Quercetin Loaded Cationic Solid Lipid Nanoparticles in a Mucoadhesive In Situ Gel – A Novel Intravesical Therapy Tackling Bladder Cancer

The study aim was to develop an intravesical delivery system of quercetin for bladder cancer management in order to improve drug efficacy, attain a controlled release profile and extend the residence time inside the bladder. Either uncoated or chitosan coated quercetin-loaded solid lipid nanoparticles (SLNs) were prepared and evaluated in terms of colloidal, morphological and thermal characteristics. Drug encapsulation efficiency and its release behaviour were assessed. Furthermore, cytotoxicity of SLNs on T-24 cells was evaluated. Ex vivo studies were carried out using bovine bladder mucosa. Spherical SLNs (≈250 nm) ensured good entrapment efficiencies (EE > 97%) and sustained drug release up to 142 h. Cytotoxicity profile revealed concentration-dependent toxicity recording an IC₅₀ in the range of 1.6–8.9 μg/mL quercetin. SLNs were further dispersed in in situ hydrogels comprising poloxamer 407 (20%) with mucoadhesive polymers. In situ gels exhibited acceptable gelation temperatures (around 25 °C) and long erosion time (24–27 h). SLNs loaded gels displayed remarkably enhanced retention on bladder tissues relative to SLNs dispersions. Coated SLNs exhibited better penetration abilities compared to uncoated ones, while coated SLNs dispersed in gel (G₁₀C-St-QCT-SLNs-2) showed the highest penetration up to 350 μm. Hence, G₁₀C-St-QCT-SLNs-2 could be considered as a platform for intravesical quercetin delivery.

Introduction

Bladder cancer (BC) is among the top ten most frequent cancer types worldwide, where about 550,000 new cases are diagnosed with bladder cancer each year. It is considered as one of the most lethal cancer types, particularly in older males. BC is defined as abnormal cells proliferating in the lining of the bladder. It is classified into 5 stages (from 0 to IV) indicating the progression of the disease. In general, BC is divided into two types: non-muscle invasive bladder cancer (NMIBC) and muscle invasive bladder cancer (MIBC). At diagnosis, 70% of the cases would present with NMIBC, while in the other 30% the disease would have progressed to MIBC [1,2]. The standard therapy for NMIBC is the transurethral resection of bladder tumour (TURBT) in addition to intravesical chemotherapy and immunotherapy such as bacillus Calmette–Guérin (BCG), as well as systemic chemotherapy such as Mitomycin C (MMC) to have better control on tumour recurrence and progression [3].

Unfortunately, 70% of patients will experience tumour recurrence, with 25% advancing to MIBC within five years following TURBT [2]. The oral bioavailability of BC therapeutic agents is lowered by the gastric acid and the degradative hepatic enzymes, whereas the poorly vascularized urothelial cells would render systemic therapy less effective [4,5]. To enhance the therapeutic efficacy and achieve high drug levels within the bladder, higher doses of the systemically administered agents are required, which results in increased systemic adverse effects and high toxicity to healthy cells [3].

On the other hand, local administration ensures high concentrations of the therapeutic agents inside the target organs, while minimizing systemic side effects [4]. Because of the position and anatomy of the bladder, it is regarded as a perfect organ for intravesical drug delivery (IDD), which is defined as direct administration of the therapeutic agents into the bladder through a urethral catheter. Also, the presence of the permeability barrier of the bladder, with tightly packed umbrella cells and uroplakin plaques, hinders the systemic absorption of the therapeutic agents [5]. Therefore, maximum exposure of the bladder cancer cells to the chemotherapeutic agents with minimum systemic exposure could be achieved through IDD.

However, IDD has its own limitations as well. The presence of bladder permeability barrier (BPB), which hinders the penetration of the chemotherapeutic agents into the bladder tissues, in addition to frequent drug dilution and wash-out by urine diminish the efficacy of the intravesical therapy and may lead to therapeutic failure [6,7]. Thus, developing an efficient delivery system, that is able to overcome the limitations of IDD and achieve better control over bladder cancer, remains a challenge.

