Trans-Resveratrol-Loaded Nanostructured Lipid Carrier Formulations for Pulmonary Drug Delivery Using Medical Nebulizers
Abstract
Aerosolization is a non-invasive approach of delivering drugs for both localized and systemic effects, specifically pulmonary targeting. The aim of this study was to deliver trans-resveratrol (TR) as an anti-cancer drug entrapped in a new generation versatile carriers nanostructured lipid carrier (NLC) to protect degradation and improve bioavailability via medical nebulizers. Twelve TR-NLC (i.e., F1-F12) formulations were prepared using different combinations and ratios of formulation ingredients via hot high-pressure homogenization. Upon analysis, formulations F1 and F2 demonstrated a particle size of <185 nm, a polydispersity index (PDI) <0.25, Zeta potential values of ∼30 mV and an entrapment efficiency >94%. The aerosolization performance of the F1 and F2 formulations was performed via a next generation impactor (NGI), using medical nebulizers. The air jet nebulizer demonstrated lower drug deposition in the earlier stages (1-2) and significantly higher deposition in the latter stages 3-5 (for both formulations), targeting middle to lower lung deposition. Moreover, the air jet nebulizer exhibited significantly higher emitted dose (ED) (87.44 ± 3.36%), fine particle dose (FPD) (1652.52 ± 9.68 µg) fine particle fraction (FPF) (36.25 ± 4.26%), and respirable fraction (RF) (93.41 ± 4.03%) when the F1 formulation was used as compared to the F2 formulation. Thus, the TR-NLC F1 formulation and air jet nebulizer were identified as the best combination for the delivery and targeting peripheral lungs.
Introduction
The pulmonary system remains a sought route of drug delivery. This is attributed to the large surface area (100 m2) offered by the lungs, protection from 1st pass metabolism and non-invasive nature of administration. At around one in six fatalities, cancer is one of the main causes of death globally. Lung cancer has one of the worst survival rates when compared to other cancer types, accounting for about 1.8 million deaths per year.5 Based on the histology, lung cancer is divided into small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), mesothelioma, sarcoma, and carcinoid. NSCLC makes up more than 85% of all instances of lung cancer and is the most prevalent kind. The bronchi are usually where SCLC starts, but NSCLC can start in any of the many kinds of lung-lining epithelial cells. Surgery, chemotherapy, radiation therapy, and/or immunotherapy are all part of the standard treatment for lung cancer.8 Additionally, conventional treatments may harm adjacent healthy tissue, which could have a number of negative impacts on patients. New techniques for directing medications to tumour sites can be created with nanotechnology, improving the medication’s effectiveness and minimizing its negative effects on healthy tissue.
Nanoparticles possessing the ability for deep lung deposition to exert therapeutic effect, thus have achieved noticeable attention in drug delivery and cancer treatment. Nanoparticles are divided into many sub-classes, including polymeric nanoparticles, micelles, liposomes/proliposomes, ethosomes, transfersomes/protransfersomes, hybrid nanoparticles, solid lipid nanoparticles, and NLCs. These formulations can deliver both lipophilic and lipophobic drugs, demonstrating very low toxicity in comparison to conventional treatments (e.g., chemotherapy and radiation). These formulations have successfully prolonged drug action either by enhancing the drug half-life or extending the time of drug release. For targeting drug delivery, pH sensitivity is also important, so that active compounds may be released in a particular pH setting. However, it is important to note that each formulation differs with respect to its composition and method of preparation. Among these drug delivery systems, most of the suspension formulations are not stable due to the oxidation and hydrolysis of their phospholipids and are attributed to the agglomeration and leakage of entrapped drugs, resulting in a shorter shelf-life. However, their dry powder formulations are more stable and can be converted into suspensions upon hydration. SLNs possess higher drug entrapment, but due to their perfect crystalline matrix, they may cause drug leakage. Therefore, amongst these nanoparticles, NLC is a new generation formulation, possessing both solid and liquid lipid in the internal core and surfactant to form the outer layer. Moreover, the presence of both the liquid lipid and solid lipid may form an imperfect internal core, which is advantageous in terms of high drug loading and long-term stability as compared to counterpart nanoparticles.
