Molecules Adsorption on Hydroxyapatite: an Experimental-Theoretical Synergy. Gianmario Martra, University of Torino Department of Chemistry

Transcription

1 Molecules Adsorption on Hydroxyapatite: an Experimental-Theoretical Synergy Gianmario Martra, University of Torino Department of Chemistry

2 Nano-HA: reason of interest P. Xe et al., RSC Adv., 2014,4,

3 From cells toward biomaterials to biomaterials towards cells cortical bone dental implant medullary bone drug delivery dental fixture patented by the University of Torino (Nanopro project) nano-ha is used for both applications

4 Nano-HA: reason of interest P. Xe et al., RSC Adv., 2014,4,

5 Synthetic nano-ha: interaction with host living bodies

6 Synthetic nano-ha: interaction with host living bodies

7 From cells toward biomaterials to biomaterials towards cells 1. surface + water different bonding orientations and strengths water 2. surface + water + proteins Time = seconds native proteins water denatured proteins adsorbs on material surface displacing water molecules 3. surface + water + proteins + cells native cells proteins water denatured conformation, density and orientation of adsorbed proteins affect nature of bioactive sites in interaction with cell receptors

8 Molecules on biomaterials: the tool-box of spectroscopic methods Mid-IR CD-UV Raman PL structure of adsorbed biopolymers biomaterial/biopolymer interface biomaterial selected: hydroxyapatite, in nanoform NMR EPR (labels) Near-IR

9 Bone apatite nanocrystals: shape and relevant surfaces {01-10} 50 nm

10 A source of bio-inspiration for nano-ha science

11 Citrates at work in the synthesis of nano-ha

12 Nano-hydroxyapatite: tailoring of shape and structure 5Ca(OH) 2 + 3H 3 PO 4 Ca 5 (PO 4 ) 3 OH + 9H 2 O L.Bertinetti et al. T = 25 C HA-am needles T = 95 C T = 40 C cnr {01-10} platelets Y. Sakhno et al. c HA-cry b

13 What about surfaces of nano-ha? {01-10} Cross-section view of (010) c axis =btcp Chiatti, F., Sakhno, Y. et al. J. Phys. Chem. C, 117 (2013)

14 What about surfaces of nano-ha? {01-10} Cross-section view of (010) c axis =btcp Chiatti, F., Sakhno, Y. et al. J. Phys. Chem. C, 117 (2013) Coexistence of stochiometric, Ca-rich and P-rich terminations is confirmed by modeling and UHR- TEM B A P-rich Ca-rich P-rich P-rich Ca-rich Pavlo Ivanchenko - Torino, September, 5-9, 2016

15 What about surfaces of nano-ha? {01-10} Cross-section view of (010) c axis =btcp Chiatti, F., Sakhno, Y. et al. J. Phys. Chem. C, 117 (2013) And in the case of nanosize HA, where a careful inspection and analysis by UHR-TEM can be very difficult? Pavlo Ivanchenko - Torino, September, 5-9, /32

16 What about surfaces of nano-ha? {01-10} Cross-section view of (010) c axis =btcp Chiatti, F., Sakhno, Y. et al. J. Phys. Chem. C, 117 (2013) H Ca O P Pavlo Ivanchenko - Torino, September, 5-9, /32

17 The finest tip for surfaces? Molecules! chemical and physical resolution seen by molecular spectroscopies

18 When HR-TEM fails, can CO recognise different HA terminations? YES! Chiatti, F., Sakhno, Y., Corno M., Martra G, Ugliengo P., et al. J. Phys. Chem. C, 117 (2013)

19 Revealing HA nanoplatelets surface structure by IR of adsorbed CO {010} (2 CO)/ (010)_P-rich (4 CO)/ (010)_Ca-rich Chiatti, F., Sakhno, Y. et al. J. Phys. Chem. C, 117 (2013) The only combination reproducing the experimental spectrum indicates that the (010)_Ca-rich : (010) P-rich ratio should be 1:3

20 Biomaterial surfaces, water, biomolecules too complex too heavy too.. biomaterial/water biomaterial/biomolecules (small)

21 Biomaterial surfaces, water, biomolecules too complex too heavy too.. biomaterial/water biomaterial/biomolecules (small)

