PENTOSAN POLYSULFATE-LOADED NANOPARTICLES FOR THE ORAL TREATMENT OF INTERSTITIAL CYSTITIS

INTRODUCTION Interstitial cystitis or bladder pain syndrome is a chronic disease that significantly impairs quality of life. It is characterized by dysuria, urinary urgency and frequency, and pelvic pain due to the degradation of the protective glycosaminoglycan (GAG) lining of apical cell layer in the urothelium, which protects it against harmful stimuli. Pentosan polysulfate sodium salt (PPS), a GAG administered orally at a dose of 300 mg/day, is the only oral treatment approved by World Health Organization (WHO) helping the regeneration of GAG layer [1,2,3]. However, the negative charge and hydrophilic nature of PPS limit significantly its bioavailability and restrict its action to epithelial and endothelial surfaces due to its inability to cross the lipid bilayer [4]. The poor bioavailability is accompanied by significant gastrointestinal side effect (diarrhea, bleeding). To overcome these limitations a formulative strategy is mandatory to unleash its therapeutical potential. On this premise, chitosan-PPS polyelectrolyte nanoparticles (NPs) were prepared to enhance upper intestinal absorption and reduce colonic side effect. DEVELOPMENT AND CHARACTERIZATION OF NPS Formulation and characterization of PPS loaded nanoparticles PPS solution (1 mg/mL in ultrapure water) was added dropwise at a rate of 4 mL/min into a chitosan solution (1 mg/mL in 0.12 % v/v aqueous acetic acid) at 40 °C under high-shear homogenization (16,000 rpm), at a volume ratio of 4:5 (Nanosuspension 1X, NP1X). Then, the formulation, NP1X was concentrated 10-fold by tangential flow filtration (Nanosuspension 10X, NP10X). NPs were characterized for size by Dynamic Light Scattering (DLS), surface charge (Z-potential), osmolarity and pH. Encapsulation efficiency (EE%) of PPS and chitosan was determined by dimethylmethylene blue and reactive red 4 assays, respectively. In vitro evaluation of nanoparticles behavior in simulated gastric fluid (SGF) With the aim of investigating the nanoparticles behavior after oral administration, a preliminary characterization of this formulation was conducted by exposing NP10X to SGF containing pepsin, to assess the ability of the formulation to protect PPS from environmental degradation. One mL of NP10X was added to 10 mL of SGF containing pepsin at 37 °C, under magnetic stirring for six hours. In vitro permeation test on a Caco-2 cells transwell model An in vitro permeation study on Caco-2 cells transwell model was performed to evaluate the ability of the NPs to cross the intestinal barrier with respect to free PPS. Caco-2 cells were grown in the upper compartment until a tight monolayer was formed, as confirmed by transepithelial resistance after 3 weeks of culture (TEER> 900 ohm•cm2). An aqueous solution of PPS (0.45 mg/mL) and NP10X, diluted in ultrapure water to get the same PPS concentration, were completed with mannitol at 50 mg/mL (to adjust osmolarity) and 0.01% v/v of 70 mg/mL of potassium hydrogen phosphate (K2HPO4) solution, to raise the pH and increase the cell tolerability. Therefore, 500 µL of each sample were applied to the apical compartment and 1.5 mL of 0.9% w/v NaCl to the basolateral compartment. The system was incubated 4 h, at 37 °C, 5% CO2. After incubation, samples from apical and basolateral compartments, as well as cell lysates were collected. To quantify PPS in the NPs samples, PPS was separated from chitosan by treatment with 0.01% v/v of 40% w/v NaOH solution, followed by 30 minutes of sonication and centrifugation for 10 minutes at 20,000g. The resulting supernatants were analyzed by SEC-ESI-MS. The method was linear in the concentration range 5-200 ug/mL, with a limit of detection (LOD) of 1.3 ug/mL and a limit of quantitation of (LOQ) of 4.3 ug/mL. CHARACTERIZATION OF NANOSUSPENSION Characterization of NPs by DLS, Z-potential, and EE% DLS revealed an average size of 185.6 ± 98.0 nm and 203.8 ± 109.5 nm for NP1X and NP10X, respectively. Z-potential was 44.1± 5.69 mV for NP1X and 46.7 ± 3.91 mV for NP10X. Encapsulation efficiency (EE%) of PPS in NP1X or in NP10X did not change (Table 1) while after tangential flow filtration free chitosan was reduced. Table 1. Encapsulation efficiency (EE%) of PPS and free chitosan in the nano suspension. Sample EE% PPS Free Chitosan NP1X 99.8 ± 0.2 23.8 ± 2.5 NP10X 99.6 ± 0.7 5.6 ± 0.4 Characterization of NPs behavior under SGF conditions DLS revealed a progressive increase in nanoparticles size and polydispersity index up to 4 hours, with no further increase in the following two hours. The Z-potential decreased to 34.1 ± 3.9 mV in the first 30 minutes, with no further variations. The PPS encapsulation efficiency remained unchanged. Characterization of NPs in in vitro permeation study on Caco-2 cells transwell model The osmolarity of NPs suspension was 2 mOsm/L and the pH 4.5: these parameters were not considered adequate for application on cell culture and were for this reason corrected before in vitro tests. The addition of mannitol brought the osmolarity to 300 mOsm/L while with K2HPO4 the pH values raised to 5.3 and 7.3 for NPs and PPS solution, respectively, following the addition of K2HPO4. A significant increase in NPs size (321.8 ± 252.1 nm) was observed, probably attributable to the increase in ionic strength of the vehicle. Quantitation of PPS Due to a limited sensitivity of the analytical method, it was not possible to recover the whole amount of PPS applied. Anyway, the distribution in the different compartments of PPS in solution or formulated in NPs was different. No PPS was found in cell lysate or basolateral compartment if applied as a solution, while about 16% of PPS was recovered in the cell lysate if NPs were used, suggesting that encapsulation promoted its internalization by the epithelial cells, probably mediated by the interaction of chitosan with cell surface. CONCLUSION A nanosuspension based on polyelectrolyte complexes between chitosan and PPS was developed, with particles having an average size below 300 nm, and an encapsulation efficiency of PPS close to 100% PPS. The positive charge of this formulation, revealed by Z-potential, afforded a strong colloidal stability and suggests the presence of chitosan on the outer layer, that could aid adhesion to cells and PPS uptake to improve its therapeutic action. When NPs suspension was exposed to simulated gastric conditions, an increase in nanoparticle size was observed that deserves further investigation. Despite this, a preliminary permeation study on Caco-2 cells transwell model revealed an increase in cell internalization of PPS only if applied in form of nanosuspension. REFERENCES 1. Nandwana, D.; Zhang, Y.; Feng, N. Contribution of the Microbiome to Interstitial Cystitis/Bladder Pain Syndrome: A Mini Review. European Urology Focus, 10, 893-897 (2024). 2. Kasyan, G.; Kupriyanov, Y.; Karasev, A.; Baibarin, K.; Pushkar, D. Safety and efficacy of pentosan polysulfate in patients with bladder pain syndrome/interstitial cystitis: a multicenter, double-blind, placebo-controlled, randomized study. Central European Journal of Urology, 74, 201-207 (2021). 3. Arikan, M.G; Çakiroğlu, B. Efficacy of Pentosan Polysulfate Treatment in Patients with Interstitial Cystitis/Bladder Pain Syndrome. Bladder, 10, e21200007 (2023). 4. 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