Biodistribution and pharmacokinetics of dapivirine-loaded nanoparticles after vaginal delivery in mice.

PURPOSE: To assess the potential of polymeric nanoparticles (NPs) to affect the genital distribution and local and systemic pharmacokinetics (PK) of the anti-HIV microbicide drug candidate dapivirine after vaginal delivery. METHODS: Dapivirine-loaded, poly(ethylene oxide)-coated poly(epsilon-caprolactone) (PEO-PCL) NPs were prepared by a nanoprecipitation method. Genital distribution of NPs and their ability to modify the PK of dapivirine up to 24 h was assessed after vaginal instillation in a female mouse model. Also, the safety of NPs upon daily administration for 14 days was assessed by histological analysis and chemokine/cytokine content in vaginal lavages. RESULTS: PEO-PCL NPs (180-200 nm) were rapidly eliminated after administration but able to distribute throughout the vagina and lower uterus, and capable of tackling mucus and penetrate the epithelial lining. Nanocarriers modified the PK of dapivirine, with higher drug levels being recovered from vaginal lavages and vaginal/lower uterine tissues as compared to a drug suspension. Systemic drug exposure was reduced when NPs were used. Also, NPs were shown safe upon administration for 14 days. CONCLUSIONS: Dapivirine-loaded PEO-PCL NPs were able to provide likely favorable genital drug levels, thus attesting the potential value of using this vaginal drug delivery nanosystem in the context of HIV prophylaxis.

In vitro and ex vivo evaluation of polymeric nanoparticles for vaginal and rectal delivery of the anti-HIV drug dapivirine.

Prevention strategies such as the development of microbicides are thought to be valuable in the fight against HIV/AIDS. Despite recent achievements, there is still a long road ahead in the field, particularly at the level of drug formulation. Drug nanocarriers based on polymers may be useful in enhancing local drug delivery while limiting systemic exposure. We prepared differently surface-engineered poly(ε-caprolactone) (PCL) nanoparticles (NPs) and tested their ability to modulate the permeability and retention of dapivirine in cell monolayers and pig vaginal and rectal mucosa. NPs coated with poly(ethylene oxide) (PEO) were shown able to reduce permeability across monolayers/tissues, while modification of nanosystems with cetyl trimethylammonium bromide (CTAB) enhanced transport. In the case of coating NPs with sodium lauryl sulfate (SLS), dapivirine permeability was unchanged. All NPs increased monolayer/tissue drug retention as compared to unformulated dapivirine. This fact was associated, at least partially, to the ability of NPs to be taken up by cells or penetrate mucosal tissue. Cell and tissue toxicity was also affected differently by NPs: PEO modification decreased the in vitro (but not ex vivo) toxicity of dapivirine, while higher toxicity was generally observed for NPs coated with SLS or CTAB. Overall, presented results support that PCL nanoparticles are capable of modulating drug permeability and retention in cell monolayers and mucosal tissues relevant for vaginal and rectal delivery of microbicides. In particular, PEO-modified dapivirine-loaded PCL NPs may be advantageous in increasing drug residence at epithelial cell lines/mucosal tissues, which may potentially increase the efficacy of microbicide drugs.

Interactions of microbicide nanoparticles with a simulated vaginal fluid.

The interaction with cervicovaginal mucus presents the potential to impact the performance of drug nanocarriers. These systems must migrate through this biological fluid in order to deliver their drug payload to the underlying mucosal surface. We studied the ability of dapivirine-loaded polycaprolactone (PCL)-based nanoparticles (NPs) to interact with a simulated vaginal fluid (SVF) incorporating mucin. Different surface modifiers were used to produce NPs with either negative (poloxamer 338 NF and sodium lauryl sulfate) or positive (cetyltrimethylammonium bromide) surface charge. Studies were performed using the mucin particle method, rheological measurements, and real-time multiple particle tracking. Results showed that SVF presented rheological properties similar to those of human cervicovaginal mucus. Analysis of NP transport indicated mild interactions with mucin and low adhesive potential. In general, negatively charged NPs underwent subdiffusive transport in SVF, i.e., hindered as compared to their diffusion in water, but faster than for positively charged NPs. These differences were increased when the pH of SVF was changed from 4.2 to 7.0. Diffusivity was 50- and 172-fold lower in SVF at pH 4.2 than in water for negatively charged and positively charged NPs, respectively. At pH 7.0, this decrease was around 20- and 385-fold, respectively. The estimated times required to cross a layer of SVF were equal to or lower than 1.7 h for negatively charged NPs, while for positively charged NPs these values were equal to or higher than 7 h. Overall, our results suggest that negatively charged PCL NPs may be suitable to be used as carriers in order to deliver dapivirine and potentially other antiretroviral drugs to the cervicovaginal mucosal lining. Also, they further reinforce the importance in characterizing the interactions of nanosystems with mucus fluids or surrogates when considering mucosal drug delivery.

