Supplementary material

Pluronic/gelatin composites for actives controlled

release

Duccio Tatini, Paolo Tempesti, Francesca Ridi, Emiliano Fratini, Massimo Bonini, and

Piero Baglioni*

Artificial saliva composition.....................................................................................................................2

Sample setup for release experiments....................................................................................................3

CMT vs Pluronic F127 concentration....................................................................................................4

Absorption spectra of azorubine and gelatin.......................................................................................5

Protocol for the analysis and fitting of the release experiments...................................................6

Release experiments at 25 ºC....................................................................................................................7

Graphical results of the fitting with different models......................................................................8

Results of the release curves fittings......................................................................................................9

1

Artificial saliva composition

Table S1. Artificial saliva composition for release experiments of azorubine from gelatin, PGC-I and PGC-II in simulated oral cavity and stomach conditions. This represents a modified version of the recipe reported by Poggio et al. (doi:10.1016/j.dent.2010.07.010)

Artificial Saliva pH 2 Artificial Saliva pH 7NaCl (mg) 42.1 42.1KCl (mg) 60.0 60.0CaCl2 (mg) 55.5 55.5K2HPO4 (mg) / 33.1HCl 0.1M (mL) 5 /H2O (mL) 45 50

2

Sample setup for release experiments

Figure S1. Picture showing the sample setup used for the investigation of the kinetics of release. A small piece of sample (PGC-I, PGC-II or gelatin hydrogel) is loaded with azorubine (responsible of the red color) and supported into the optical cuvette using a metallic grid of U.S. mesh size 10. The sample is put into contact with artificial saliva at pH 2 or 7.

3

CMT vs Pluronic F127 concentration

The red dots in Figure S2 represent the data obtained from fitting DSC thermograms with EMG functions as reported in the paper, while the black dots represent data taken from Bohorquez et al. (doi:10.1006/jcis.1999.6273). From the linear regression of literature data we obtained the concentration of Pluronic F127 in PGC-I, PGC-II and unmixed sample. Since the initial concentration of Pluronic F127 is 20% w/w, the results from the fitting of the DSC thermograms highlight the dilution effect on the Pluronic phase when in mixtures with gelatin hydrogels.

Figure S2. Critical micellar concentration (CMT) of Pluronic F127 solutions versus Pluronic concentration.

4

Absorption spectra of azorubine and gelatin

Figure S3. Absorption spectra of azorubine and gelatin as a function of pH and temperature.

5

Protocol for the analysis and fitting of the release experiments

The absorption spectra of the samples were analysed in the hypothesis that gelatin and azorubine additively contribute to total absorption.Each spectrum (I s ( λ )) was modeled as a linear combination of the individual spectra of gelatin and azorubine:

I s ( λ )=A ∙ I gelatin ( λ )+B ∙ I azorubine ( λ )+C (1) where A is the amplitude of the gelatin spectrum contribution (I gelatin ( λ )), B is the amplitude of the azorubine spectrum contribution (I azorubine ( λ )¿, and C accounts for a flat background. The I gelatin ( λ ) and I azorubine ( λ )spectra were collected on individual gelatin and azorubine samples whose concentration equals that obtained through the full dissolution of a azorubine-loaded PGC sample in the release medium. For this model to be reliable, A and B values should not exceed 1, while the C contribution should be close to zero. An example of this approach is shown in Figure S4:

Figure S4. Example of the absorption spectrum of an azorubine-loaded PGC sample (block solid line) and relative fitting using eq. (1) (red solid line). The dashed and the dotted lines represent the contribution to the overall fitting of gelatin and azorubine, respectively.

The values found for B and C satisfy the aforementioned conditions, while in most of the samples the A values at long release times exceed 1. This is explained by taking into account that gelatin dissolution (in absence of stirring) is accompanied by the formation of opaque regions in the sample, caused by the scattering from inhomogeneities whose size is comparable to the wavelength of visible light. As a consequence, only Bvalues

6

obtained in time ranges in which 0 < A < 1 were taken into account for the modelling of the release kinetics.

7

Release experiments at 25 ºC

The release of azorubine from PGCs was investigated by UV/Vis spectroscopy. Figure S5 shows the absorption spectra of the azorubine release in artificial saliva both at pH 2 and 7. Spectra were collected at increasing time at 25 °C.

PGC I pH 2 PGC I pH 7

PGC II pH 2 PGC II pH 7

Figure S5. Absorbance as a function of time accounting for the release of azorubine from PGCs at 25 °C.

8

Graphical results of the fitting with different models

9

Figure S6. Azorubine release from gelatin (red markers), PGC-I (green markers) and PGC-II (blue markers) and relative fitting using different models.

10

Results of the release curves fittings

Table S2. Fitting results according to the proposed models (Higuchi, Korsmeyer-Peppas

and Weibull).

Higuchi Korsmeyer - Peppas WeibullkH R2 kKP nKP R2 kW nW R2

Gelatin

25°C

pH 2 0.0033 ± 0.0001 0.942

0.0194 ± 0.011

0.4627 ± 0.0738

0.952 0.00421 ± 0.00391

0.727 ±

0.1210.913

pH 7 0.0088 ± 0.0002 0.687

0.1738 ± 0.0379

0.2290 ± 0.0308

0.976 0.0308 ± 0.0176

0.605 ±0.08

60.970

37°C

pH 20.070549

± 0.000409

0.6670.5296 ± 0.0254

0.139± 0.011

0.645 2.18e-006 ± 2.99e-

006

4.185 ±

0.4460.997

pH 70.067987

± 0.000295

0.4590.6202 ± 0.0194

0.09814 ±

0.00678

0.548 9.5e-006 ± 1.0e-

006

4.265 ±

0.3310.998

PGC-I

25°C

pH 20.007347

± 0.000738

0.8930.0721 ± 0.0111

0.3197 ± 0.0197

0.971 0.0051 ±0.0032

0.767 ±

0.1010.999

pH 7 0.01026 ± 0.00014 0.779

0.1376 ± 0.0249

0.2604 ± 0.0254

0.977 0.01790 ±0.00844

0.679 ±

0.0710.974

37°C

pH 20.093948

± 0.000353

0.7660.39984 ± 0.0134

0.20004 ±

0.00763

0.727 0.001244 ±

0.000429

2.0541 ±

0.1030.996

pH 70.081635

± 0.000267

0.704

0.3904 ± 0.0107

0.18914 ±

0.00581

0.632 0.000310 ±

0.000102

2.2119 ±

0.0879

0.995

PGC-II

25°C

pH 20.006174

± 0.000081

0.9940.01215± 0.00304

0.5277 ± 0.0315

0.993 0.00091 ± 0.00042

0.946 ±0.06

00.956

pH 7 0.00852 ± 0.00015 0.885

0.09723 ± 0.0247

0.3096 ± 0.0355

0.985 0.00707 ± 0.00494

0.814 ±

0.1060.977

37°C

pH 2 0.10319 ± 0.00034 0.826

0.30776 ±

0.00911

0.25174 ±

0.00669

0.819 0.003662 ±

0.000813

1.697 ±

0.0650.998

pH 70.098693

± 0.000243

0.6700.41426

± 0.00805

0.17419 ±

0.00413

0.671 0.000445 ±

0.000122

2.236 ±

0.0770.993

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