Abstract

Nanocarriers, utilized as transport mediums, encompass a variety of nanomaterials including micelles, polymers, carbon-based materials, and liposomes, with diameters ranging from 1 to 1000 nm. Their diminutive size enables drug delivery to otherwise inaccessible bodily sites, though producing substantial doses poses challenges due to their minute scale. Within medicine, nanosized particles exhibit selective applications. Melatonin, crucial for regulating sleep, is naturally secreted by the pineal gland and synthesized by most cells in a non-circadian manner. When combined with nanostructured formulations, melatonin exhibits heightened efficacy and safety, proving effective in treating diverse diseases and pathological conditions. The extraction of melatonin from raw milk involves quantitative analysis via precolumn derivatization. Through a series of mild reactions including diazotization ring closure, hydrolysis decarboxylation, nucleophilic addition, and acetylation, melatonin is obtained with high output. Nanocarriers, prepared through various techniques such as solvent evaporation, spray drying, nanoprecipitation, ionic gelation, and salting out, incorporate melatonin to form Melatonin-Loaded Nanocarriers. Storage is recommended in a labeled, airtight container in a cool, well-ventilated room, shielded from sunlight, with lyophilization serving as an alternative long-term storage method. Both in vitro and in vivo evaluations are conducted. Melatonin, renowned for its role in regulating circadian rhythms and sleep-wake cycles, possesses diverse pharmacological properties including antioxidant, anti-inflammatory, and neuroprotective effects. However, its clinical utility is hampered by poor aqueous solubility, low bioavailability, and rapid metabolism. Nanotechnology offers promising remedies by employing nanocarriers such as polymeric nanoparticles, liposomes, and micelles for melatonin encapsulation and delivery. This review delves into the development and characterization of melatonin-loaded nanocarriers, discussing various nanocarrier types, preparation methods, physicochemical properties, and in vitro/in vivo evaluations. It explores the therapeutic applications of melatonin-loaded nanocarriers across different disease models, suggesting their potential to advance clinical interventions and enhance patient outcomes. Ultimately, this review sheds light on the current landscape, challenges, and future prospects of leveraging nanotechnology for melatonin delivery, holding implications for personalized medicine and precision therapeutics.

Keywords

Introduction

Melatonin, a hormone predominantly known for its role in regulating circadian rhythms and sleep-wake cycles, has garnered significant attention in recent years due to its diverse pharmacological properties. Its antioxidant, anti-inflammatory, and neuroprotective effects have sparked interest in utilizing melatonin as a therapeutic agent for various diseases and pathological conditions beyond sleep disorders. However, the clinical translation of melatonin faces challenges, primarily related to its poor aqueous solubility, low bioavailability, and rapid metabolism.

To overcome these limitations and enhance the therapeutic potential of melatonin, nanotechnology offers promising solutions. Nanocarriers, particularly polymeric nanoparticles, liposomes, micelles, and other nanostructures, serve as efficient delivery systems to encapsulate, protect, and deliver melatonin to targeted sites within the body. By encapsulating melatonin within nanocarriers, its stability can be improved, its release can be controlled, and its bioavailability can be enhanced, thereby maximizing its therapeutic efficacy while minimizing potential side effects.

In this review, we focus on the development and characterization of melatonin-loaded nanocarriers as innovative drug delivery systems. We discuss the various types of nanocarriers utilized for melatonin delivery, their preparation methods, physicochemical properties, and in vitro/in vivo evaluations. Furthermore, we explore the therapeutic applications of melatonin-loaded nanocarriers across different disease models and pathological conditions, highlighting their potential in advancing clinical interventions and improving patient outcomes. This comprehensive overview aims to provide insights into the current status, challenges, and future directions of utilizing nanotechnology for melatonin delivery, with implications for personalized medicine and precision therapeutics.