Polymeric nanoparticles affect the intracellular delivery, antiretroviral activity and cytotoxicity of the microbicide drug candidate dapivirine.

PURPOSE: To assess the intracellular delivery, antiretroviral activity and cytotoxicity of poly(ε-caprolactone) (PCL) nanoparticles containing the antiretroviral drug dapivirine. METHODS: Dapivirine-loaded nanoparticles with different surface properties were produced using three surface modifiers: poloxamer 338 NF (PEO), sodium lauryl sulfate (SLS) and cetyl trimethylammonium bromide (CTAB). The ability of nanoparticles to promote intracellular drug delivery was assessed in different cell types relevant for vaginal HIV transmission/microbicide development. Also, antiretroviral activity of nanoparticles was determined in different cell models, as well as their cytotoxicity. RESULTS: Dapivirine-loaded nanoparticles were readily taken up by different cells, with particular kinetics depending on the cell type and nanoparticles, resulting in enhanced intracellular drug delivery in phagocytic cells. Different nanoparticles showed similar or improved antiviral activity compared to free drug. There was a correlation between increased antiviral activity and increased intracellular drug delivery, particularly when cell models were submitted to a single initial short-course treatment. PEO-PCL and SLS-PCL nanoparticles consistently showed higher selectivity index values than free drug, contrasting with high cytotoxicity of CTAB-PCL. CONCLUSIONS: These results provide evidence on the potential of PCL nanoparticles to affect in vitro toxicity and activity of dapivirine, depending on surface engineering. Thus, this formulation approach may be a promising strategy for the development of next generation microbicides.

Rheological properties of vaginal hydrophilic polymer gels.

The objective of this work was to investigate the main theological features of vaginal hydrophilic polymer gels and to elucidate about the relationship between these characteristics and gels composition, and their general influence in therapeutic/usage purpose. Flow and dynamic oscillatory properties of four commercially available (Conceptrol, Gynol II, RepHresh, and Replens) and two investigational vaginal gels were determined by cone-and-plate rheometry, at body temperature. Several parameters (apparent viscosity, complex viscosity, storage modulus, loss modulus, critical oscillatory stress, tan delta, thixotropy and yield stress) were measured and/or calculated. Gels presented non-Newtonian, pseudoplastic, thixotropic behavior, with yield stress. Overall viscosities varied between 13500 Pa.s and approximately 80 Pa.s within a biologically relevant shear rate interval (0.01-100 s(-1)). Yield stress values were variable between different determination methods but coherent in terms of ranking. Also, tested gels showed viscoelastic properties, being characterized by predominant elastic solid-like behavior. Rheological behavior of vaginal gels strongly depended on the type of gelling agent used, which potentially influences their spreading and retention properties when administered in the vaginal canal. Small variations in gels composition can result in substantial changes in their features, namely viscosity, yield stress and thixotropy. Rheological properties of tested gels appeared to be correlated with their therapeutic/usage purpose.

Performance of an in vitro mucoadhesion testing method for vaginal semisolids: influence of different testing conditions and instrumental parameters.

The purpose of this work was to develop an in vitro mucoadhesion testing method for vaginal semisolid formulations. The proposed method was based on the measurement of the force (detachment force, Fdt) and the work (work of adhesion, Wad) needed to detach a sample of cow vaginal mucosa from a semisolid formulation, using a commercially available texture analyzer. Several testing conditions and instrumental parameters were tested in order to evaluate the mucoadhesive potential of a model vaginal semisolid formulation (1% Carbopol 974P gel). Also, mucoadhesive potential of several commercially available vaginal semisolid products was evaluated. Obtained results showed that the method is reproducible even when the same cow mucosa sample is used up to six times. The similarity of the fluid used to bathe the vaginal mucosa to the one naturally occurring in the vagina influenced considerably the performance of the test, advising that simulation of vaginal fluid properties is important when measuring mucoadhesive properties. Also, temperature of experiment was an important fact to be considered, as results showed slight but significant differences between body (37 degrees C) and room (20 degrees C) temperature. Fdt and Wad increased with increasing instrumental parameters while a plateau region was observable at higher values of probe speed, probe force, and mucosa/sample contact time. Comparison between results for Fdt and Wad demonstrated that although both parameters are generally in agreement, Wad seems to be more reliable and reproducible when evaluating mucoadhesion. Evaluation of commercially available formulations confirmed that experimental conditions are important features that can influence significantly the determination of mucoadhesive potential, being the proposed method an interesting and useful tool in the in vitro evaluation of vaginal semisolids.