22 Water molecules on hydroxyapatite: energetic & structural features H 2 O on HA: enthalpy of adsorption IR of adsorbed CO ~ 300 K ~ 100 K 10 - ads H (kj/mol) ads. I run ads. II run Int (H 2 O) (from IR data) R 2 = Int (OH) - LIQ H(H 2 O) CO= 42 cm -1 ; - ads H ~ 45 kj/mol Ca 2+ cus: moderate Lewis acid strength n ads (H 2 O molecules/ca 2+ ) strong H 2 O Ca 2+ cus interaction strong H 2 O H 2 O interaction

23 Water molecules on hydroxyapatite: energetic & structural features H 2 O on HA: enthalpy of adsorption IR of adsorbed CO ~ 300 K ~ 100 K IV III II I - ads H (kj/mol) ads. I run ads. II run Int (H 2 O) 10 (from IR data) R 2 = Int (OH) - LIQ H(H 2 O) n ads (H 2 O molecules/ca 2+ ) strong H 2 O Ca 2+ cus interaction strong H 2 O H 2 O interaction CO= 42 cm -1 ; - ads H ~ 45 kj/mol Ca 2+ cus: moderate Lewis acid strength H 2 O on HA: IR IV III II IR H 2 O bulk I

24 Revealing HA nanoplatelets surface structure by IR of adsorbed CO {01-10} (2 CO)/ (010)_P-rich (4 CO)/ (010)_Ca-rich Chiatti, F., Sakhno, Y. et al. J. Phys. Chem. C, 117 (2013) The only combination reproducing the experimental spectrum indicates that the (010)_Ca-rich : (010) P-rich ratio should be 1:3

25 Revealing HA nanoplatelets surface structure by IR of adsorbed CO {01-10} (2 CO)/ (010)_P-rich (4 CO)/ (010)_Ca-rich Chiatti, F., Sakhno, Y. et al. J. Phys. Chem. C, 117 (2013) The only combination reproducing the experimental spectrum indicates that the (010)_Ca-rich : (010) P-rich ratio should be 1:3

26 Shape and surface structure of nano-ha: effect of preparation conditions Source of Calcium: from Ca(OH) 2 to CaAc 2 : effect on particle length BUT {01-10} facets remain the most abundant HA-2 HA-3

27 Shape and surface structure of nano-ha: effect of preparation conditions Source of Calcium: from Ca(OH) 2 to CaAc 2 : effect on particle length BUT {01-10} facets remain the most abundant HA-2 Ca_rich/ P_rich 1/1.4 Ca_rich/ P_rich 1/5 HA-3

28 Water on nano-ha: meterials and methods Ca-rich:P-rich = 1:1.4 (HA2: Ca(OH) 2, T=25 o C) Ca-rich:P-rich = 1:3 (HA1: Ca(OH) 2, T=95 o C) Ca-rich:P-rich = 1:5 (HA3: CaAc 2, T=25 o C) water/ha interaction Conditions considered: measure of the amount of adsorbed water microgravimetry structural arrangement of adsorbed water molecules at different coverage Mid- and Near -IR spectroscopy

29 Water on nano-ha: combined quantitative microgravimetric and Mid-IR study HA-3 HA-2 HA H 2 O: - anharmonicity; - coupling - H-bond HOH: interaction through H: up-shift interaction through O: down-shift H 2 O H 2 O Wavenumber (cm -1 ) 3400 HA-2 HA-1 HA ca. 4 H 2 O molecules H 2 O molecules Wavenumber, cm -1 Limitation of Mid-IR: complexity of OH signal and low sensitivity of H 2 O signal to intermolecular structure Pavlo Ivanchenko - Torino, September, 5-9, 2016

30 Water on nano-ha: combined quantitative microgravimetric and Near-IR study HA-2 Ca_rich/ P_rich 1/1.4 Surface coverage: ca. 4 molecules/nm 2 = 1 molecule per surface Ca 2+ site HA-1 Ca_rich/ P_rich 1/3 Spectral patterns divided in two main parts: HA-3 Ca_rich/ P_rich 1/ O-H vibrations of free OHgroups H-bonded OH-groups asym HOH+ HOH Wavenumber, cm -1 surface A. Burneau, et.al, The surface properties of silicas (1998) 199 Assignment on the basis of literature data on other materials first principle assigment for H 2 O on HA

31 Water on nano-ha: Combined theoretical and IR study HA-2 Ca_rich/ P_rich 1/1.4 Ca-rich P-rich HA-1 Ca_rich/ P_rich 1/3 asym HOH+ HOH HA Wavenumber, cm -1 Ca_rich/ P_rich 1/5 Pictures and simulated spectra are contribution of Dr. Chiatti Near-IR asym + mode of adsorbed water: - possibility to obtain insights on the orientation of H 2 O molecules on nano-ha surfaces - limited differences in the spectral pattern in dependence on the (010)_Ca-rich/P-rich ratio Thus, is the Ca-rich/P-rich ratio effective or ineffective for the interaction of HA with water?

