1. Martin-Luther University Halle-Wittenberg, Germany
Evaluation of in vitro degradation rate of Hyaluronic acid -based hydrogels cross-linked with 1,4-butanediol diglycidyl ether (BDDE)
Mohammed AL-Sibani
15th April 2017

2. Theoretical background
(C14 H21NO11)n
Hyaluronic acid (HA)
Hyaluronic acid (HA) is a high-molecular weight polymer composed of repeating disaccharide units of glucuronic acid and N-acetyl glucosamine.

3. Theoretical background
Applications
HA has been used in a wide range of medical and cosmetic applications including treatment of arthritis, dermal filler, moisturizer, cream and oil.

4. Theoretical background
Drawback
Native HA is found in linear chains.
It exhibits poor mechanical properties and fast in- vivo degradation.
The half-life of native HA after injection into the skin is no longer than 1 day

5. Theoretical background
HA cross-linking
HA should be cross-linked via a chemical cross-linker.
The cross-linked HA forms a 3-dimensional network scaffold (hydrogel).
The cross-linked hydrogels exhibit a higher resistance against enzymatic degradation than native HA.

6. Aim of study
How is the degree of cross-linking estimated ?

7. Evaluation of in-vitro degradation rate of the cross-linked hydrogel
Aim of study
How is the degree of cross-linking estimated ?
Evaluation of in-vitro degradation rate of the cross-linked hydrogel

8. Evaluation of in-vitro degradation rate of the cross-linked hydrogel
Aim of study
How is the degree of cross-linking estimated ?
Evaluation of in-vitro degradation rate of the cross-linked hydrogel
Weight loss method

9. Aim of study How is the degree of cross-linking estimated ?
Evaluation of in-vitro degradation rate of the cross-linked hydrogel
HPLC
UV-Visible spectrometer
Weight loss method

10. Evaluation of hydrogel degradation rate
Chromatographic method (HPLC)
Preparation
A cross-linked BDDE-HA hydrogel was prepared following a method described in literature.
Measurements
Two samples (A & B) equivalent in weight were taken from the prepared hydrogel and incubated with 500µl of hyaluronidase BTH solution at 37 C. Sample A was digested for 24 h and sample B was kept until complete digestion.
The reactions were stopped by boiling for 10 min. The extracts were removed by centrifuging and then diluted with purified water in 100 ml volumetric flasks.

11. Evaluation of hydrogel degradation rate
Chromatographic method
Measurements
The content of N-acetyl-glucosamine (NAG) in the extracts was assayed using HPLC (Agilent 1220). The mobile phase was composed of two solutions A: 96% (water 96% + acetonitrile 4%), B: methanol 4%.
Analysis was performed in isocratic mode ,injection volume of 2 µl, flow rate of 0.4 ml/min, wavelength was set up at 195 nm.
Quantification was carried out against series of standard solutions between 20 and 300 µg/ml.
The degradation rate was evaluated by calculating the relative difference (%) of NAG found in the extract after one day digestion to the total amount of NAG found in the extract after complete digestion.
Statistics: Each analysis was carried out three times. The result was expressed as mean ± 95% confidence interval.

12. Evaluation of hydrogel degradation rate
Chromatographic method
Data finding
Sample A
NAG after one day digestion
Sample B
NAG after complete digestion

13. Evaluation of hydrogel degradation rate
Chromatographic method
Data finding
Chromatograms of standards
Calibration curve

14. Evaluation of hydrogel degradation rate
Chromatographic method
Data finding
Trial
Amount of (NAG) in samples (A) extracts.
Amount of (NAG) in samples (B) extracts
Degradation rate
(A /B) X 100 1
18.5 mg
29.8 mg
62% 2
14.7 mg
25.4 mg
57.9% 3
20.0 mg
29.5 mg
67.8%
Average degradation rate with 95% confidence interval =
62.6% ±12.3 w/w
Precision value % R.S.D =7.95
Limit of detection (LOD) = µg/ml
Average recovery = 81.0%
Trial
Weight of swollen hydrogel
Theoretical value of HA in 5.0g sample
Actual value obtained
Recovery 1
153
32.7 mg
29.8 mg
91.0 % 2
144
34.7 mg
25.4 mg
73.2% 3
139
35.9 mg
29.5 mg
82.2%

15. Evaluation of in vitro degradation rate
Colorimetric method (UV-Visible)
Preparation
A cross-linked BDDE-HA hydrogel was prepared following a method described in literature.
Measurements
Two samples (A & B) equivalent in weight were taken from the prepared hydrogel and incubated with 500µl of hyaluronidase BTH solution at 37 C. Sample A was digested for 24 h and sample B was kept until complete digestion.
The extracts were centrifuged and then transferred into 10 ml volumetric flasks and diluted up to the mark.

