Molecules Adsorption on Hydroxyapatite: an Experimental ... · Gianmario Martra, University of...

Gianmario Martra,

University of Torino

Department of Chemistry

Molecules Adsorption on Hydroxyapatite:

an Experimental-Theoretical Synergy

Nano-HA: reason of interest

P. Xe et al., RSC Adv., 2014,4, 39704-39724

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

Nano-HA: reason of interest

P. Xe et al., RSC Adv., 2014,4, 39704-39724

Synthetic nano-HA: interaction with host living bodies

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

{01-10}

Bone apatite nanocrystals: shape and relevant surfaces

50 nm

A source of “bio-inspiration” for nano-HA science

Citrates at work in the synthesis of nano-HA

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


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


=bTCP

15/32


Chiatti, F.,

Sakhno, Y. et al.

J. Phys. Chem.

C, 117 (2013)

25526-25534

c axis

Ca

O

P

H

{01-10}


Cross-section view of (010)

16/32

=bTCP


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 …..

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

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


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…..



complex

heavy (QM)

…..

298 K 423 K

Glycine adsoprtion in controlled and model conditions

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



complex

heavy (QM)

…..

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

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


What about the origin of the opposite behavior of HA-LT and HA-HT, both pure HA?

BSA/HA-HT

BSA/HA-LT


opposite

ζ-potential trends

for the two types of

HA particles


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


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



…a non conventional conclusion

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

Molecules Adsorption on Hydroxyapatite: an Experimental ... · Gianmario Martra, University of...

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