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 *
a
Department of Bioengineering, University of Utah, Salt Lake City, Utah, 84112, USA
b
Department 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:
patrick.kiser@utah.edu
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
19
F 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
F NMR spectrum (400 MHz spectrometer, Varian) was collected using 10 L of
19
,,-trifluorotoluene (TFT) as an internal standard added accurately using a 25L syringe.
Similarly, Mn of PTMEG utilized for degradable polymeric diol synthesis was also determined
according to the equation below
Mn 
m
 x

 N c
F ,s


 y

N
F
,
i


where
x  19 F peak area of trifluoro acetic ester of sample at - 75 ppm
Equation 1
y  19 F peak area of TFT at - 63 ppm
c  mM of TFT added  0.0815 mM
N F,s  number of fluorine atoms per molecule of sample
N F,i  number of fluorine atoms per molecule of internal standard TFT
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 (Error! Reference source not found.). Furthermore, a method for determination of end
hydroxyl content was developed by slight modification of the ASTM method utilizing
(ASTM 4273). The Mn of degradable polymeric diol as determined by the
19
F NMR
19
F 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,
19
F 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:
Q
(M  M )
i
f
M
i
where
Equation 2
Q  swelling ratio
M  mass of ring or rod segments before the release study
i
M  mass ring or rod segments after the release study
f
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 B
0.0
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.