Formulation and In Vitro evaluation of liposomal drug delivery system of pregabalin

 Corresponding author: T Malyadri Copyright © 2025 Author(s) retain the copyright of this article. This article is published under the terms of the Creative Commons Attribution Liscense 4.0. Formulation and In Vitro evaluation of liposomal drug delivery system of pregabalin T Malyadri *, Anjaneyulu Devarakonda, Bollineni Divya, Perla Pravallika SK Mosin, Thatiparthi Vanisri and Medarametla Kishore Babu QIS Group of Institutions, QIS College of Pharmacy, Prakasam (Dt.), Ongole-A.P. World Journal of Biology Pharmacy and Health Sciences, 2025, 21(03), 161-174 Publication history: Received on 08 January 2025; revised on 15 February 2025; accepted on 18 February 2025 Article DOI: https://doi.org/10.30574/wjbphs.2025.21.3.0194 Abstract In this study that Pregabalin was successfully prepared as a liposomal drug delivery system by using two different techniques such as physical dispersion method and ether injection method. In this liposome’s preparations, cholesterol ratio was constant and soya lecithin concentrations were gradually increased (like 1:1, 1:2 and 1:3). The liposomes prepared by physical dispersion method showed better percentage drug entrapment when compared with ether injection method. The morphological characters of prepared liposomes were determined with the help of optical microscope. The particle size was analyzed by Malven particle size analyzer. The results of the particle size showed, when the concentration of soya lecithin was increased the size of the particle was reduced. The in vitro release showed that as the concentration of soya lecithin was increased the release rate of drug was retarded. Among the two-methods ether injection method showed prolonged action when compared to physical dispersion method. The stability studies for all the formulations were performed by keeping the formulations at two different temperatures 4ºC±2ºC and 25ºC±2ºC for a period of 30 days. After the stability period the formulations were tested for morphological analysis, percentage drug entrapment and in vitro drug release and compared with before stability study. There was no change in morphological characters at 4ºC±2ºC, but there was a slight reduced in particles size at 25ºC±2ºC. The percentage drug entrapment was reduced in all the formulations at both the conditions. The in vitro drug release was reduced for all the formulations. Liposomes prepared by physical dispersion method showed better stability compared with ether injection method. Keywords: liposomes; Pregabalin; Drug delivery; In vitro; In vivo 1. Introduction 1.1. Novel Drug Delivery System Novel Drug Delivery system (NDDS) refers to the approaches, formulations, technologies, and systems for transporting a pharmaceutical compound in the body as needed to safely achieve its desired therapeutic effects. NDDS is a system for delivery of drug other than conventional drug delivery system. NDDS is a combination of advance technique and dosage form which are far better than conventional dosage form. The aim of NDDS is to provide a therapeutic amount of drug to the appropriate site in the body to accomplish promptly and then maintain the desired drug concentration. NDDS combining polymer science, pharmaceutics and molecular biology1. 1.2. LIPOSOMES-An Introduction Liposomes are colloidal, vesicular structure composed of one or more bilayers surrounding an equal number of aqueous compartment. Liposomes are small artificial vesicles of spherical shape that can be created from cholesterol and natural nontoxic phospholipids. Due to their size and hydrophobic and hydrophilic character (besides biocompatibility), World Journal of Biology Pharmacy and Health Sciences, 2025, 21(03), 161-174 162 liposomes are promising systems for drug delivery. The sphere like shell encapsulated a liquid interior which contain substances such as peptides, protein, hormones, enzymes, antibiotics, anti-fungal and anti-cancer agents2. 1.3. Application of Liposomes Liposomes for Brain Targeting Liposome in Eye Disorders Liposome for Respiratory Drug Delivery System Liposomes in parasitic diseases and infections Macrophage activation and vaccination Liposomes in anticancer therapy Liposomes in bioengineering Liposomes in agro-food industry 1.4. Mechanism of Liposome Formation3 In aqueous medium, the lipid molecules in self-assembled structures are oriented in such a way that the polar portion of the molecule remains in contact with the polar environment and at the same time shields the non-polar part. Among the amphiphiles used in the drug delivery, viz. soap, detergents, polar lipids, the latter (polar lipids) are often employed to form concentric bilayer structures. However, in aqueous mixtures these molecules are able to form various phases, some of them are stable and others remain in the metastable state. At high concentrations of these polar lipids, liquidcrystalline phases are formed that upon dilution with an excess water can be dispersed into relatively stable colloidal particles. 