Types of nanocarriers:

The nanocarriers are of different types, they are;

• Solid Lipid Nanocarriers.
• Carbon Nanomaterials.
• Magnetic Nanoparticles.
• Inorganic Nanoparticles.
• Liposome.
• Dendrimer Nanocarriers.
• Polymeric Micelles.

Sources of melatonin:

According to available research, these six foods are good sources of melatonin:

• Milk
• Goji Berries
• Tart Cherries
• Eggs
• Nuts
• Fish

Benefits:

1. Provide better sleep. 
2. Promote eye health.
3. Age-related macular degeneration.
4. Melatonin protects the mitochondria in cells and inhibits cell death.

Side effects:

• Feeling sleepy in the daytime.
• Headache.
• Stomach ache.
• Nausea.
• Dizziness.
• Feeling irritation.
• Dry mouth.

Therapeutic application:

• Melatonin incorporated into lipid based nanosystems (liposomes) for transdermal melatonin delivery and improved drug skin permeation for cancer therapy.
• Melatonin incorporated into non-ionic surfactant based vesicles (niosomes) for anti-inflammatory activity, fungicidal effect with an anti-candidiasis reaction, better permeation and antioxidant activity in skin.
• Melatonin loaded silica based nanoparticles used in the treatment of colorectal cancer.
• Sustained release of melatonin from GelMA liposome reduce osteoblast apoptosis and postmenopausal osteoporosis.
• Melatonin loaded chitosan nanoparticles can enhance the intestinal mucosal barrier, alter the composition of intestinal bacteria with anti-inflammatory properties.
• Nanofibres and nanocapsules as biomaterials for melatonin controlled release for treatment of insomnia
• Melatonin, a hormone with multifaceted pharmacological properties, holds immense therapeutic potential for a wide range of diseases and disorders. However, its clinical efficacy is often hindered by challenges such as poor aqueous solubility, low bioavailability, and rapid degradation. To address these limitations and harness the full therapeutic benefits of melatonin, nanotechnology has emerged as a promising approach.

Nanocarriers, including polymeric nanoparticles, liposomes, micelles, and other nanostructures, offer versatile platforms for encapsulating and delivering melatonin with enhanced efficacy and targeted delivery. The development of melatonin-loaded nanocarriers involves meticulous design considerations to optimize drug loading capacity, release kinetics, stability, and biocompatibility. The process begins with the selection of suitable materials for nanocarrier fabrication, considering factors such as biodegradability, biocompatibility, and controlled release properties. Various methods, including solvent evaporation, nanoprecipitation, emulsion techniques, and self-assembly, are employed for the preparation of melatonin-loaded nanocarriers, each offering unique advantages in terms of scalability, reproducibility, and control over particle size and morphology..

Characterization of melatonin-loaded nanocarriers

Characterization of melatonin-loaded nanocarriers is essential to assess their physicochemical properties and performance characteristics. Techniques such as dynamic light scattering, transmission electron microscopy, atomic force microscopy, and Fourier-transform infrared spectroscopy are utilized to evaluate particle size distribution, surface morphology, drug encapsulation efficiency, and stability under physiological conditions.

Preparation of melatonin:

The laboratory preparation of melatonin involves the following steps:

• Synthesis of the First Compound: Phthalimide, 1,3-dichloropropane, ethyl acetoacetate, and sodium iodide react in a solvent under alkaline conditions to obtain ethyl-2-acetyl-5-(1,3-dioxisoindolin-2-yl)pentanoate.
• Formation of the Second Compound: A ring-closing reaction is carried out on the first compound using p-methoxyphenyl diazonium salt in the presence of alkali and a solvent, resulting in ethyl 3-(2-(1,3-dioxisoindolin-2-yl)ethyl)-5-methoxy-1H-indole-2-carboxylate.
• Hydrolysis and Decarboxylation: The second compound undergoes hydrolysis under alkaline conditions, followed by decarboxylation under acidic conditions, to yield 2-(5-methoxy-1H-indol-3-yl)ethan-1-amine.
• Acetylation to Form Melatonin: The third compound is subjected to an acetylation reaction to produce melatonin.

