Engineering vaginal film platform for mucoadhesion and sustained drug release for HIV-1 prevention

2.2.1. Synthesis and chemical characterization of PLACL

Three PLACLs with different molar ratios of L-lactide (LA) and ε-caprolactone (CL) were synthesized using ring-opening polymerization based on the previously published method [23]. Briefly, the synthesis was carried out in a 50 ml flask placed in a temperature-controlled oil bath. Premeasured amounts of LA and CL were simultaneously added and melted. The flask was purged with nitrogen for 30 min and then stannous octoate (0.05 mol% versus total monomers) was added into the flask under stirring at 100 rpm and reacted at 140 °C. The reaction between monomers was stopped after 24 h by cooling the mixture to room temperature and addition of methylene chloride. The product was dissolved in chloroform and precipitated in an excess of methanol to remove the catalyst impurities and unreacted monomers. The product was dried at 35 °C under vacuum until it reached the constant weight. The copolymer was named according to the feeding molar ratios of LA to CL, as PLACL 70, 80 and 90, respectively.

The molecular weight of PLACL samples was determined by gel permeation chromatography (GPC). PLACLs were dissolved in tetrahydrofuran to a concentration of 5 mg/ml and samples were filtered using a 0.45 μm nylon membrane. Molecular weights and dispersities were determined using a Waters GPC system with Jordi 500 Å, 1000 Å and 10,000 Å divinylbenzene columns and a refractive index detector (Waters) which was calibrated using polystyrene standards of 62,300 Da, 30,000 Da, 9000 Da and 2500 Da. The injection volume for each sample was 20 μL and the flow rate was 0.500 ml/min. The chemical composition of PLACL samples was analyzed using proton nuclear magnetic resonance (¹H NMR) spectroscopy (Bruker 400). All samples were dissolved in deuterated chloroform and the spectra were recorded at 400 MHz. Chemical bond formation of the PLACL was studied using attenuated total reflectance-Fourier-transform infrared spectroscopy (ATR-FTIR). The FTIR spectra were recorded in absorption mode with a resolution of 4 cm⁻¹ using Bruker Vertex 70 FTIR spectrometer. Differential Scanning calorimetry (DSC, Mettler Toledo) was utilized to study the thermal properties of PLACL copolymers. Sample (~5 mg) was sealed in an aluminum pan and first heated from room temperature to 250 °C (1st cycle), then cooled to −70 °C (2nd cycle), and finally reheated to 250 °C (3rd cycle) at a heating/cooling rate of 10 °C/min and a nitrogen flow rate of 50 ml/min. All the processes were carried out under nitrogen atmosphere. Glass transition temperature (T_g) and melting temperature (T_m) were obtained from the heating cycle (−70 °C to 250 °C, 3rd cycle). DSC data was analyzed using STARe software.

2.2.2. Film formulation development

The polymeric film was manufactured using a modified solvent casting method [35]. Thiolated chitosan used in this study was synthesized and characterized using the method described in our previous work [22]. Briefly, chitosan (190–310 kDa) was dissolved in 1 % v/v acetic acid and pH adjusted to 5.0 using sodium hydroxide. This mixture was reacted with 2-iminothiolane HCl and stirred for 24 h. The solution was then dialyzed against 5 mM HCl, 5 mM HCl containing 1 % NaCl, and 1 mM HCl using a dialysis tube (10 kDa MWCO). The obtained solution was lyophilized before utilizing in film formulation development. Degree of thiolation was determined using Ellman’s assay as previously described [22]. The degree of thiolation was found to be 182.94 +/− 31.44 μmol/g.

The film formulation was composed of PLACL, thiolated chitosan, PVA 40–88, METHOCEL E5, propylene glycol, and glycerin. All films utilized in this study were manufactured using the same method with slight variations to formulation composition. To manufacture a 20 g liquid blend of the formulation tested in the macaque study, thiolated chitosan (0.2 g) was wetted in a portion of Milli-Q water overnight and PLACL (0.8 g) was dissolved in 1 g of methylene chloride separately. PVA 40–88 (1 g) was added to a beaker containing water and heated to 90 °C in a water bath for 15–20 min under overhead stirring (100 rpm) to allow solubilization of PVA, followed by stirring for 4 h. The PVA mix was added to PLACL containing dispersion and stirred overnight. After 24 h, the wetted thiolated chitosan solution and PVA/PLACL solution were mixed until a uniform dispersion was formed (at least 6 h). This blend was mixed in the laboratory fume hood for evaporation of methylene chloride. MK-2048 was dispersed in a mix of propylene glycol (0.8 g) and glycerin (0.8 g), and then added to the film dispersion containing all other excipients. Once the excipients and drug were mixed uniformly, the solution was cast onto a polyester substrate using a 4″ doctor blade and dried on an automatic film applicator (Elcometer^® 4340) with temperature set at 60 °C for 30 mins. The film sheet was separated from the substrate and cut into 1″ × 1″ individual unit doses. PVA and thiomer films were prepared as controls. PVA film, representing baseline mucoadhesion and lack of sustained release, was composed of PVA 40–88, METHOCEL E5, propylene glycol, and glycerin. Thiomer film, which possesses mucoadhesiveness, but lacks sustained release, was composed of thiolated chitosan, PVA 40–88, METHOCEL E5, propylene glycol, and glycerin. Thiomer PLACL film equipped with mucoadhesion and sustained release properties was composed of thiolated chitosan, PVA 40–88, METHOCEL E5, propylene glycol, glycerin, and PLACL 70, 80 or 90.

