFORMATIONAL CHANGES
Structure of proteins in solution: CD-UV
BSA native
BSA 100 °C
n π*
π π*
transition n π* signal at 222 nm
transition π π* signals at 208 and 195 nm BSA after thermal treatment:
1) ↓CD222 (n → π* transition) ↓ in a-helix content*
*W. Norde, Colloids Surf, 64 (1992), 87-93
BSA native
BSA 100 °C
BSA/HA LT
BSA/HA HT
adsorbed BSA:
1) ↓CD222 (n → π* transition)
complete coverage of
HA agglomerates
↓ in a-helix content*
*W. Norde, Colloids Surf, 64 (1992), 87-93
**D.M. Charbonneau, J. Phys. Chem. B 2010, 114, 1148
2) ↓ CD208/CD222↑ b-sheet –like structures**
Structure of adsorbed proteins: CD-UV
PROTEIN CONFORMATIONAL CHANGES
transition n π* signal at 222 nm
transition π π* signals at 208 and 195 nm
ζ-potential of BSA adsorbed onto HA surface
CHANGE IN SURFACE CHARGE: ζ-potential
pure HA, main surfaces: {01-10}
opposite
ζ-potential trends
for the two types of
HA particles
pure HA, main surfaces: { 01-10}
BSA/HA-HT
BSA/HA-LT
ζ-potential of BSA adsorbed onto HA surface
What about the origin of the opposite behavior of HA-LT and HA-HT, both pure HA?
BSA/HA-HT
BSA/HA-LT
pure HA, main surfaces: {01-10}
opposite
ζ-potential trends
for the two types of
HA particles
pure HA, main surfaces: { 01-10}
CHANGE IN SURFACE CHARGE: ζ-potential
HA LThexagonal hydroxyapatite
Ca10(PO4)6(OH)2 T = 25 °C
…and the protein
Bovine Serum Albumin (BSA) secondary structure ≈ 67% a-helix
MW = 66 KDa
583 residues
3 homologous domains
pI = 4.7
negatively charged at pH 7.4
• the most aboundant in blood (60%);
• relevant physiological importance;
• high structural homology with Human
Serum Albumin (75%);
• relative ease of isolation/purification;
• intensively studied and characterized.
HA HThexagonal hydroxyapatite
Ca10(PO4)6(OH)2 T = 95 °C
the biomaterials…
SSABET = 76 m2/g SSABET = 73 m2/g
XRD
20 25 30 35 40 45 50 55 60
2
LT
(104)
(213)
(222)
(310)
(300)
(202)
(211)
(210)
(102)
Inte
nsit
y (
a.u
.)
(002)HT
…back to the nano-HA materials
Chiatti, F., Sakhno, Y. et al. J. Phys. Chem. C, 117 (2013) 25526-25534
Ca-rich : P-rich
2 : 1
Ca-rich : P-rich
1 : 2
2 CO molecules
Slab (010) P-rich
4 CO molecules
Slab (010) Ca-rich
modeling and theoretical spectra from a collaboration
with Prof. P. Ugliengo, University of Torino
theoretical spectra
experimental
spectra
HA-HT
HA-LT
combining quantum mechanical modeling with
experimental IR using the CRYSTAL09 program
{01-10} SURFACE TERMINATIONS OF HAs
CHANGE IN SURFACE CHARGE and EFFECT on PROTEIN CONFORMATION
ζ-potential of BSA adsorbed onto HA surface
BSA/HA-HT
BSA/HA-LT
{01-10} Ca-rich
prevailing
{01-10} P-rich
prevailing
from CD data…
ζ-potential of native BSA
ζ-potential of BSA treated at 100°C
pure HA, main surfaces: {01-10}
pure HA, main surfaces: { 01-10}
The princess and the pea
(when nanoscience meets imagination….
…and by chance Hans Christian Andersen)
a readapted tale by F. Catalano
≈
Main characters
Bovine serum albumin (BSA)
(Soft protein, can change conformation
depending on the surface properties)
The princess
HA LT T = 25 °C
hexagonal
hydroxyapatite
HA HT T = 95 °C
hexagonal
hydroxyapatite
≈ The bed(s)
Cross-section view of {010}
≈ The pea
What a
beautiful
dream!
We didn’t sleep
a wink,
changing
conformation all
the time!
Shut
up !!!
{01-10} Ca-rich
prevailing
{01-10} P-rich
prevailing
ACKNOWLEDGEMENTS
Pavlo Ivanchenko
Federico Catalano
Marta Corno
Piero Ugliengo
……the real Experimental-Theoretical Synergy
Yuriy Sakhno