Quercetin (QCT) is a natural polyphenolic flavonoid found in variety of vegetables and fruits such as onions, apples and berries. It is chemically known as 3,3′,4′,5,7-pentahydroxyflavone (C₁₅H₁₀O₇) [8]. Thanks to its good anticancer activity, low toxicity and wide accessibility, QCT was chosen as a model drug for intravesical therapy of bladder cancer. QCT has a wide array of pharmacological applications, including antioxidant [9], anti-inflammatory [10], antidiabetic [11] and anti-cancer properties [12]. Most importantly, QCT hinders the progression of many types of cancers, including cervical, colon, breast, liver, prostate and lung cancer [13]. Various mechanisms are thought to be responsible for the anticancer properties of QCT such as its ability to suppress enzymes involved in carcinogens activation and cellular signalling. Based on its ability to bind to cellular receptors and proteins, QCT exhibits a wide range of anticancer effects [14,15]. Furthermore, the combination of QCT with conventional chemotherapeutic agents displayed synergistic effects, which may further enhance the efficiency of the conventional chemotherapy [16]. Research carried out by Oršolić and colleagues showed that QCT has genotoxic and cytotoxic activity against T-24 cells (human bladder cancer cells). The chemotherapeutic effects of QCT were explained by its ability to expand the DNA damage of human bladder cancer cells, preventing cell propagation and colony formation [17]. However, clinical use of QCT is restricted by its poor water solubility and consequently poor bioavailability [18].

Smart drug carriers for IDD offer several advantages over conventional formulations, such as improved drug solubility and prolonged adhesion of the drug carriers to the urothelial surfaces, in addition to high penetration into malignant tissues. For instance, liposomes and polymeric nanoparticles have been widely used for IDD [19,20,21,22,23,24].

Solid lipid nanoparticles (SLNs) are considered as one of the most appropriate carriers for the delivery of lipophilic drugs as they ensure high drug encapsulation and controlled release properties, together with low toxicity. They have been widely employed for the delivery of several chemotherapeutic agents, such as docetaxel [25], etoposide [26] and doxorubicin [27], due to the high solubility of such lipophilic agents in the solid lipids [28].

Compared to liposomes, SLNs display improved entrapment efficiency for various lipophilic drugs and ensure higher stability of the loaded drugs due to the rigid core lipid matrix [3]. Furthermore, SLNs are considered safer drug carriers relative to polymeric nanoparticles as they can be synthesized using various techniques not involving the use of organic solvents [29]. Despite the abovementioned advantages of SLNs, they have scarcely been used for intravesical applications.

Improved response to intravesical chemotherapy was attained in patients who received positively charged ions with MMC compared to patients treated with MMC alone, which may be explained by the enhanced penetration of MMC into the bladder tissues in the presence of positively charged ions [30]. In spite of the promising outcomes, the implementation of electromotive drug administration into general practice is difficult and nearly restricted to the European academic centres [23]. Meanwhile, positively charged nanocarriers can improve drug delivery into urothelial layers without the need for such complex administration devices [23,24,31]. Being mucoadhesive, these cationic nanocarriers are able to efficiently attach to the mucosal surfaces, allowing prolonged contact of the formulations with the diseased cells and, hence, improved cellular uptake [5]. Furthermore, the use of cationic mucoadhesive polymers is believed to encourage paracellular transport through rearrangement of the tight junctions between cells and, hence, improves therapeutic agent penetration into the bladder wall [32].

Usually drug solutions are washed out by urination within 2 h following intravesical administration [33]. Administration of frequent doses of the drugs requires repeated catheterization which causes patient inconvenience and puts them at increased risk of infection. Therefore, development of an IDD system that can withstand wash-out by urine and remain inside the bladder for a prolonged period of time, allowing for continuous drug release, is essential. Various approaches have been developed to achieve this goal, among them the use of mucoadhesive thermosensitive in situ gels for IDD [34].