16. Evaluation of hydrogel degradation rate
Colorimetric method
Measurements
The content of NAG in the extract was quantified using a single beam UV-Visible spectrophotometer (Spekol 1500). The absorbance was recorded at 585 nm. Quantification was carried out against series of standard solutions.
The degradation rate was obtained by calculating the relative difference (%) of NAG found in the extract after one day digestion to the total amount of NAG found in the extract after complete digestion.
Approximately 1.0 ml from the diluted extract was mixed with 0.1 ml of 0.25 M sodium carbonate and boiled for 1 min in a water bath. Amounts 6:1 of glacial acetic acid and Ehrlich's reagent were then added. The mixture was left until a violet color was produced.
Statistics: Each analysis was carried out three times. The result was expressed as mean ± 95% confidence interval.

17. Evaluation of hydrogel degradation rate
Colorimetric method
Data finding
Sample A NAG after one day digestion
Sample B
NAG after complete digestion

18. Evaluation of hydrogel degradation rate
Colorimetric method
Data finding
Color gradient of standards
Calibration curve

19. Evaluation of hydrogel degradation rate
Colorimetric method
Data finding
Trial
Amount of (NAG)in samples (A) extracts.
Amount of (NAG) in samples (B) extracts
Degradation rate
(A /B) X 100 1
17.8 mg
27.3 mg
65.2 % 2
16.4 mg
28.7 mg
57.0 % 3
17.4mg
25.7 mg
67.7 %
Average degradation rate with 95% confidence interval =
63.3 ± 13.9 w/w
Precision value % R.S.D = 8.83
Limit of detection (LOD) = µg/ml
Average recovery = 83.1%,
Trial
Weight of swollen hydrogel
Theoretical value of HA in 5.0g sample
Actual value obtained
Recovery 1
138
36.2 mg
27.3 mg
75.4% 2
164
30.48 mg
28.7 mg
94.2% 3
155
32.25 mg
25.7 mg
79.7 %

20. Evaluation of hydrogel degradation rate
Gravimetric method (balance)
Preparation
A cross-linked BDDE-HA hydrogel was prepared following the procedure described previously.
Measurements
This method is widely described in literature and it has been commonly used to follow-up the in-vitro degradation rate of cross-linked hydrogels. It is also called “ the weight loss method ”.
A sample (5.0 g) was taken from the prepared hydrogel and incubated with 500µl of hyaluronidase BTH solution at 37 C for 24 h.

21. Evaluation of hydrogel degradation rate
Gravimetric method
Measurements
The degradation rate was evaluated due to the change in hydrogel weight before and after enzymatic incubation based on following equation:
Measurements were carried out using 4-decimal point analytical balance from Adam Equipment Inc.
Statistics: Each analysis was carried out three times. The result was expressed as mean ± 95% confidence interval.
Data finding
Average degradation rate with 95% confidence interval =
73.4%±5.7 w/w
Precision value % R.S.D = 3.13
Trial
Initial weight
Remaining weight after one-day enzymatic digestion
Weight lost
Degradation rate =
(weight lost) /(Initial weight) x 100% 1
5.0 g
1.2 g
3.8 g
76% 2
1.37 g
3.63 g
72.6% 3
1.42 g
3.58 g
71.6%

22. Conclusion

23. Conclusion
The HPLC and UV methods could both offer ideal alternatives for the evaluation of the degradation rate of BDDE-HA cross-linked hydrogels and provide more information for designing the future HA hydrogel. However, UV method is much easier and cheaper than HPLC.
The application of chromatographic method is more challenging than the colorimetric method particularly when dealing with cross-linked polymer. The stationary phase may become saturated or blocked over the course of analysis. The viscoelastic properties of HA could affect separation process and retention time.

24. Conclusion

25. Conclusion
200 µg /ml
100 µg /ml
150 µg /ml
250 µg /ml

26. Thank you