Ceramic core@shell nanospheres - PowerPoint Presentation

Slide1

Ceramic core@shell nanospheres as vaccine carriers

Livia Naszályi Nagy

Department of Organic and Macromolecular chemistry

NMR and

Stuctural

analysis research group

Slide2

vaccines

Targeted delivery may be achieved

Simultaneous delivery of antigen and adjuvant

NP elicits boosted immune response due to it’s size

2

Nanovaccines

2011-2020

Decade

of vaccines (WHO)

Demand

for new vaccines

Slide3

Immune stimulator:

CpG

ODN 1826 (

phosphorothioate

backbone)

s

ilica

@z

irconia

vaccine carriers

have to bind immune stimulator (adjuvant)

for certain biological experiment have to be fluorescent

be colloidally stable and do not release cargo at pH 7.4

3

Nanoparticulate carriers with

same

surface chemistry – different size

5’-tccatga

cg

ttcctga

cg

tt-3’

silica

zirconia

immune

stimulator

NMR

In NMR4Nanos MIF project

Slide4

Z. Hens et J.C. Martins, Chem. Mater., 2013, 25 (8), pp 1211–1221

NMr Toolbox

Line broadening in 1D

1

H NMR

2D

1H-1H DOSY: Decrease in the diffusion coefficient

2D

1

H-

1

H NOESY: difference in crosspeaks sign and intensity

4

Slide5

Material preparation5

Formation of ZrO

2

shell

on

SiO

2

NPs

dropwise addition of

tetrabutyl

zirconate

50°C, 3 h,

Ar

atmosphere

Surface modification of SiO

2

@ZrO

2

NPs

Deoxynucleoside

monophosphate mixture

(

dAMP

,

dCMP, dGMP and TMP) 2 mg/ml in waterCpG ODN 1826 oligonucleotide 2.5 mg/ml in water5(6) carboxyfluorescein 2.0 mg/ml in ethanolDialysis

W. Stöber et al., J. Colloid Interface Sci. 26, 62-69 (1968).

hydrolysisunder basic conditionsSN2condensation:

-H2O

SiSiSi

SiSiSihydrolysisunder slightlybasic conditionscondensation

-H2OOH

SiSiZr

ZrZrSynthesis of Stöber

silica nanoparticles Controlled hydrolysis and condensation of tetraethyl orthosilicateethanol solvent, RT, 24h;evaporation of ammoniaJ. M. Kim et al., Ceram. Int. 35, 1243–47 (2009).

Slide6

Investigation of adsorption6

FTIR spectroscopy

1D 1H NMR and

2D

1H-1

H NMR NOESY

and DOSYSimultaneous measurement of zeta potential of NPs and

UV absorbance

of supernatant

vs.

pH

Set suspension pH

Measure zeta potential

Transfer sample to Epp

Settle out particles

Dilute supernatant for UV-vis

1 mg/ml

Slide7

7

Sample

SiO

2

volume mean diameter

(nm)

SiO

2

@ZrO

2

volume mean diameter

(nm)