2. Classification of liposomes4 2.1. Liposome classification based on structural features Multilamellar Large Vesicles (MLV) • Unilamellar vesicles (UV) • Oligolamellar vesicles (OLV) • Multivesicular vesicles (MVV) 2.2. Passive loading techniques In these passive loading technique, the drug is encapsulated by incorporating an aqueous phase of a water-soluble (hydrophilic) drug or an organic phase of a lipidsoluble drug initially or at predetermined stage during the preparation of the liposomes. The huge drug encapsulation efficiency can be achieved with the help of these passive loading technique which is more suitable for lipid-soluble drugs with a high resemblance to the lipid membrane. Different methods discuss under this class start with a lipid solution in organic solvent and end up with lipid dispersion in water. The a choice of component are typically combined by co-dissolving the lipids in an organic solvent and the organic solvent is then separated by film deposition under vacuum. when residual solvent is removed, the solid lipid mixture is hydrated with the help of aqueous buffer. The lipids spontaneously swell and hydrate to form liposome. Liposomal encapsulation technology (LET) is the latest delivery method used by medical researcher to transmit drugs that act as healing promoters to the definite body organs. LET is state of art method of preparing sub-microscopic bubbles called liposome5. 2.2.1. Mechanical Dispersion Method In these method variety components are mainly combined by co-dissolving the lipids in an organic solvent and after that the organic solvent is then separated by film deposition under vaccum. When all the solvent is evaporated, the solid lipid mixture is hydrated using aqueous phase. The lipids spontaneously swell and hydrate to form liposomes6. The following are types of mechanical dispersion methods 2.2.2. Lipid film hydration method The lipid-film hydration procedure is the most common and simple method for preparation of MLV by dissolving the phospholipids in the organic solvents: dichloromethane, chloroform, ethanol and chloroform-methanol mixture (2:1 v/v; 9:1 v/v; 3:1 v/v). A thin and homogeneous lipid film is formed when solvent is evaporated under vacuum at the temperature: 45-60 ºC. Nitrogen gas is involved in order to completely remove the residual solvent. A solution of distilled World Journal of Biology Pharmacy and Health Sciences, 2025, 21(03), 161-174 163 water, phosphate buffer, phosphate saline buffer at pH 7.4 and normal saline buffer are used in hydration step. The time for the hydration process varied from 1 h to 2 h at the temperature 6070 ºC. In order to obtain full lipid hydration, the liposomal suspension is left overnight at 4 ºC. The lipid-film hydration method can be used for all different kinds of lipid mixtures7. 2.2.3. Micro-emulsification method An equipment called as microfluidizer is used to prepare small vesicle from concentrated lipid suspension. The lipids can be introduced into the fluidizer as suspension of large MLVs. This equipment pumps the suspension at very high pressure through the 5 mm screen. Then it is forced long micro channel, which direct two streams of fluid collide together at right angle and very high velocity. The fluid collected can be recycled through the pump and interaction chamber until vesicles of spherical dimension are obtain8. 2.3. Sonication Sonication is perhaps the most extensively used method for the preparation of SUV. Here, MLVs are sonicated either with a bath type sonicator or a probe sonicator under a passive atmosphere. The main disadvantages of this method are very low internal volume/encapsulation efficacy, possible degradation of phospholipids and compounds to be encapsulated, elimination of large molecules, metal pollution from probe tip, and presence of MLV along with SUV. There are two sonication techniques9. 2.3.1. Probe sonication The tip of a sonicator is directly engrossed into the liposome dispersion. The energy input into lipid dispersion is very high in this method. The coupling of energy at the tip results in local hotness; therefore, the vessel must be engrossed into a water/ice bath. Throughout the sonication up to 1 h, more than 5% of the lipids can be de-esterified. Also, with the probe sonicator, titanium will slough off and pollute the solution10. 