These steps involve nucleophilic addition, diazotization ring closure, hydrolysis, decarboxylation, and acetylation reactions. The reaction conditions are relatively mild, leading to a high yield of melatonin.

Preparation of nanocarriers:

1. Solvent evaporation technique:

The solvent evaporation technique is employed for the preparation of polymeric nanoparticles. First, the polymer is dissolved in an organic solvent along with the drug. This resulting solution is then added to an aqueous phase containing a surfactant or emulsifying agent to form an emulsion. After forming a stable emulsion, the organic solvent is evaporated or removed by either increasing the temperature under reduced pressure or by continuous stirring. The resulting nanosuspension is then freeze-dried using 5% mannitol as a cryoprotectant to obtain a fine powder of nanoparticles.

2. Spray drying technique:

One well-established method for producing a drug powder from a liquid phase involves dissolving the drug and polymer in ultrapure water with a surfactant (Tween 80) and filtering the solution through a 0.45-micrometer syringe filter. The filtered solution is then spray-dried at 30-55°C, during which millions of precisely sized droplets are formed by the vibration of a mesh. A novel electrostatic particle collector is used to collect the nanocarriers.

3. Nano precipitation technique:

The drug and polymer are dissolved in a water-miscible organic solvent and then added to the aqueous phase containing a stabilizer under stirring. The decrease in interfacial tension between the aqueous and organic phases results in the rapid diffusion of the organic solvent into the aqueous phase. The resulting nanosuspension is freeze-dried using 5% mannitol as a cryoprotectant to obtain a fine powder of nanocarriers.

4. Ionic gelation technique

The drug and polymer are dissolved in a weak acidic medium or water. The resultant solution is then added dropwise to a solution containing counter ions and a stabilizer under constant stirring. Spherical-shaped particles form due to the complexation of oppositely charged species, resulting in gelation and precipitation. The resulting nanosuspension is freeze-dried using 5% mannitol as a cryoprotectant to obtain a fine powder of nanocarriers.

5. Salting out technique:

This method is primarily used for preparing polymeric nanoparticles. The polymer and drug are dissolved in an organic solvent, and the resulting solution is then added to an aqueous phase containing a surfactant or emulsifying agent to form a stable emulsion. The organic solvent is then evaporated by increasing the temperature under reduced pressure. The resulting nanosuspension is freeze-dried using 5% mannitol as a cryoprotectant, yielding a fine powder of nanoparticles.

Preparation of melatonin loaded nanocarriers

Quantitative analysis using a precolumn derivatization method is employed for extracting melatonin from raw milk. Raw milk from 43-month-old Holstein cows is incubated with papain at 45°C for 30 minutes. The enzyme reaction is halted by adding trichloroacetic acid, and the reaction mixture is centrifuged. Melatonin in the supernatant is extracted with chloroform, then the chloroform layer is washed with water and evaporated to dryness. For melatonin derivatization, 150 ?L of 2 mM Na2CO3, 100 ?L of 100 mM H2O2, 50 ?L of 1 mM CuSO4, and 700 ?L of water are added to the residue, and the mixture is heated in water at 94-96°C for 6 minutes. The melatonin derivative is then extracted with ethyl acetate, evaporated to dryness, dissolved in aqueous 15% (v/v) CH3CN, and subjected to HPLC analysis.

Storage:

The products should be stored in an airtight, well-closed and labelled container in a cool, well-ventilated room, away from sunlight. Lyophilisation is a method for long-term storage of nanoparticles.

EVALUATION

1. In-vitro evaluations.

1. Yield of nano particles:

The yield of nanoparticles was determined by comparing the whole weight of nanoparticles formed against the combined weight of the co polymer and drug.

