INTRODUCTION:

Researchers that use nanotechnology to build new formulations always focus on improving bioavailabilityand manufacturing method, with a particular emphasis on changing the drug delivery mechanism. Fundamentally, a liposomeis a vesicle that contains at least one phospholipid and cholesterol lipid bilayer, which is used to carry out the desired delivery of nutrients or medications.

Out of all of these lipid-based nanocarriers, cochleates liposomes with a specific location for drug delvery that results in fewer side effects rose to prominence as a novel nanocarrier multi-layered system for hydrophilic and hydrophobic drugs with better animproved stability, efficacy, improved drug permeability, and reduced drug dosage.¹²

Fig. 1: Nanocochleates: a potential drug delivery

History:

Cochleates are used to transfer antigen and peptides for vaccine delivery. They were discovered in 1975 by Dr. Dimitrious Papahadjoupoulos and his colleagues as precipitates generated by the interaction of negatively charged phosphatidylserine with calcium. The cylindrical structures were given the Greek term COCHLEATE meaning SHELL due to their rolled-up shape. Cochleate structures do not form consistently, which leads to the dialysis process forms enormous needle-like structures, or aggregation of stacked sheets. In 1999, cochleates were added to the hydrogel isolation process in order to produce particles that were smaller yet more uniform. Using a binary phase system, like non-miscible hydrogels, it is possible to construct cochleates with a mean particle size of less than 500 nm. These nanocochleates were excellent candidates for hydrophobic drug encapsulation.^1-4

Advantage:

1. Cochleates are more stable due to their non-aqueous inner core, which lessens the lipids susceptibility to oxidation.

2. The lyophilization procedure increases the shelf life of the formulation and enables longer periods of room temperature storage.

3. Pharmaceuticals that are encapsulated are shielded from deterioration caused by external factors including sunlight, water, temperature, or digestive enzymes

4. Parenterally administered drugs are often administered orally as cochleates. Take Amphotericin B³⁵, for instance.

5. They increase the oral bioavailability of a wide range of substances, including biopharmaceuticals that are difficult to administer and high lipophilicity medications, genes, vaccines, proteins, and peptides. Take ibuprofen and artemisinin, for instance.

6. Because the basic, naturally occurring lipids that make up the lipid bilayer of nanocochleates is composed of simple naturally occurring lipids that are nontoxic, non inflammatory, they are safer and more biocompatible.³⁰

Fig. 2: Nanocochelates: a potential drug delivery

Structure and Composition:

By adding negatively charged phospholipid bilayers (liposomes), which are rolled up by the interaction with multivalent counter ions of metal (Ca2+ or Zn2+) as bridging agents between the phospholipid bilayers, Cochleate and Nanocochleate are produced into cigar-like spiral rolls. In this process, the phospholipid head groups dryness is necessary for the bilayers to approach closely. To reduce their contact with the water, they roll up. They have a negligible or absent aqueous phase. A cochleates bilayers are stacked incredibly precisely at 54 Angstroms, which is an extremely near repeating distance. Instead of producing larger liposomes, little ones produce cochleates.²⁸

Method of preparation:

Fig. 3: method of preparation of nanocochleates

Liposomes suspended in an aqueous two-phase polymer solution are used to create nanocochleates, which enable differential partitioning and phase separation of structures based on polar molecules. Liposomes containing a polymer solution in two phases that has been treated with molecules that are positively charged, such as Zn2+ or Ca2+. A precipitate of sodium cochleate with particles smaller than one millimeter develops as a result. The technique could be applied to create nanoclusters with compounds that are significant to biologically important moleimportant.²²

1. Hydrogel Method:

The hydrogel method involves the following steps to produce nanocochleates:

Step 1: Small unilamellar liposomes or molecule-loaded liposomes that are biologically relevant are made by standard procedures such as sonication, microfluidization, and other comparable processes.

Step 2: Polymer A such as Dextran (mol.wt 2, 00,0005, 00,000), Polyethylene glycol (mol.wt 3400 8000), or Phosphatidyl serine is added to the liposome solution.

