Supplement
Engineering a Degradable Polyurethane Intravaginal Ring for Sustained Delivery of Dapivirine
Manpreet Kaura, Kavita M. Guptaa, Azadeh E. Poursaida, Prasoona Karraa, Alamelu Mahalingamb, Hyder A. Aliyara, Patrick F Kisera,b *
aDepartment of Bioengineering, University of Utah, Salt Lake City, Utah, 84112, USA
bDepartment of Pharmaceutics and Pharmaceutical Chemistry, University of Utah, Salt Lake City, Utah, 84112, USA
*Corresponding author: Tel: +1-801-505-6881; Fax: +1-801-585-5151; email:
Unreacted -caprolactone and number average molecular weight
The degradable polymeric diol synthesized was analyzed for the presence of unreacted -caprolactone content through: 1) 1H NMR of the product (Mercury 400 MHz spectrometer, Varian); and 2) extraction and quantification by HPLC. Briefly, about 100 mg of weighed reaction mixture was mixed with 2 mL of 90:10 v/v mixture of acetonitrile:dioxane. The mixture was vortexed followed by centrifugation to remove the undissolved polymer, and the supernatant was analyzed by HPLC using the eluents: acetonitrile with 0.1 % TFA (eluent A) and water with 0.1% TFA (eluent B). A C16 alkyl amide column (Supelcosil TM ABZ + Plus 3µm, 3.3 cm x 4.6 mm ID, Supelco, Bellefonte, PA) was utilized with a gradient of 70:30 v/v A:B to 30:70 v/v B:A in 6 minutes. The retention time of -caprolactone monomer was 1.1 minutes ( = 230 nm). A calibration curve was generated using known standards of -caprolactone and used for calculating the unreacted -caprolactone content.
To obtain polyurethanes with good mechanical properties, it is critical to add the diols and diisocyanate in the right stoichiometric ratio (NCO/OH = 1 to 1.1). Therefore, it is important to determine the molecular weight of the polymeric diol. The number average molecular weight of the product was determined by 19F NMR of the trifluoroacetic ester of the product. About 50 mg of weighed dried diol was reacted with excess trifluoroacetic anhydride (200 L) in 1 mL of dry DCM. The solvent and excess TFAA were removed. The dried product was then dissolved in CDCl3 and 19F NMR spectrum (400 MHz spectrometer, Varian) was collected using 10 L of ,,-trifluorotoluene (TFT) as an internal standard added accurately using a 25L syringe. Similarly, Mn of PTMEG utilized for degradable polymeric diol synthesis was also determined according to the equation below
Equation 1
The reaction product was evaluated for the presence of unreacted caprolactone and end hydroxyl content to determine the number average molecular weight (Mn). The results of HPLC analysis of extracted unreacted caprolactone demonstrated that less than 2% of starting caprolactone was present in the final reaction product after 48 h, suggesting that the reaction went to 98% completion. Additionally, unreacted caprolactone was not detected in 1H NMR spectrum of the polymeric diol providing further confirmation that the reaction went to completion (Figure 3). Furthermore, a method for determination of end hydroxyl content was developed by slight modification of the ASTM method utilizing 19F NMR (ASTM 4273). The Mn of degradable polymeric diol as determined by the 19F NMR method was 1390 + 25 (n = 3, mean SD) while that of PTMEG was 958 39 (n = 3, mean SD). The theoretical Mn of the degradable polymeric diol based on the stoichiometry of the reaction was 1408. Together, the results of the 1H, 19F NMR method and quantification of unreacted caprolactone content suggested that the reaction went to completion and that the degradable polymeric diol was successfully synthesized.
Figure 1: 1H NMR spectrum of degradable polymeric diol. No peaks for unreacted caprolactone monomer were seen. The protons on carbon adjacent to end hydroxyls (a) were clearly seen. The ratio of integrations of the peaks corresponding to caprolactone (d) and PTMEG (g + 0.5e) suggested that approximately 13 units of tetramethylene glycol and four units of caprolactone were present per molecule of the degradable polymeric diol adding up to a Mn = 1392, which corresponded with the reaction stoichiometry.
Swelling calculation
At the end of 30 days, the segments were wiped dry and the masses and dimensions of the segments were recorded. At the end of each release study, the swelling ratio was calculated by using the following equation:
Equation 2
HPLC method for quantifying dapivirine from tissue extracted samples
An Agilent 1200 Series high performance liquid chromatography (HPLC) system was used for Dapivirine and Norethindrone quantification. The area of the analyte was computed using ChemStation 32 software. A C-18 column (Kromasil C18, 10 μm, 250mm X 4.6 mm) was used with acetonitrile containing 0.1% (v/v) TFA (A) and water containing 0.1% (v/v) TFA (B) (both containing 0.1% (v/v) TFA) as eluents at a flow rate of 1 mL/min. A gradient method of 30:70 A: B to 70:30 A: B (details in Table 1) was applied for 18 min. The retention time of dapivirine was 10.2 min (detection wavelength of 286 nm) and the retention time of Norethindrone was 12.8 min (detection wavelength of 245 nm). Reference solutions were prepared in acetonitrile to prepare the standard curves.
Table 1: The gradient for the HPLC method to quantify dapivirine and norethindrone. Mobile phase (A) acetonitrile and (B) water (both containing 0.1% (v/v) TFA).
Time (min) / % Mobile Phase A / % Mobile Phase B0.0 / 30 / 70
3.0 / 45 / 55
13.0 / 55 / 45
16.0 / 70 / 30
17.0 / 30 / 70
18.0 / 30 / 70
Mechanical Testing Apparatus
Figure 2: Experimental setup for evaluation of force vs. compression/retraction profile of intravaginal rings (IVR). A load cell (1 kg) probe was applied on top of the ring with a compression rate of 1 mm/sec () to a particular deformation (50% compression) and released back () to original position with the same rate.