2.2.3. Physicochemical characterization and stability of films

The films were characterized for mass using analytical balance (Mettler Toledo XS105), thickness using thickness gauge (Mitutoyo Corporation), moisture content using Karl Fisher apparatus (Metrohm, 758 KFD Titrino) in accordance with the titration method specified by the manufacturer, and puncture strength using texture analyzer (TA. XT. Plus^®, New York, NY). The drug content in films was determined by extracting the drug using 40 % acetonitrile and analysis by high-performance liquid chromatography (HPLC) method. The mechanical properties of films containing different polymer compositions were evaluated using uniaxial tensile testing with ADMET MTEST Quattro mechanical testing system (n = 4). A previously investigated PVA based film and marketed film product VCF^® were used as control groups. The casted films were cut into rectangular shape (10 mm × 7 mm). Samples were stretched until failure at a constant jogging speed of 10 mm/min. The stress (MPa) was obtained by dividing the applied force (N) with cross-section area (mm²) and % elongation (strain) was obtained from the displacement using ((L–L0)/L0*100), where L0 was initial gauge length and L was instantaneous gauge length. Elastic modulus was calculated from the linear stress–strain curve between 5 % and 15 % strain. Films were placed in the humidity chamber at 25 °C / 60 % relative humidity (RH) and 40 °C / 75 % RH, and the drug content was monitored for 6 months.

2.2.4. In vitro drug release

To determine the kinetics of drug release from MK-2048-loaded films in different media, an in vitro release study was conducted using a USP dissolution apparatus I with basket (Distek dissolution system model 2100; North Brunswick, NJ). Films measuring 1”x1” were utilized in this study. The dissolution was performed at 37 °C and a rotation speed of 10 rpm. 50 ml of 0.5 % w/v of sodium dodecyl sulfate (SDS) in acetate buffer (pH 4.2) or 0.5 % w/v of SDS in Milli-Q water was used as the media for screening film formulations. The test was carried out for up to 7 days. At predetermined time intervals, 1 ml aliquots were withdrawn and replaced by 1 ml of fresh media.

MK-2048 in collected samples was analyzed using HPLC. Dionex Ultimate 3000 HPLC system (Thermo fisher) with diode array detector (Pump DGP-3600 A, Autosampler WPS-3000TSL, Photometer PDA-3000) and Chromeleon data acquisition system was used for analytical method development. An Xbridge C18 HPLC column (5 μm, 2.1 × x 50 mm, waters) with guard column was used to separate MK-2048. The mobile phase consisted of (A) 0.1 % of formic acid in Milli-Q water and (B) acetonitrile. The gradient elution consisted of 10 % B at 0–2 min, 10 % B at 2–6 min with linear change to 50 % B at 7 min and then goes to 10 % B at 8 min with linear change and stays at 10 % B from 8 to 11 min. The flow rate was 1 ml/min. Sample injection volume was 20 μl and MK-2048 was determined at wavelength of 334 nm.

2.2.5. Ex vivo mucoadhesiveness

The ex vivo mucoadhesion testing of films was carried out using texture analyzer (TA. XT. Plus^®, New York, NY). The film was attached to the head of a cylindrical probe (1″ radius, TA-57R) by double-sided adhesive tape. Porcine intestinal tissue was fixed in the tissue holder with an exposure area of 0.785 cm². Before the probe with the film contacted the tissue, 15 μl of the vaginal fluid simulant (VFS) was added to the surface of the tissue and left for equilibration for 15 s. Following equilibration, the probe attached with the film applied a constant force of 150 g force on the tissue for 60 s. The probe was then withdrawn to detach the tissue at a speed of 0.5 mm/s. The change of the withdrawal force involved in detaching the film from tissue and the debonding distance were recorded by the Exponent software. The work of adhesion calculated from the area under the force vs. distance curve was used for comparing the mucoadhesiveness of different film formulations.