Step 3: The liposome/polymer A solution is combined with another polymer B, such as Polyvinyl pyrrolidone, Polyvinyl alcohol, Ficoll (mol.wt 30,000 50,000) and Polyvinyl methyl ether (PVMB) (mol.wt 60,000 1,60,000), resulting in an aqueous two-phase polymer system. This could be achieved mechanically using a syringe pump positioned to the right.

Step 4: To induce the development of miniature cochleates, a cation salt solution is added to the two-phase system of steps 3 and the cation diffuses into polymer B and then into the particles that comprise the liposome/polymer A.

Step 5: To separate the cochleate structures and eliminate the polymer solution, cochleate precipitates are repeatedly rinsed with a buffer solution containing a positively charged molecule, especially a divalent cation. ²¹

Fig: 4 - Schematic Representation of Hydrogel Method.

2. Trapping Method:

Trapping method: This method allows for the enchochelation of both hydrophilic and hydrophobic substances. It consists of a liposomal solution formulation that, in the case of hydrophilic medications, encloses the drug in the liposome’s aqueous layer or, in the case of hydrophobic drugs, intercalates the drug within the bilayers. To create liposomes, phospholipid powder could be mixed with water, or phospholipid film could be mixed with water phase.¹⁰ A cochleate assembly is produced when a liposomal suspension drop is supplemented with calcium. (as shown in fig. 8) Electron microscopy can be used to assess the amount of aggregation in cochleates generated by the trapping method after freeze-fracture, in contrast to other procedures. It involves the subsequent steps:

Step 1: Vortex the mixture for 15 minutes in order to create liposomes from phospholipids such as phosphatidyl serine

Step 2: The created liposomes are separated from the previously described solution using filtration.

Step 3: After the liposomes have been separated, the hydrophobic drug is given to them along with a capturing solvent like ethanol or dimethyl sulfoxide.

Step 4: Add a dropwise solution of calcium chloride to the solution from step 3 to precipitate crystalline cochleates.

Step 5: The cochleates are rinsed with a buffer containing calcium in order to get rid of any leftover solvent. The modified trapping method dissolves dioleoyl phosphatidyl serine (DOPS) in ethanol. After adding calcium chloride (cacl2), the mixture is homogenized for five minutes at 13,000 rpm and then stirred for an hour1.²²

Fig. 5: Schematic Representation of trapping method.

3. Liposomes before cochleates (LC) dialysis method.

Using this method, microscopic cochleates consisting of lipids, detergent, a chemical that is relevant to the body, and a cation are created. The main objective of adding detergent is to disturb the liposomes. To create the cochleates, calcium chloride is added to the mixture after it has been dialyzed using a buffer.¹² The detergent is removed with two dialysis treatments. The following are the steps involved in this process:

Step 1: An aqueous suspension is prepared using the lipid detergent combination.

Step 2: Mix polymer A with the suspension made in step 1 (such as phosphatidyl serine or polyethylene glycol, or PEG).

Step 3: Lipid/polymer A detergent. A solution containing polymer A Is mixed with polymer B, such as polyvinyl pyrrolidone (PVP), polyvinyl alcohol (PVA), and polyvinyl methyl ether (PVME). Immiscible polymers A and B combine to form a two-phase polymer system.

Step 4: A cationic moiety solution is added to the two-phase polymer system.

Step 5: The two-phase polymer system is cleaned to eliminate the polymer.¹⁶

4. Direct Calcium (DC) dialysis method.

This technique generates big cochleates. Direct dialyzation of the lipid and detergent mixture is done against a calcium chloride solution. The following are the steps involved in this process:

Step 1: Phospholipids and cholesterol (9:1) are mixed in a 9:1 weight ratio in an extraction buffer.

Step 2: An API concentration is mixed with a non-ionic detergent and vortexed for five minutes.

Step 3: The clear solution produced in Step 2 is dialyzed against three distinct buffers at room temperature.