Resveratrol (a novel anti-cancer agent) is a stilbenoid polyphenol with two phenol rings connected by an ethylene bridge. Two isomeric variants of resveratrol have been identified: cis- and trans-resveratrol. Amongst these two, trans-resveratrol (TR) notably induces cellular responses such as cell cycle arrest, differentiation, apoptosis, and enhances cancer cell anti-proliferation. A combination of NLCs and TR as an anti-cancer drug delivery system may result in better in-vivo absorption and bioavailability when compared to TR alone. It is important to know that there have been many preclinical investigations on resveratrol’s anticancer properties, but translational research and clinical trials have progressed very little. Most of the research has concentrated on its cellular processes, signal transduction pathways, and anticancer effects both in vitro and in vivo. A research study conducted by Boocock et al. demonstrated the oral administration of various doses (0.5, 1, 2.5, and 5 g) in humans, where the highest dose demonstrated a recovery of 2.4 μM in the plasma after 1.5 h. In another dose-dependent study where multiple daily doses of 0.5, 1, 2.5 and 5 g were tested in humans for 29 days, the recovered maximum plasma concentration was 4.25 µM. Moreover, in another study where Zhu et al. administered a dose of 50 mg twice a day for 12 weeks, they found a 2.9 μM mean plasma concentration and demonstrated an effect on cancer biomarkers.
Pulmonary drug delivery of NLC formulations may be achieved through the use of nebulizers. Upon nebulization, the drug is inhaled through a mouthpiece or facemask with normal tidal breathing, depositing formulations into the deep lungs. There are three main types of medical nebulizers available that have been extensively used for the delivery of lipid-based formulations: air jet, vibrating mesh, and ultrasonic nebulizers. Each nebulizer type consists of many sub-types, possessing the same working principle but with minor modifications in terms of their design.
Air jet nebulizers are employed with pressure or compressor systems from which high velocity compressed air is passed through a nebulizer and converts aqueous suspensions or solutions into aerosols. Upon passing the compressed air through the venturi nozzle of the air jet nebulizer, it develops a negative pressure over the formulation, generating inhalable aerosol droplets (which is also called the Bernoulli effect). Moreover, surface tension of formulations and impaction on the baffle may also help droplet formation in the air jet nebulizers, followed by droplets passing through the mouthpiece for inhalation.
Ultrasonic nebulizers have an incorporated piezoelectric crystal, which, upon connecting to a power supply, generates vibration at high frequencies (1-3 MHz) creating energy. This energy either generates a fountain (capillary wave formation) or bubble (cavitational bubble formation) in the stagnant formulation to produce inhalable droplets. Vibrating mesh nebulizers contain a perforated mesh plate, piezoelectric crystal, and a horn transducer. Piezoelectric crystals create vibrations that are transmitted to the connected horn transducer in order to extrude formulations from the perforated mesh plate to produce inhalable droplets.The deposition of aerosol particles/droplets is pertinent to the combined effect of inertial impaction, sedimentation and Brownian diffusion, providing a representative simulation of lung deposition. The in-vitro assessment of the aerodynamic diameter of aerosol particles released from the nebulizers can be analysed via a next generation impactor (NGI).
This work aimed to combine formulation excipients in various ratios to prepare NLC formulations. This was followed by their characterization and nebulization performance using medical nebulizers (air jet and ultrasonic) for drug deposition in an in-vitro lung model. Physicochemical properties (particle size, PDI, Zeta potential and entrapment efficiency) were examined and compared between freshly prepared and stored formulations (25°C for two months). The superior formulations identified based on their physicochemical properties were selected for aerosolization studies, where nebulizers were employed for drug deposition in the NGI stages. Through this process, an ideal formulation and nebulizer combination were identified for maximal ED, FPD, FPF and RF. Lastly, the sustained release profile of the best formulation was also determined in different pH media (mirroring various physiological media) at room temperature.
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Materials
Trans-resveratrol (TR) was purchased from Manchester Organics (Chesire, UK). Tripalmitin (Dynasan 116) was kindly provided by IOI Oleochemicals (Witten, UK). Glycerol monostearate was obtained from Alfa Aesar, UK. Gelucire 50/13, Compritol 888 ATO and glycol monocaprylate type II (Capryol 90) were generously gifted by Gattefose (Birkshire, UK). Soya phosphatidylcholine (Lipoid; S-75) was purchased from Lipoid, Switzerland. Tween 80 was procured from Sigma Aldrich, UK. HPLC grade acetonitrile, ammonium molybdate, formic acid, and tetrahydrofuran were purchased from Fischer Scientific Ltd., UK.
Iftikhar Khan, Maria Sabu, Nozad Hussein, Huner Omer, Chahinez Houacine, Wasiq Khan, Abdelbary Elhissi, Sakib Yousaf, Trans-Resveratrol-Loaded Nanostructured Lipid Carrier Formulations for Pulmonary Drug Delivery Using Medical Nebulizers, Journal of Pharmaceutical Sciences, 2025, 103713, ISSN 0022-3549, https://doi.org/10.1016/j.xphs.2025.103713.
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