Thermosensitive polymers, including poloxamers, exist as liquid formulations during storage and administration and transform into gels inside the body. Previous literature reported the use of poloxamers for preparation of intravesical in situ gels with extended drug release properties [35]. Men K et al. introduced a composite system, wherein cationic nanocarriers were incorporated into an in situ poloxamer 407 (P407) based gel. The system offered extended retention of the formulations inside the bladder cavity as well as higher permeation into the bladder wall [31]. In another study, it was found that the use of P407 hydrogel together with mucoadhesive polymers led to remarkable effect in sustaining release of drug. However, the formulation exhibited very low gelation temperature which may cause a problem during handling and administration [36]. By optimizing gel composition, an easily administered in situ gel with rapid and efficacious adhesion to bladder tissues could be obtained.

Our aim was to develop a novel intravesical system for QCT delivery to enhance QCT performance in bladder cancer management and address the drawbacks of systemic drug delivery by direct local delivery into the bladder. Cationic QCT-loaded SLNs were synthesized and characterized in terms of their colloidal and thermal properties, encapsulation efficiency, and in vitro release behaviour. In addition, their in vitro cytotoxicity against the bladder cancer cell line was studied. Cationic SLNs were further incorporated into mucoadhesive in situ gels, as appropriate IDD systems, to extend the residence time and enhance treatment efficiency. The composite system of cationic nanoparticle and thermo-sensitive hydrogels was evaluated ex vivo in terms of its retention on bladder mucosa, penetration into the bladder wall, and safety on bladder tissues.

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Materials

Quercetin was obtained from M/s Sisco Research Laboratories (Maharashtra, India). Precirol was a sample gift from Gattefossé (Saint Priest, France). Stearic acid was kindly provided by Pharco Pharmaceuticals Company (Alexandria, Egypt). Poloxamer188 (P188, Pluronic-F68TM) was obtained from BASF (Ludwigshafen, Germany). Tween 80 was purchased from Chemtech (Alexandria, Egypt). Chitosan (Cs) for coating (Mw 60–120 kDa, Degree of deacetylation 85%, viscosity 27 CPS) was purchased from Sigma-Aldrich (Steinheim, Germany). Poloxamer 407 (Pluronic F127) was a sample gift from Borg Pharmaceutical Companies (Alexandria, Egypt). Carbopol 974P (Cb) was a sample gift from Lubrizol (Oevel, Belgium). Chitosan for gel preparation (Mw 161.116, degree of deacetylation 93%) was obtained from Oxford Lab Chem (Mumbai, India). Hydroxy propyl methyl cellulose (HPMC) was purchased from Cairo Pharmaceuticals Co., (Cairo, Egypt). T-24: Urinary Bladder cancer cell line (transitional cell carcinoma) was obtained from Nawah Scientific Inc., (Cairo, Egypt). Dulbecco’s Modified Eagle Medium (DMEM) high glucose, Roswell Park Memorial Institute (RPMI) 1640 high glucose, Penicillin/streptomycin, Trypsin and EDTA were purchased from Lonza GmbH (Köln, Germany). Fetal bovine serum (FBS) was purchased from Gibco (Grand Island, NY, USA). Coumarin-6 dye (cou) and Sulforhodamine B (SRB) were purchased from Sigma-Aldrich (Steinheim, Germany). Trichloroacetic acid (TCA) was purchased from Merck (Kenilworth, NJ, USA). Tris (hydroxymethyl) aminomethane (TRIS) was obtained from Chem-Lab (Zedelgem, Belgium). All other used chemicals were of analytical grade.

Shawky, S.; Makled, S.; Awaad, A.; Boraie, N. Quercetin Loaded Cationic Solid Lipid Nanoparticles in a Mucoadhesive In Situ Gel—A Novel Intravesical Therapy Tackling Bladder Cancer. Pharmaceutics 2022, 14, 2527. https://doi.org/10.3390/pharmaceutics14112527

excipients formulation