SiO

2

@ZrO

2

PdI

S2Z

123

 34

109

 29

0.021S3Z75

 25134  530.133

S4Z35  1347

 130.043S6Z

6  325  60.021SiO2@ZrO2 nPs

Slide8

Results of surface modification8

Slide9

Adsorption of Nucleoside MPs

Nu mixture: whitening + stabilizing effect

FTIR: base-pair formation,

Phosphate-group binding

Zeta potential of dialyzed samples

S2Z

d

A

MP

-

0.2

±

0.0

mV

d

C

MP

-

6.3

±

0.6 mV

dGMP

-53.0 ±

1.0 mV

TMP-

30.3 ± 0.5 mVNu-22.2 ± 0.3 mVnative11.9 ± 0.2when added to ethanolic sol

9

Slide10

Adsorption of Nu

10

2D

1

H-

1

H DOSY NMR on S6ZNu

2D

1

H-

1

H

NOESY NMR on

S6ZNu

Surface desorption experiment in NMR

pH 6.5

pH 6.9

+ Nu

+ Nu

+ Nu

pH 7.7

Surface coverage

for S2ZNu

0.62 mg Nu/ NP m

2

when added to ethanolic sol

Slide11

Adsorption of Nucleoside MP

s and ODN

Adsorption occurs below pH 7

Investigation of concurrent adsorption

of

immune

stimulator and fluorophore

Nu and ODN bind stronger than CF

ODN stays at the NP surface at pH 7.4, CF and Nu are likely to desorb

use of dye labelled ODN

11

when added to aqueous sol

0.66 mg/m

2

for ODN

0.66-0.88 mg/m

2

for Nu

0.30 mg/m

2

for ODN

0.15 mg/m

2

for CF

0.66 mg/m2 for Nu0.33 mg/m2 for CF

Slide12

CF pushed off the surface by ODN in NMR investigation

12

Adsorption of CF

and

ODN

when added to aqueous sol

Detail of

1D

1

H NMR

spectra

ODN

S4Zd + ODN

S6Z CF

S6Z CF

+ ODN, washed, +ODN

Slide13

13Effect of Buffer on adsorption

Buffer

Adsorption of Nu

Remark

K-phosphate

Confined

Charged surface

HEPES*

No interference

Complexation

MES**

No interference

pH 6.0- 6.7

*4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, **2-(N-

morpholino

)

ethanesulfonic

acid

*

**

Evaluation of MES analogues of

higher

working

pH (MOPSO)

when added to aqueous sol

Slide14

14

B

road signals

in 1D

1

H NMRThe sharp lines give negative NOEs at 500 ms mixing time

when added to aqueous sol

Adsorption of Nucleoside MP

s and ODN

S6Zd + Nu in

basic water

0.1 mg Nu / m

2

NP

S4Zd + ODN in basic water

0.6 mg ODN / m

2

NP

H

2

O

S6Zd + Nu in

basic water

H

2

O

Slide15

Summary15

in water

Pure adsorption of ligands

Lower amount of Nu adsorbed

in ethanol

Ligands precipitate onto the NP surface: very little “real” adsorption

Higher cargo

Surface modification of SiO

2

@ZrO

2

NPs

Addition of ligands in DMSO

Use FITC-

labeled

ODN

Find

non-

interfering

buffer

Slide16

Acknowledgement

Synthetic and analytical laboratory facilities to Prof. Isabel Van Driessche (SCRIPTS group), Prof. Peter Dubruel (PCN group),

Prof. Stefaan de Smedt (GeRN group)

Promotor: José C. Martins

Mentor: Krisztina Fehér

This project has received funding from the European Union’s Horizon 2020 research and innovation

programme

under the Marie

Sklodowska

-Curie grant agreement No 703374.

TEM

analysis

to Katrien De

Keukeleere

and Evert

Dhaene

Slide17

Livia Naszályi NagyMarie Curie postdoctoral researcherdepartment of Organic and Macromolecular Chemistry

E Livia.NaszalyiNagy@ugent.be

T +32 9 264 44 88

M +32 487 126 129

www.ugent.be

Ghent

University@ugentGhent University

Thank you for your attention!

Slide18

Nu mixture 1H NMR

18

2D

1

H-

1

H DOSY

Slide19

Fluorophore adsorption to NP19

Slide20

20

Slide21

SiO2@ZrO2 – HEPES interaction

Appearance

of new UV absorbance peak at 238 nm

related to

particle – HEPES interaction

21

Slide22

A.F. Martins et al. Contrast Media Mol. Imaging 2013 (8) 265

22

S

i

O

2

@ZrO2 – HEPES interaction

S6Z Nu + 10 mM HEPES

S6Z Nu + 10 mM HEPES

HEPES

HEPES

HEPES

Ga

3+

+ HEPES

Slide23

Base pair formation in Nu mixture:adsorption to the surface through phosphate group, charged surface

23

Adsorption of

d

GMP

when added in aqueous solution to ethanolic sol

Ethanol

Butanol

Strong interaction between dGMP/TMP and

SiO

2

@ZrO

2

surface? Titration: yes

NMR: no sign of adsorption

FTIR:

transformation of silica network

formation of Si-phosphate bonds

gel formation: dGMP stacking*

*Neurohr and Mantsch, Can. J. Chem. 1979 (57) 1986

Slide24

Adsorption of dGMP to S2Z24

Spontaneous stacking of dGMP in aqueous solution

Slide25

Adsorption of dGMP to S2Z25

Slide26

Effect of HEPES on ODN adsorption26