Nanoparticle-releasing nanofiber composites for enhanced in vivo vaginal retention

Abstract

Current approaches for topical vaginal administration of nanoparticles result in poor retention and extensive leakage. To overcome these challenges, we developed a nanoparticle-releasing nanofiber delivery platform and evaluated its ability to improve nanoparticle retention in a murine model. We individually tailored two components of this drug delivery system for optimal interaction with mucus, designing (1) mucoadhesive fibers for better retention in the vaginal tract, and (2) PEGylated nanoparticles that diffuse quickly through mucus. We hypothesized that this novel dual-functioning (mucoadhesive/mucus-penetrating) composite material would provide enhanced retention of nanoparticles in the vaginal mucosa. Equivalent doses of fluorescent nanoparticles were vaginally administered to mice in either water (aqueous suspension) or fiber composites, and fluorescent content was quantified in cervicovaginal mucus and vaginal tissue at time points from 24 h to 7d. We also fabricated composite fibers containing etravirine-loaded nanoparticles and evaluated the pharmacokinetics over 7d. We found that our composite materials provided approximately 30-fold greater retention of nanoparticles in the reproductive tract at 24 h compared to aqueous suspensions. Compared to nanoparticles in aqueous suspension, the nanoparticles in fiber composites exhibited sustained and higher etravirine concentrations after 24 h and up to 7d, demonstrating the capabilities of this new delivery platform to sustain nanoparticle release out to 3d and drug retention out to one week after a single administration. This is the first report of nanoparticle-releasing fibers for vaginal drug delivery, as well as the first study of a single delivery system that combines two components uniquely engineered for complementary interactions with mucus.

Keywords: Electrospinning; Microbicides; Nanofibers; Nanoparticles; Pharmacokinetics; Vaginal drug delivery.

Figure 1. Fiber composites successfully formed with similar nanoparticle and nanofiber diameter

(A) TEM micrograph of blank PLGA nanoparticles. (B) SEM micrograph of PVP composite fibers containing Rho-NP. (C) Confocal imaging of Rho-NP electrospun within PVP composite fibers. (D) Diameter (mean ± S.D.) of Rho-NP, Rho-NP-PVP composite fibers, Rho-NP-PVA composite fibers, and ETR-NP-PVA composite fibers. NP diameter was measured using DLS; fiber diameter was measured from SEM images. (E) SEM micrograph of PVA composite fibers containing Rho-NP. (F) Confocal imaging of Rho-NP electrospun within PVA composite fibers.

Figure 2. Fiber composites dissolve quickly in vitro to release nanoparticles

(A) Composite fiber dissolution on black agar hydrogels used to simulate low fluid volume conditions. White dotted circle represents original area of fiber mesh. Representative images from n=3 replicates per group. (B) Cumulative Rho-NP release from composite fibers under in vitro sink conditions (4 mg composite fibers in 2 mL water). Dotted line at 0.5 h indicates time when both PVA and PVP fibers had fully dissolved. Data plotted as mean ± S.D., with n=3 per group.

Figure 3. Significantly less external leakage of nanoparticles is observed for PVA and PVP composite fibers compared to aqueous suspensions

(A–D) Xenogen imaging of black paper circles was used to measured external leakage for 1 h immediately after administration, with n=4 mice per group. In (A), the top two circles represent mice receiving blank PVA composite fibers, and the bottom two circles represent untreated mice. All images were set to the same scale of minimum and maximum radiance. (E) Maximum radiance quantified from Xenogen images in (A-D), mean ± S.D.

Figure 4. Fiber composites result in increased nanoparticle dose retention in the reproductive tract and association with vaginal tissue at 24h

(A) Whole reproductive tracts imaged after dissection at 24 h, pre-wash. (B) Total dose recovery defined as the sum of dose recovered at 24h in wash buffer, vaginal tissue (post-wash), and uterine horns. (C) Opened vaginal tracts imaged after wash (tissues placed in 5 mL PBS for 30 minutes). (D) Dose recovery of fluorescent nanoparticles at 24h in wash buffer or homogenized vaginal tissue, post-wash. Data plotted as mean ± S.D., with n≥7 per group, n=5 for blank). ***p<0.001 relative to all other groups (one-way ANOVA comparing all four groups within the same tissue type).

Figure 5. Nanoparticles are retained in the reproductive tract out to three days after a single PVA composite fiber administration

(A) Whole reproductive tracts, pre-wash. (B) Opened vaginal tracts post-wash, cut open to the cervix. Minimum and maximum radiance were set to the same values for all Xenogen images taken in (A) and (B). (C) Dose recovery of fluorescent nanoparticles at 1d, 2d, 3d, 5d, and 7d, measured by tissue homogenization methods, plotted as mean ± S.D. For each time point, n=3 animals received PVA composite fibers and n=1 untreated mouse was used as a control (1d time point from Fig. 4, different study with same methods; n=7 mice per group).

Figure 6. PVA composite fibers result enhanced ETR retention over 7 days in the cervicovaginal lavage and vaginal tissue relative to aqueous suspensions

(A) ETR content in 200 μL cervicovaginal lavage, including any undissolved fibers present. (B) ETR content in homogenized vaginal tissue, post-lavage. Line represents mean of n=5 mice per group at each time point out to 72h, with fiber group only extended to 5d and 7d. Statistical outliers were identified with Grubb’s test (alpha = 0.01) and were not included for PK analysis or displayed in this figure. **p<0.01, ***p<0.001.

Figure 7. Low systemic drug exposure was observed for both delivery platforms, with more sustained release observed for the PVA composite fibers

(A)–(D) ETR content detected by LC-MS/MS in homogenized tissues. *p<0.05, **p<0.01, **p<0.001. Line represents mean of n=5 mice per group at each time point out to 72h, with fiber group only extended to 5d and 7d. Statistical outliers were identified with Grubb’s test (alpha = 0.01) and were not included for PK analysis or displayed in this figure.