Pelvic organ prolapse (POP) is the downward herniation of one or more pelvic organs and causes discomfort reducing quality of life of many women. Risk factors for POP include increased number of vaginal deliveries of children and advanced age (1). For many years the gold standard for treatment was pelvic mesh, but due to a high rate of complications, FDA warnings, and unavailability of mesh, new treatments are now needed.
Clinical studies have demonstrated changes in the extracellular matrix of connective tissues of the pelvic floor in women with POP, suggesting their involvement in the POP pathophysiology (2). Lysyl oxidase-like 1 (Loxl-1) is a key cross-linking enzyme in elastin matrix remodeling and repair. The pelvic floor undergoes elastin matrix remodeling during pregnancy. In addition, there are changes in Loxl-1 expression in women with POP and menopause. A mouse knockout (KO) model of Loxl-1 has been shown to prolapse after pup delivery in a reliable manner that reproduces risk factors seen in women. 
One option for treatment of POP are nanoparticles (NPs), to deliver elastogenic therapeutic agents to the pelvic floor with or without mesh implantation. Biodegradable polylactic-co-glycolic acid (PLGA) NPs  are frequently used for targeted and controlled drug release (3). Surface functionalization of these NPs with cationic amphiphiles, e.g., didodecyldimethylammonium bromide (DMAB) has been shown to enhance their binding to disrupted elastic fibers by hydrophobic interactions and upregulate activity of the elastin cross-linking enzyme, lysyl oxidase (3). The aim of this study was to assess biodistribution of these NPs when implanted with mesh in the LOXL1 KO mouse model and long-term effects on elastic matrix in the vaginal wall. We hypothesized that NPs administered at the time of mesh implantation would remain local for at least 6 weeks and would improve elastic matrix homeostasis in the vaginal wall.

PLGA (acid 50:50; Durect Corporation) NPs were formulated by double emulsion solvent evaporation and conjugated with an infrared probe (Vivotag 800™, Perkin Elmer) for detection with an IVIS Spectrum rodent in vivo imaging system. Briefly, PLGA and Vivotag 800™ were dissolved in chloroform, sonicated (30 s, 4C) on ice to form a water-in-oil emulsion. This was emulsified with aqueous DMAB (0.5% w/v) to form a double water-in-oil-in-water emulsion and stirred for 16 h at room temperature, then desiccated for 1 h under vacuum to remove any residual chloroform. NPs were recovered by ultracentrifugation and washed twice with nanopure water  to remove residual DMAB. Mean hydrodynamic diameter and zeta potential of the NPs were determined using a dynamic light scattering technique (Malvern, Zetasizer NanoZS). NPs were then encapsulated with Vivotag 800™ for detection of fluorescence in the IVIS Spectrum system.

Twenty-four age matched prolapsed female Loxl-1 KO mice were divided into two groups: polypropylene mesh with saline treatment (S) and polypropylene mesh with fluorescent NP treatment (NP). For mesh implantation, mice were anesthetized with isoflurane (2%v/v) after the rectovaginal space was shaved and disinfected. A 1-cm incision was made between the vaginal and rectal openings. A space was created between the vagina and rectum and a 4 x 8 mm piece of polypropylene mesh was implanted. Fluorescent NPs (100 µl; 200mg NPs/ml) were then infused into this space and the incision closed with liquid suture (Vetbond, 3M). Mice were administered buprenorphine twice daily for 48h for pain management.
Mice were imaged in vivo using the transillumination mode on an IVIS Spectrum imager at 1h, and 1, 2, 4, 7, 21, and 42 days after mesh implantation and NP treatment. One S group mouse was included in each in vivo imaging session. Seven or 42 days after mesh implantation, the animals were imaged and euthanized, and the heart, lungs, liver, spleen, kidneys, and rectum, vaginal, and urethral (RVU) complex were collected. 
The organs were imaged ex vivo using the IVIS Spectrum. Organs from one S group animal included in each ex vivo imaging session. Imaging settings were identical to in vivo imaging except for the closer camera position. After imaging, the organs were fixed in formalin, embedded, and sectioned to localize fluorescent NPs using an Odyssey imager and to perform histology analysis following staining with H&E and modified Hart’s stain (for elastic matrix). IVIS imaging data from S animals were compared to NP animals using a rank sum test to determine significance (p<0.05). NP distribution and histology were assessed qualitatively. As a comparison for elastic matrix, RVU complexes from control age-matched mice with POP were analyzed.

PLGA NPs formulated via double-emulsion method exhibited a mean hydrodynamic diameter of 334±14 nm and a zeta potential of +30.2±1.6 mV.  In vivo imaging showed Vivotag 800™-associated fluorescence signal (radiant efficiency) only in the treated area in the NP-group mice. In the S-group mice, no signal was detected. In the treated areas, radiant efficiency in the NP group during in vivo imaging was significantly higher than the S group 1 hour (p < 0.001), 1 day (p < 0.001), 2 days (p = 0.007), 4 days (p = 0.003), and 7 days (p < 0.001), but not at 21 or 42 days after mesh implantation and infusion of NPs. 
The RVU complex exhibited significantly higher radiant efficiency than the S group upon ex vivo imaging of the organs at both 7 days (p = 0.016) and 42 days (p = 0.035) after implantation, but not in any of the other organs (Fig. 1). Odyssey images confirmed higher NP fluorescence around the mesh in NP treated animals versus the S group (Fig. 2). Hart’s staining showed clumps of disorganized elastin in the S group versus control animals, while the NP group did not show disorganized elastin (Fig. 2).

The results of in vivo imaging indicate lack of migration of the NPs away from the pelvic region demonstrating that a significant number of injected NPs are retained in the injection area and can be detected up to 7 days after injection. This is supported by ex vivo imaging of the RVU complex. The ex vivo data and Odyssey data of the RVU complex at 42 days demonstrates that NPs are still present at the injection site 6 weeks later, corroborating the elastin-binding properties of our NPs. Harts staining indicates improved elastin homeostasis compared to the S group, upon NP treatment.

This study demonstrates that NPs delivered to the pelvic floor will reside in the pelvic floor and not migrate to other areas, indicating that an NP-based therapy can be targeted to the pelvic floor. The presence of NPs 42 days after treatment indicates that NP drug delivery can have a long-lasting release at the pelvic floor to re-establish elastin morphology. This study shows the potential of a NP treatment for the treatment of POP. Future studies can be designed to investigate the use of NP impregnated with different therapeutic options to improve POP and the long term effects of NPs on POP mitigation.

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