32 Water on nano-ha: buiding-up interfacial water multilayers (by Near-IR) Ca_rich/ P_rich 1/ molecules/nm 2 HA-2 +water Ca_rich/ P_rich 1/3 Ca_rich/ P_rich 1/5 HA-1 HA-3 Wavenumber, cm -1 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

33 Water on nano-ha: buiding-up interfacial water multilayers (by Near-IR) Ca_rich/ P_rich 1/1.4 also molecules/nm water (multylayers) 2 has (have) a surface.. HA-2 +water Ca_rich/ P_rich 1/3 Ca_rich/ P_rich 1/5 HA-1 HA-3 Wavenumber, cm -1 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

34 Biomaterial surfaces, water, biomolecules complex heavy (QM).. biomaterial/water biomaterial/biomolecules (small)

35 Glycine adsoprtion in controlled and model conditions 298 K 423 K

36 Glycine adsoprtion in controlled and model conditions step-wise adsorption procedure control of the adsorbed amount more than 1 component c

37 Gly on HA: a step toward a surface science model for biology state-of-the-art our targets 1) nano HA with defined surfaces 2) adsoprtion of biomolecules in highly controlled conditions no identification of experimental surfaces MM, DM: - FFs accuracy - occurrence of chemical reactions? 3) in-situ spectroscopy (IR) 4) first principle QM simple biomolecule: glycine

38 Chemisorption of Gly on HA: QM calculations B3LYP/6-31G(d,p) CRYSTAL09 reacted with water (-OH formation) for each adduct: optimum structure IR spectrum + co-adsorbed H 2 O (W)

39 Recognition of components in the Exp spectrum A. Rimola, Y. Sakhno et al. J. Phys. Chem. Lett., 2 (2011) minor contributions from other species on less abundant different terminations

40 Biomaterial surfaces, water, biomolecules complex heavy (QM).. biomaterial/water biomaterial/biomolecules (small)

41 Biomaterial surfaces, water, biomolecules complex heavy (QM).. biomaterial/water biomaterial/biomolecules (small)

42 Biomaterial surfaces, water, biomolecules complex heavy (QM).. biomaterial/water biomaterial/biomolecules (small)

43 MATERIALS HA HT hexagonal hydroxyapatite Ca 10 (PO 4 ) 6 (OH) 2 T = 95 C the biomaterials XRD HA LT hexagonal hydroxyapatite Ca 10 (PO 4 ) 6 (OH) 2 T = 25 C Intensity (a.u.) HT LT (002) (102) (210) (211) (300) (202) (310) (222) (213) (104) Bovine Serum Albumin (BSA) secondary structure 67% a-helix MW = 66 KDa 583 residues 3 homologous domains pi = 4.7 negatively charged at ph SSA BET = 76 m 2 /g SSA BET = 73 m 2 /g and the protein 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.

44 BSA ADSORPTION: PROCEDURE Amount of adsorbed proteins: adsorption isotherms in HEPES (ph 7.4) HA suspension (25 mg) in HEPES BSA solution of different concentrations ( mg/ml) incubation (15 min) Primary isotherm centrifugation Secondary isotherm Measure of adsorption intensity of supernatant (λ= 280 nm) resuspension/centrifugation cycles of precipitates in HEPES total amount of BSA adsorbed precipitate recovery: BSA irreversibly adsorbed on HA Measure of adsorption intensity of supernatants (λ= 280 nm) irreversibly adsorbed fraction

45 ADSORPTION ISOTHERMS NP agglomeration: from theoretical to actual surface coverage (1) (theoretical monolayer) HA HT HA LT BSA BSA [BSA] incubation (mg/ml) overstimated! theoretical monolayer [BSA] incubation (mg/ml) ABOVE BELOW HEPES (ph 7.4) HEPES, BSA (ph 7.4) decrease of the surface actually accessible to BSA for adsorption MULTILAYERS OF IRREVERSIBLY ADSORBED PROTEINS: PROTEIN HARD CORONA