2.3.2. Bath sonication The liposome dispersion in a cylinder is placed into a bath sonicator. Controlling the temperature of the lipid dispersion is usually easier in this method, in contrast to sonication by dispersal directly using the tip. The material being sonicated can be protected in a sterile vessel, dissimilar the probe units, or under an inert atmosphere11 2.3.3. French pressure cell French pressure cell involves the extrusion of MLV through a small orifice. An important feature of the French press vesicle method is that the proteins do not seem to be significantly pretentious during the procedure as they are in sonication. An interesting comment is that French press vesicle appears to recall entrapped solutes significantly longer than SUVs do, produced by sonication or detergent removal. The method involves gentle handling of unstable materials. The method has several advantages over sonication method. The resulting liposomes are rather larger than sonicated SUVs. The drawbacks of the method are that the high temperature is difficult to attain, and the working volumes are comparatively small (about 50 mL as the maximum)12. 2.3.4. Membrane extrusion In this method, MLVs is reduced by passing them through a membrane filter of defined bore size. There are two types of membrane filter. The tortuous bath type and the nucleation track type. The former is used for sterile filtration. In this random bath arises between the cross fiber in the matrix. Liposomes that are larger than the channel diameter get struck when one tries to pass them though such membrane. The nucleation track is composed of thin continuous sheet of polycarbonate. They will offer less resistance to passage of liposomes as these consist of straight sided pore holes off exact diameter bored from one side to another. This method can be used to process both LUVs and MLVs13. 2.3.5. Dried reconstituted vesicles In DRV method freeze drying of a dispersion of empty SUVs are to be done and then dispersion of it with the aqueous fluid containing the material to be entrapped. This leads to a hydration of solid lipids in finely reduced sized form. Though, the step of freeze-drying is introduced to freeze and lyophilize a performed SUVs dispersion rather than to dry the lipids from an organic solution. This leads to an ordered membrane structure as compared to random matrix structure, which on addition of water can rehydrate, fuse and reseal to form vesicles with a high encapsulation efficiency. The water soluble hydrophilic materials to be entrapped are added to the dispersion which are empty SUVs and they are dried World Journal of Biology Pharmacy and Health Sciences, 2025, 21(03), 161-174 164 together, so the material for inclusion is present in the dried precursor lipid before the final step of addition of aqueous medium14. 2.3.6. Freeze-thawed liposome SUVs are rapidly frozen and thawed slowly. The short-lived sonication disperses aggregated materials to LUV. The creation of UV is as a result of the fusion of SUV throughout the processes of freezing and thawing. This type of synthesis is strongly inhibited by increasing the phospholipid concentration and by increasing the ionic strength of the medium. The encapsulation efficacies from 20% to 30% were obtained15. 2.3.7. Solvent Dispersion Method • Ether injection (solvent vaporization) • Ethanol injection • Double emulsion method • Reverse phase evaporation method • Stable plural lamellar vesicles 2.3.8. Detergent Removal Method16: In this method the phospholipids are brought into close contact with the aqueous phase via detergents, which associate with phospholipids molecules. The structures formed as a result of this association are known as micelles. They are composed of several hundreds of component molecules. The concentration of detergent in water at which micelles start to form is called CMC. Below CMC the detergent molecule exist in free solution. As the detergent molecule is dissolved in water at concentration higher than the CMC, micelle form in large amounts. As the concentration of detergent added is increased more amount of detergent is incorporated into the bilayer, until a point is reached where conversion from lamellar form to spherical micellar form take place. As detergent concentration is further increased, the micelles are reduced in size. • Dialysis • Column Chromatography • Dilution ACTIVE LOADING17 The exploitation of liposomes as drug delivery system is encouraged with the advancement of well-organized encapsulation procedures. The membrane from the lipid bilayer is in general impermeable to ions and larger hydrophilic molecules. Ions transport can be synchronized by the ionophores though permeation of neutral and weakly hydrophobic molecule can be inhibited by concentration gradients. A few weak acid or bases yet, can be transported throughout the membrane because of various transmembrane gradient, such as electric, ionic (pH) or specific salt (chemical potential) gradient. Some method exist for improved incorporation of drugs, including remote (active) loading method which load drug molecules into preformed liposome using pH gradient and potential difference across liposomal membrane. A concentration variation in proton concentration across the membrane of liposomes can drive the loading of amphipathic molecule. Active loading methods have the following benefit over passive encapsulation Technique • It will lead to high encapsulation efficiency and