2.2.6. Ex vivo tissue epithelial integrity

The human ectocervical tissues used in this study were obtained through the University of Pittsburgh Health Sciences Tissue Bank as per approved IRB protocol PRO09110431. All tissues were de-identified and collected through an Honest Broker. The human ectocervical tissues were collected from three premenopausal women (30–55 years old) undergoing hysterectomy with benign health conditions. The excised tissues were transferred to the lab in Dulbecco’s Modified Eagle Medium (DMEM). Tissues were snap frozen and then stored in −80 °C for later use.

The impact of the thiomer containing films on the integrity of human ectocervical tissue was evaluated with transepithelial electrical resistance (TEER) using Ussing Chamber system (Physiologic Instruments, Inc. San Diego, CA) [36]. A frozen tissue was thawed in 37 °C water bath for 5 mins. The stromal tissue was removed from the excised ectocervical tissue using a Thomas-Stadie-Riggs tissue slicer (Thomas Scientific, Swedesboro, NJ). Thickness of the tissue was measured using a digital micrometer by mounting between the tissue two glass slides. The excised tissue was placed in the Ussing Chamber slider and equilibrated for 30 mins with exposure to PBS in apical and basal chambers. The tissue exposure area was 0.26 cm². TEER was monitored by Acquire and Analyze Rev. II software, version 3.1 after adding thiomer film or thiomer PLACL80 film into the apical hemichamber. Both hemichambers were bubbled with continuous oxygen to maintain tissue viability. 0.6 % (w/v) of formulated Nonoxynol (N-9, GYNOL II^®) was used as a positive control and PBS as a negative control for validation of this TEER assay. Haemotoxylin and Eosin (H&E) staining was performed post TEER study to assess post exposure morphological changes in the tissue.

2.2.7. Ex vivo tissue permeability

The tissue permeability of thiomer containing films was evaluated using the In-Line Cell system (PermeGear, Hellertown, PA). Tissue procurement and processing were conducted as previously described for the TEER study. Excised human ectocervical tissue was placed between donor and receptor compartments. The tested articles introduced into the donor compartment were unformulated MK-2048 or single 6 mm biopsy punches of MK-2048 PVA base film, MK-2048 thiomer film, or MK-2048 thiomer PLACL80 film with 450 μL VFS. The receptor medium used was DMEM at a flow rate of 50 μl/min and the cells were water jacketed to maintain a temperature of 37 °C. Excised human ectocervical tissue was exposed to each film for 6 h. A 3 ml receptor sample was collected throughout the experiment every hour. After the exposure period, the tissue was collected for H&E staining and drug quantitation. Apparent partition coefficients (P_app) were calculated using the following equation:

where dX is the cumulative amount of MK-2048 (ng) in the receptor chamber, A is the surface area of the cervicovaginal tissue (cm²) and Cd is the initial concentration of MK-2048 in the donor compartment (ng/ml).

2.2.8. In vitro anti-HIV activity

Cellular toxicity and in vitro activity studies were conducted using TZM-bl cell assay as previously described [37]. TZM-bl cells were seeded in a 96-well plate. 1″ × 1″ Films were dissolved in 1 ml saline solution with 10–10⁶ dilution using cell culture medium. 100 l of film solution was added into TZM-bl cells for incubation. For cytotoxicity study, film solutions were incubated with TZM-bl cells overnight and media was replaced with 100 μl of CellTiter-Glo^®. After 10 mins incubation, luminescence was determined by plate reader. The % viability of cells treated with film solution was calculated by the luminescence of film after treatment over that of cells without any treatment. For HIV inhibition efficacy, film dilutions were added to TZM-bl plates followed by adding HIV-1_BAL to the cells. Cells were incubated for 48 h and then media was replaced with 100 μl of Bright-Glo^®. After 2 mins incubation, luminescence was determined by plate reader. The % of HIV inhibition was calculated by using the equation: (1 - cells with film treatment/cells with HIV-1 infection only) * 100.

2.2.9. Compatibility with Lactobacillus

The standard microbicide safety test was used as previously described [38] to assess Lactobacillus compatibility with 1″ × 1″ MK-2048 thiomer PLACL80 film. Briefly, bacterial suspensions were prepared in ACES buffer and the films were then dissolved and mixed in the suspensions. The suspensions were incubated for 30 mins at 37 °C. Samples were taken at time zero and again after 30 mins. Viability was determined by standard plate count. A sample was compatible with Lactobacillus if the reduction in viability was <1 log₁₀.