46 ADSORPTION ISOTHERMS NP agglomeration: from theoretical to actual surface coverage (1) (theoretical monolayer) HA HT HA LT BSA BSA [BSA] incubation (mg/ml) overstimated! theoretical monolayer [BSA] incubation (mg/ml) ABOVE BELOW HEPES (ph 7.4) HEPES, BSA (ph 7.4) decrease of the surface actually accessible to BSA for adsorption MULTILAYERS OF IRREVERSIBLY ADSORBED PROTEINS: PROTEIN HARD CORONA

47 PROTEIN CONFORMATIONAL CHANGES Structure of proteins in solution: CD-UV transition n transition π π* signal at 222 nm π* signals at 208 and 195 nm BSA after thermal treatment: π π* BSA native BSA 100 C 1) CD 222 (n π* transition) in a-helix content* *W. Norde, Colloids Surf, 64 (1992), n π*

48 PROTEIN CONFORMATIONAL CHANGES Structure of adsorbed proteins: CD-UV transition n transition π π* signal at 222 nm π* signals at 208 and 195 nm adsorbed BSA: BSA native BSA 100 C 1) CD 222 (n π* transition) in a-helix content* *W. Norde, Colloids Surf, 64 (1992), BSA/HA LT BSA/HA HT 2) CD 208 /CD 222 b-sheet like structures** complete coverage of HA agglomerates **D.M. Charbonneau, J. Phys. Chem. B 2010, 114, 1148

49 CHANGE IN SURFACE CHARGE: ζ-potential ζ-potential of BSA adsorbed onto HA surface BSA/HA-HT pure HA, main surfaces: {01-10} opposite ζ-potential trends for the two types of HA particles BSA/HA-LT pure HA, main surfaces: { 01-10}

50 CHANGE IN SURFACE CHARGE: ζ-potential ζ-potential of BSA adsorbed onto HA surface BSA/HA-HT 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-LT What about the origin of the opposite behavior of HA-LT and HA-HT, both pure HA?

51 back to the nano-ha materials HA HT hexagonal hydroxyapatite Ca 10 (PO 4 ) 6 (OH) 2 T = 95 C the biomaterials XRD HA LT hexagonal hydroxyapatite Ca 10 (PO 4 ) 6 (OH) 2 T = 25 C Intensity (a.u.) HT LT (002) (102) (210) (211) (300) (202) (310) (222) (213) (104) Bovine Serum Albumin (BSA) secondary structure 67% a-helix MW = 66 KDa 583 residues 3 homologous domains pi = 4.7 negatively charged at ph SSA BET = 76 m 2 /g SSA BET = 73 m 2 /g and the protein 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.

52 {01-10} SURFACE TERMINATIONS OF HAs combining quantum mechanical modeling with experimental IR using the CRYSTAL09 program theoretical spectra 4 CO molecules Slab (010) Ca-rich HA-HT experimental spectra Ca-rich : P-rich 2 : 1 2 CO molecules Slab (010) P-rich HA-LT Ca-rich : P-rich 1 : 2 Chiatti, F., Sakhno, Y. et al. J. Phys. Chem. C, 117 (2013) modeling and theoretical spectra from a collaboration with Prof. P. Ugliengo, University of Torino

53 CHANGE IN SURFACE CHARGE and EFFECT on PROTEIN CONFORMATION ζ-potential of BSA adsorbed onto HA surface ζ-potential of native BSA BSA/HA-HT pure HA, main surfaces: {01-10} {01-10} Ca-rich prevailing ζ-potential of BSA treated at 100 C pure HA, main surfaces: { 01-10} BSA/HA-LT {01-10} P-rich prevailing from CD data

54 a non conventional conclusion

55 The princess and the pea (when nanoscience meets imagination. and by chance Hans Christian Andersen) a readapted tale by F. Catalano

56 Bovine serum albumin (BSA) (Soft protein, can change conformation depending on the surface properties) Main characters The princess HA HT T = 95 C hexagonal hydroxyapatite HA LT T = 25 C hexagonal hydroxyapatite The bed(s) Cross-section view of {010} The pea

57 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

58 ACKNOWLEDGEMENTS Piero Ugliengo Pavlo Ivanchenko Federico Catalano Yuriy Sakhno the real Experimental-Theoretical Synergy Marta Corno

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