**1. Introduction**

Rutin, also known as vitamin P, is a polyphenolic flavonoid found in plants such as buckwheat, green tea, citrus fruits (e.g., orange, grapefruit, lemon, lime), and apples [1,2]. Chemically, rutin is known as (2-(3,4-dihydroxyphenyl)-4,5-dihydroxy-3-[3,4,5-trihydroxy-6-[(3,4,5-trihydroxy-6-methyl-oxan-2-yl)oxymethyl]oxan-2-yl]oxy-chromen-7-one) or quercetin-3-rutinoside. Rutin has several attractive features as a drug such as its natural source, safety, cost-effectiveness, and wide spectrum of pharmacological actions [3–5]. Moreover, several reports have shown rutin to have antioxidant, antidiabetic, anticancer, anti-inflammatory, antibacterial, anti-arthritic, and neuroprotection activities [5–8]. In addition, rutin was also shown to have antihypertensive, cardioprotective, antispasmodic, anti-thrombotic, and anti-hyperlipidemia actions [9–12]. Despite these interesting pharmacological properties, rutin suffers from some drawbacks such as poor aqueous solubility and stability which result in poor oral bioavailability [13,14]. This poses some challenges

**Citation:** Hassan, A.S.; Soliman, G.M. Rutin Nanocrystals with Enhanced Anti-Inflammatory Activity: Preparation and Ex Vivo/In Vivo Evaluation in an Inflammatory Rat Model. *Pharmaceutics* **2022**, *14*, 2727. https://doi.org/10.3390/ pharmaceutics14122727

Academic Editor: Ana Isabel Fernandes

Received: 3 November 2022 Accepted: 1 December 2022 Published: 6 December 2022

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for the formulation of oral dosage forms of rutin whether for its application as a nutritional supplement or a therapeutic agent [2]. To overcome these shortcomings and improve rutin's therapeutic efficacy, several formulation strategies and drug delivery systems have been exploited. For instance, dried rutin nanocrystals have been prepared and incorporated into tablets [2]. The nanocrystal tablets achieved complete rutin dissolution in 30 min compared to only 71% and 55% dissolution from the microcrystal tablets and the commercialized tablets, respectively. Rutin-loaded silver nanoparticles were fabricated and their antithrombotic activity was evaluated [12]. Rutin-silver nanoparticles had prolonged activated partial thromboplastin time and prothrombin time. Quinoa and maize starch nanoparticles were also used to encapsulate rutin and improve its bioavailability [15]. In a simulated in vitro digestion test, the nanoparticles were able to increase rutin's bioavailability and improve its antioxidant activity. Rutin was also loaded into liquid crystalline nanoparticles (LCNs) and its anticancer activity against non-small cell lung cancer was tested in an A549 human lung epithelial carcinoma cell line [16]. Rutin-LCNs showed promising anti-proliferative and anti-migratory activities. In addition, they induced apoptosis in the A549 cells and inhibited colony formation.

Nanocrystals (NCs) are sub-micron colloidal dispersions composed of 100% drug material. They were first introduced in the 1990s as a means of improving dissolution rate, aqueous solubility, and thus the bioavailability of poorly water-soluble drugs [17]. NCs have a particle size in the nanometer range and are typically produced by the milling of bulk drug material [18]. They are stabilized by surfactants, polymers, or both [19]. The enhanced dissolution rate and solubility are believed to be due to the decrease in particle size, which in turn increases the surface area available for dissolution according to the Noyes–Whitney equation [20]. The enormous increase in surface area and saturation solubility result in improved drug oral bioavailability and permeability through biological membranes making them an attractive drug delivery approach for poorly water soluble drugs [18,19,21]. These interesting features culminated in the approval of Rapamune® (sirolimus, Pfizer), Megace ES® (megestrol acetate, Elan/Par Pharm), Emend® (aprepitant, MSD), Tricor® (fenofibrate, AbbVie), and Invega Sustenna® (paliperidone palmitate, Elan/Johnson and Johnson) for oral administration [18,22]. Nanocrystals have also found interesting applications for dermal and transdermal drug delivery since they were reported to increase drug penetration through the skin [18,23]. This is usually achieved by virtue of nanocrystals' ability to create a higher concentration gradient across the skin due to increased drug saturation solubility leading to increased passive diffusion [24]. They also increase drug delivery through the hair follicles due to particle size reduction, as well as adhesion to the skin [23]. However, no nanocrystal-based topical products have been approved for clinical use so far thus showing that further research is still needed in this area.

The literature shows a very limited number of reports on using nanocrystals to improve the dermal and transdermal delivery of rutin. Thus, Pelikh et al. prepared rutin and hesperetin nanocrystals with a size in the range of 160–700 nm and studied the effect of particle size and vehicle type on the skin penetration of the drugs [18]. The results showed that oleogels and creams were better than hydrogels in improving rutin nanocrystals skin penetration. In addition, smaller particles achieved better skin penetration. Furthermore, Pyo et al. showed that particle size also influenced the antioxidant efficacy of rutin nanocrystals where the nanocrystals with a mean dimeter of 300 nm had the highest antioxidant capacity compared to the drug microparticles (33 μm) and commercial cosmetic drug products [25]. In another study, rutin nanocrystals were suspended in Carbopol gel and tested as an anti-photoaging agent [26]. The nanocrystal gel achieved 3-fold higher drug permeability through mice skin compared with the coarse drug gel and prevented UV irradiation-induced photoaging and tissue damage. For cosmetic applications, rutin nanocrystal formulation is commercially available under the tradename Juvedical® (Juvena) [27].

To date no reports, however, have studied the effects of nanocrystal formulation on rutin's anti-inflammatory properties following topical application. Therefore, the aim of this study was to prepare rutin nanocrystals using different stabilizers such as Pluronic F-17, Tween 80, hydroxypropyl beta-cyclodextrin, and PEG 6000 and test their ability to enhance rutin's permeability through mice skin. In addition, the in vivo anti-inflammatory properties of the selected rutin nanocrystal formulations were examined using a carrageenaninduced rat paw edema model.

### **2. Materials and Methods**

Rutin (RT) (purity > 95%) was purchased from Oxford Lab Fine Chem LLP, Vasai, India. Hydroxypropyl beta-cyclodextrin (HP-β-CD), and Pluronic F-127 was obtained from Sigma-Aldrich (St. Louis, MO, USA). Potassium dihydrogen phosphate, PEG 200, PEG 6000, Tween 80, hydroxypropyl methyl cellulose (HPMC), disodium hydrogen phosphate, and sodium hydroxide were obtained from United Company for Chem. and Med. Prep., Cairo, Egypt.