2.2.10. Film evaluation in pigtail macaques

Animal handling: Reproductive aged, female Macaca nemestrina, 11–16 years old, and weighing 8–10.5 kg were utilized in the animal experiment. Animals were not hormonally synchronized for studies described here. Monkeys were housed in ABSL-2 facilities (general colony housing) at the Washington National Primate Research Center (WaNPRC) located at the University of Washington in Seattle, Washington. The animals were housed and cared for under conditions that meet NIH standards as stated in the Guide for the Care and Use of Laboratory Animals (National Academy of Sciences, 2011), ILAR recommendations, and AAALAC accreditation standards for animals of this species. The University of Washington, including the Primate Center, is fully accredited by AAALAC.

PK study: The lead ER film (thiomer PLACL80 film) loaded with 24 mg MK-2048 was evaluated in macaques for film retention and pharmacokinetics. Each animal had one film intravaginally administered by a trained technician. Colposcopy-aided photography was used to evaluate the vaginal fornix and face of the cervix, where films were placed. At each visit, the appearance of the cervicovaginal environment was documented using digital photography integrated with the colposcope and the status of film residue in situ was noted. Findings generated were used to describe the intravaginal dissolution time of the ER film under investigation and to note impact of films on mucosal integrity within the field of view. Vaginal pH was monitored for the duration of the study to assess the effects of prolonged film retention and drug delivery to the vaginal milieu. Vaginal pH was measured by a colorimetric test strip to detect large shifts in pH. Vaginal secretions were sampled with a polyester tipped swab, rolled and held against the vaginal wall for approximately 20 s. The moistened tip of the swab was dabbed on the colorimetric pH (4.0–9.0) indicator strip (resolution 0.5 pH units) and the result was determined by comparing the strip to the color chart provided by the pH test manufacturer. Blood plasma, cervicovaginal fluids and local tissues were collected to monitor MK-2048 drug delivery and describe the pharmacokinetics of MK-2048. A 2–5 ml blood sample was collected into a K₂EDTA tube, processed and plasma drawn off. Plasma samples were stored at ultra-low temperature (−70 °C) until batched for overnight shipment to the analytic lab for pharmacokinetic testing. Vaginal fluid samples were collected with a polyester tipped swab, rolled and held against the vaginal wall for approximately 20 s. Each swab was weighed (pre/post collection) to determine sample weight. The swab head was snapped off and stored in a 3.6 ml cryovial and frozen (−70 °C) for batched shipment to the Anderson Lab (PK) for processing. Cervical and vaginal tissue biopsies were collected with 1.5 mm Silcut biopsy forceps. Each sample was weighed (target ≥4 mg) and store at −70 °C in a cryotube for bioanalysis.

Bioanalysis: UPLC-MS/MS was used to quantify MK-2048 in collected biosamples. Chromatographic separation was achieved using an Acquity^® UPLC BEH Shield RP18; 1.7 μm 2.1 × 100 mm reversed-phase analytical column and two mobile phases utilizing a gradient consisting of 40:60 acetonitrile:10 mM ammonium formate pH 3 and 90:10 acetonitrile:10 mM ammonium formate pH 3 at 0.5 ml/min. This method utilized stable labeled isotopic internal standard analogues for MK-2048. Detection of the analytes was achieved by protonated ESI ionization tandem mass spectrometry. Linearity was displayed over the range of 25.0 pg/ml to 50,000 pg/ml for MK-2048 when 200 μl of plasma was analyzed. Due to the high concentrations associated with samples taken near the drug delivery device, and the propensity for nonspecific binding, cervical vaginal fluid and cervical and vaginal tissue biopsy samples were extracted into and diluted with human plasma. This resulted in a matrix >90 % human plasma, which allowed for the analysis of these samples using the same methodology described above. Cervical vaginal fluid was determined to have a density of 1 and was reported in pg/ml (1 mg of fluid was equivalent to 1 μl of fluid). Cervical and vaginal tissue was reported as pg/g. Assay accuracy and precision met established validation guidelines.

2.2.11. Statistical analysis

All values are reported as mean ± standard deviation (SD). Statistical data analyses were performed using the Student’s t-test between two groups and one-way ANOVA with Tukey’s post hoc test for the analysis among three or more study groups, with p < 0.05 as the minimal level of significance. In vivo maximum concentration (Cmax) and half-life in biophases, reported as geometric mean and 95 % confidence intervals (95 % CI), were used to describe drug delivery in the animals that received MK-2048 thiomer films. All tests were performed using the GraphPad Prism software version 7.