Step 4: A 6 mM Ca2+ solution is used for the final dialyses.¹⁴

5. Binary aqueous-aqueous emulsion system.

Binary aqueous-aqueous emulsion system: This method is based on the incompatibility of two-phase systems of polymer solutions that are both aqueous and immiscible with one another. It is not necessary to utilize an organic solvent while using this method. It is used to create cochleates that are less than a millimeter in diameter.³¹The following are the steps involved in this process:

Step 1: A high PH or a film method are used to generate liposomes.

Step 2: Polymer A, such as Dextran, is mixed with liposomes.

Step 3: Next, a non-miscible polymer, like PEG, is mixed with the dextran/liposome phase.

Step 4: After adding calcium to the solution from step 3, nanocochleates softly diffuse from one phase to the next. Next, the gel is then rinsed in physiological buffer.²⁶

6. Solvent drip method:

The solvent drip technique involves the preparation of an amphipathic or hydrophobic cargo moiety solution and a liposomal suspension from Soy PS independently. For hydrophobic medicines, the solvent of choice should be dimethyl sulfoxide or dimethyl formamide.¹⁵ The solution is then added to the liposomal suspension after that. The solubility of the cargo moiety is decreased due to the solvents miscibility with water, which is partially attributed to the lipid hydrophobic liposomal bilayers. Chloride addition results in the formation of cochleates. Washing the extra solvent is a good idea.²³

Meachnism of action:

Fig: 6 Nanocochleates: a novel carrier for drug transfer

Nanocochleates are absorbed from the gut following oral ingestion. The cargo molecule is delivered into the blood vessel via nanocochleates, which penetrate the intestinal epithelium. If they enter the bloodstream by a different route besides intravenous, they pass through the related cell. They are given to the intended cell once they enter circulation. Figure – Nanocochleates Absorption from intestine.

Delivery to targeted cells:

Delivery to specific cells Numerous studies have been conducted on the relationship between negatively charged lipids and calcium. The majority of natural binding processes necessitate the interaction of a negatively charged phospholipid (typically phosphatidylserine or phosphatidylglycerol) with calcium. An essential mechanism in many natural membrane fusion processes is calcium-induced membrane rupture, which involves negatively charged lipids and leads to subsequent membrane fusion events. As a result, the cochleate may be seen as a membrane fusion in between.¹¹

Delivery by cell membrane fusion:

Fig: 7 Nanocochlates a novel way of drug delivery

Delivery by fusing of cell membranes The outer layer of the nanocochleate and the cell membrane fuse together when the nanocochleate first approaches the membrane, disrupting and rearranging it in the process. A tiny quantity of a particular material is introduced into the cells cytoplasm as a result of this combination.¹⁸ Figure - Direct Membrane fusion.

Evaluation of Nanocochleates:

1. Particle size distribution:

The particle size and size distributions are most significant parameters in innovative drug delivary system by employing scanning electron microscopy (SEM) and transmission electron microscopy (TEM) can be measured. For improved outcomes, the size evaluation of nanocochleates is assessed quantitatively using photon correlation spectroscopy and freeze fracturing microscopy. Atomic force microscopy is a sophisticated nanotechnology employed in the characterization of nanocochleates and the determination of the mean particle size of the liposomal and cochleate dispersions can be achieved by the application of laser diffraction technology.²⁰

2. Density:

Density Using a gas pycnometer, the density of nanocochleates is measured in air or helium.

3. Molecular Weight Measurement:

Molecular weight calculations Using a refractive index detector, gel permeation chromatography (GPC) can quantify the polymers overall molecular weight and dispersion within the matrix. Through the use of GPC, it was demonstrated that the entanglement of the rolling up of one or more long polymer chains is what builds polyalkylcynoacrylate nanocochleates²⁴.

4. Drug content:

Content of drugs To extract the free drug content from the supernatant, the redispersed nanocochleates suspension is centrifuged for 40 minutes at 250C while spinning at 15000 rpm. The complimentary medication After an appropriate dilution, the concentration in the supernatant can be measured using ultraviolet UV-Vis spectrophotometry.