Assessing the physical-chemical properties and stability of dapivirine-loaded polymeric nanoparticles.

Nanocarriers may provide interesting delivery platforms for microbicide drugs and their characterization should be addressed early in development. Differently surface-engineered dapivirine-loaded, poly(epsilon-caprolactone) (PCL)-based nanoparticles (NPs) were obtained by nanoprecipitation using polyethylene oxide (PEO), sodium lauryl sulfate (SLS), or cetyltrimethylammonium bromide (CTAB) as surface modifiers. Physical-chemical properties of NP aqueous dispersions were evaluated upon storage at -20-40 °C for one year. NPs presented 170-200 nm in diameter, roundish-shape, low polydispersity index (≤0.18), and high drug association efficiency (≥97%) and loading (≥12.7%). NPs differed in zeta potential, depending on surface modifier (PEO: -27.9 mV; SLS: -54.7 mV; CTAB: +42.4 mV). No interactions among formulation components were detected by differential scanning calorimetry (DSC) and Fourier transform infrared spectroscopy (FTIR), except for SLS-PCL NPs. Colloidal properties of NPs were lost at -20 °C storage. Negatively charged NPs were stable up to one year at 5-40°C; as for CTAB-PCL NPs, particle aggregation was observed from 30 to 90 days of storage depending on temperature. Colloidal instability affected the in vitro drug release of CTAB-PCL NPs after 360 days. In any case, no degradation of dapivirine was apparent. Overall, PEO-PCL and SLS-PCL NPs presented suitable properties as nanocarriers for dapivirine. Conversely, CTAB-PCL NPs require additional strategies in order to increase stability.

Development and validation of a HPLC method for the assay of dapivirine in cell-based and tissue permeability experiments.

Dapivirine, a non-nucleoside reverse transcriptase inhibitor, is being currently used for the development of potential anti-HIV microbicide formulations and delivery systems. A new high-performance liquid chromatography (HPLC) method with UV detection was developed for the assay of this drug in different biological matrices, namely cell lysates, receptor media from permeability experiments and homogenates of mucosal tissues. The method used a reversed-phase C18 column with a mobile phase composed of trifluoroacetic acid solution (0.1%, v/v) and acetonitrile in a gradient mode. Injection volume was 50µL and the flow rate 1mL/min. The total run time was 12min and UV detection was performed at 290nm for dapivirine and the internal standard (IS) diphenylamine. A Box-Behnken experimental design was used to study different experimental variables of the method, namely the ratio of the mobile phase components and the gradient time, and their influence in responses such as the retention factor, tailing factor, and theoretical plates for dapivirine and the IS, as well as the peak resolution between both compounds. The optimized method was further validated and its usefulness assessed for in vitro and ex vivo experiments using dapivirine or dapivirine-loaded nanoparticles. The method showed to be selective, linear, accurate and precise in the range of 0.02-1.5µg/mL. Other chromatographic parameters, namely carry-over, lower limit of quantification (0.02µg/mL), limit of detection (0.006µg/mL), recovery (equal or higher than 90.7%), and sample stability at different storage conditions, were also determined and found adequate for the intended purposes. The method was successfully used for cell uptake assays and permeability studies across cell monolayers and pig genital mucosal tissues. Overall, the proposed method provides a simple, versatile and reliable way for studying the behavior of dapivirine in different biological matrices and assessing its potential as an anti-HIV microbicide drug.

Development and validation of a rapid reversed-phase HPLC method for the determination of the non-nucleoside reverse transcriptase inhibitor dapivirine from polymeric nanoparticles.

The objective of this work was to develop and validate a rapid reversed-phase (RP) high-performance liquid chromatography (HPLC) method for the in vitro pharmaceutical characterization of dapivirine-loaded polymeric nanoparticles. Chromatographic runs were performed on a RP C18 column with a mobile phase comprising acetonitrile-0.5% (w/v) triethanolamine solution in isocratic mode (80:20, v/v) at a flow rate of 1 ml/min. Dapivirine was detected at a wavelength of 290 nm. The method was shown to be specific, linear in the range of 1-50 microg/ml (R(2)=0.9998), precise at the intra-day and inter-day levels as reflected by the relative standard deviation values (less than 0.85%), accurate (recovery rate of 100.17+/-0.35%), and robust to changes in the mobile phase and column brand. The detection and quantitation limits were 0.08 and 0.24 microg/ml, respectively. The method was successfully used to determine the loading capacity and association efficiency of dapivirine in poly(lactic-co-glycolic acid)-based nanoparticles and its in vitro release.