Percentage yield = Amount of nanoparticles / Amount of drug + polymer

2. Drug content / surface entrapment / drug entrapment:

Then amount of drug present in supernatant subtracted from the total amount used in the preparation of nanoparticle (W). (W-w) is the amount of drug entrapped. Percentage of drug entrapment calculated by

 Percentage drug entrapment = W-w/W X100

3. Particle size and  zeta potential:

Value of particle size and zeta potential of prepared nanoparticles is determined by using Malvern Zetasizer.

4. Surface morphology:

Surface morphology study carried out by Scanning Electron Microscopy (SEM) of prepared nanoparticles.

5. Polydispersity index :

Polydispersity index of prepared nanoparticles was carried out by using Malvern Zetasizer.

2. In-vivo evaluation 

1. Mechanism of cellular uptake:
1.          The mechanism involved behind the nanoparticles entry into the cells is of equal importance as the successive pathways to be followed inside the cells depends on it. Physicochemical characteristics of nanoparticles decide the mechanism of translocation such as phagocytosis, macropinocytosis and endocytosis.
2. Bioactivity of nanocarriers:
2.            Highest care should be taken to fully retain the strength and effectiveness of a drug to be encapsulated into the nanocarriers. Luciferase activity in terms of bioluminescence strength is determined which is proportional to ATP generated by live cell assays due to the conversion of luciferase into luciferin.
3. Animal models of tumour:
3.           After nanocarriers reveal preliminary effectiveness in vitro, these carriers are subjected to further evaluation in terms of their toxicity profile and response in biological species. Animal studies and selection of animal model is highly specific and selected on the basis of drug of investigation as well as the proposed route of administration. Cancer cells are injected (typically subcutaneously) to the immune-deficient mice which allow to grow visible tumours. In this regard, Wang has reviewed the various strategies based on nanoparticles for vaccination against cancer.
4. Oral toxicity studies in rat:
4.              Acute oral toxicity studies in male and female rat document that no death or treatment related signs in higher doses. The micro sponge’s solutions were individually administered to male and female Sprague dawley rat at a concentration 250,500 and 1000 ml/kg body weight for 28 days.  All animals survived the duration of study with no significant changes in clinical science.
5. Other evaluation studies:
5.            Oral toxicity studies in rabbit, mutagenicity in bacteria, allergenicity in guinea pig, compatibility studies by Thin Layer Chromatography (TLC).

CONCLUSION:

Incorporating melatonin into nanocarriers enhances its efficacy and safety as a targeted drug delivery system. Melatonin, naturally produced by the body and synthesized in the laboratory, is obtained through nucleophilic addition reaction, diazotization ring closure reaction, hydrolysis decarboxylation, and acetylation, yielding high yields under mild conditions. Various techniques such as solvent evaporation, spray drying, nano precipitation, ionic gelation, and salting out are utilized for nanocarrier preparation.

The preparation of melatonin-loaded nanocarriers involves mixing absolute ethyl alcohol containing melatonin with an aqueous solution containing gelatin at 50°C. This solution is slowly added into a dichloromethane solution, followed by stirring for 1 hour and ultrasonic vibration for another hour to obtain emulsion 1. Emulsion 1 is then slowly added to an aqueous solution to obtain emulsion 2, and this process is repeated to obtain emulsion 3. Finally, centrifugation at 30°C produces melatonin-loaded nanocarriers.

These nanocarriers offer a promising solution to overcome challenges associated with melatonin's clinical translation, including poor aqueous solubility, low bioavailability, and rapid metabolism. By encapsulating melatonin, nanocarriers enhance its stability, control its release, and maximize therapeutic efficacy. Extensive investigation into the development and characterization of melatonin-loaded nanocarriers, along with in vitro and in vivo evaluations, demonstrates their therapeutic potential across various disease models and conditions. Further research is required to optimize nanocarrier formulations, improve targeted delivery, and enhance clinical outcomes, ultimately advancing precision medicine and patient care. Evaluation is conducted through in vivo and in vitro studies.

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