5. Entrapment efficiency:

Non-encapsulated medication is separated from the nanocochleate dispersion by centrifugation at 5000 rpm for 30 minutes. This method determines the entrapment efficiency. The medication that was contained in the sediment vesicles was released by adding ethanol after the disodium EDTA solution broke the nanocholesteates structure and created a nanoliposome. The absorbance of produced solution determine using UV- Visible spectroscopic tech and entappment efficiency is calculation.

Entrapment efficiency = Amount of drug present in cochleates / Total amount present.²⁵

6. Stability study:

The dispersion of nanocochleates can be maintained at 2-8 0C and 25±2⁰C/60% RH for three months in order to assess the stability of the dispersion. It is established whether nanocochleates are stable. After their stability study, check the drug release by nanocochleate formulation, including the particle size and percentage of entrapment efficiency.²⁷

7. Surface charge:

When one or more nanocochleate molecules have the same surface charge, there is a significant repulsion between them, which prevents aggregation from occurring. This is why the stability of the nanocochleate formulation depends on the surface charge of the nanocochleate molecules. Strong attraction between molecules of nanochelates occurs when their charges are opposing, leading to increased aggregation. The velocities of nanocochleates are measured using laser light scattering techniques as Laser Droppler Anemometry (LDA) and Laser Droppler Velocimetry (LDA). Electrophoretic mobility of nanocochleates can also be used to quantify the surface charge of colloidal particles.

8. In vitro release study:

The in vitro release profile of nanocochleate is studied using modified ultra-filtration, diffusion cell, or standard dialysis techniques.¹⁷

Application:

1. Proteins, peptides, and DNA have been delivered using nanocochleates for application in gene therapy and vaccination.

2. Amphotericin B has been administered as an antifungal agent using nanocochleates, and the produced amphotericin B cochleates show better stability and efficacy at low dosages.

3. Administration of Antifungal Drugs An antifungal drug called ketoconazole (KCZ) is frequently used to treat fungal diseases such ringworm, candidiasis, and athletes foot.

4. Measles, mumps, polio, rubella, tetanus, and other vaccinations are among them. In order to avoid these diseases, vaccine-adjusted systems (VADS) with operators are typically utilized, along with live or inactive pathogens to protect antigens, liposomes, cochleates, viruses, etc.

5. Administration of anti-inflammatory agents.²⁹

6. Artemisia absinthium L. exhibits low solubility and instability in the volatile oil delivery system.

7. For this reason, essential oil (EO)Antibacterial agent delivery and fighting bacterial multidrug resistance.

8. The administration of insulin with magnetocolates and prepackaged insulin.

9. The discovery of using nanoliposomes for oral paclitaxel administration as an anticancer medication delivery method.

10. Drug delivery agents are incorporated into nanoparticles.

11. Nutrients including vitamins and omega fatty acids have also been delivered by nanocholesterols. Delivery of factor VIII recombinant.

12. Nasal vaccination uses nanocochleates as immunomodulators.

13. Processed foods can have their nutritional content increased by biogeod nanocochleates, which have the capacity to stabilize and preserve significant levels of micronutrients.

14. Proteins, peptides, and DNA for vaccinations and gene therapy have been delivered via nanocochleates.

15. Using cochleates to deliver antibacterial agents: This method will benefit from cochleates decreased toxicity³⁰.

CONCLUSION:

A nanocochleates are novel drug delivery system approach broadly applicable toEncapsulation of biologically important molecules such as genes, vaccine, antigen, and also Encapsulate hydrophilic and hydrophobic drugs. Encochleation helps to improve the Efficiency of the product because of the delivery increases the quality of the formulation alsoEnhance bioavailability and increase the therapeutic efficacy of therapeutic active agent. Nanocochleate are promising for oral, transdermal and dermal delivery. Many more drugCandidates can be encapsulated in to nanocochleates for their effective delivery.²⁹

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Received on 28.10.2024 Revised on 02.12.2024

Accepted on 07.01.2025 Published on 03.03.2025

Available online from March 10, 2025

Res. J. Pharma. Dosage Forms and Tech.2025; 17(1):63-68.

DOI: 10.52711/0975-4377.2025.00009

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