capacity. • Using these method leakage of the encapsulated compounds can be reduced. • “Bed side” loading of drugs therefore limiting loss of retention of drugs by diffusion, or chemical degradation while storage. • These process is flexible for constitutive lipid, as drug is loaded after the formation of carrier unit. • It also reduce the safety hazard by avoiding biologically active compounds in the preparation step during dispersion. • The transmembrane pH gradient may be occurred by various method. Based upon the nature of drug to be encapsulated18. World Journal of Biology Pharmacy and Health Sciences, 2025, 21(03), 161-174 165 3. Methodology 3.1. Preformulation studies18 Preformulation testing is an investigation of physical and chemical properties of a drug substance alone and when combined with excipients. It is the first step in the rational development of dosage form. The objective of Preformulation testing is to generate information useful to the formulation in developing stable and stable and bioavailable dosage forms. The use of Preformulation parameters maximize the chances in formulating an acceptable, safe, efficacious and stable product. • Solubility • Melting Point 3.1.1. Drug – excipients interaction studies19 FT-IR spectra were taken for the dried samples using FT-IR 8400S (Shimadzu, Japan) to determine the possible interactions between the drug and polymers. The plain drug, individual lecithin and cholesterol, combination of drug with cholesterol and lecithin in three different ratio (1:1, 1:2 and 1:3) were taken and mixed with KBr. The samples were compressed to form a pellet using a hydraulic press. The prepared pellets were transformed into disk. The disk was applied to the center of the sample holding device and scanned from 4,500 to 400 cm-1 using FT-IR spectrophotometer76. 3.1.2. Formulation of liposomes loaded with Pregabalin hydrochloride The formulation of liposomes loaded with Pregabalin was prepared by two different techniques namely, physical dispersion method and ether injection method. In both the techniques ratio of cholesterol was kept as same and the lecithin concentration was increased as 1:1, 1:2 and 1:3. 3.1.3. Physical dispersion method20 Liposomes were prepared by physical dispersion method using different ratio of soya lecithin and cholesterol was kept as constant. In this method the soya lecithin and cholesterol were dissolved in chloroform. Then it was spread over flat bottom conical flask and allowed to evaporate at room temperature for overnight without disturbing the solution for a formation of lipid film. The drug was dissolved in phosphate buffer pH 6.8. It act as an aqueous medium. Then the aqueous medium was added to the lipid film for hydration. For this the flask was inclined to one side and aqueous medium was introduced down the side of flask and flask was slowly returned to upright orientation. Then the conical flask was kept on water bath and the temperature was maintained at 37± 2ºC for 2 hours for the completion of hydration. The conical flask was gently shaken until the lipid layer was removed from wall of conical flask and formation a liposomes suspension. Then the formed liposomes suspension was stored at 4ºC for one day for the maturation of liposomes. The prepared liposome suspension was centrifuged at 15,000 rpm for 20 mins. Then the precipitate was collected and diluted with distilled water for further studies35. Different batches of liposomes were prepared as per the general method described above and composition for the preparation of liposomes is given in Table 5. 3.1.4. Ether injection method21 Liposomes were prepared by ether injection method using different ratio of soya lecithin and cholesterol was kept as constant. In this method the cholesterol and soya lecithin were dissolved in ether and methanol. The drug was dissolved in phosphate buffer pH 6.8. It act as an aqueous medium. The aqueous medium was heated to 60°C. The method involves injecting drop by drop of ether-lipid solutions into the above warmed aqueous medium. The ether vaporizes upon contacting the aqueous phase, and the dispersed lipid forms primarily unilamellar liposomes. Then the product was collected and it was stored at 4°C for maturation of liposome. Then prepared liposomal suspension was centrifuged at 15,000 rpm for 20 mins. The precipitate was diluted with distilled water for evaluation studies. Different batches of liposomes were prepared as per the general method described above and composition for the preparation of liposomes is given in Table No. 1. World Journal of Biology Pharmacy and Health Sciences, 2025, 21(03), 161-174 166 Table 1 Formulation of Pregabalin liposomes S. No. Ingredients Physical dispersion method Ether injection method F 1 F 2 F 3 F 4 F 5 F 6 1. Cholesterol 100 mg 100 mg 100 mg 100 mg 100 mg 100 mg 2. Lecithin 100 mg 200 mg 300 mg 100 mg 200 mg 300 mg 3. Pregabalin 10 gm 10 gm 10 gm 10 gm 10 gm 10 gm 4. Ether - - - 7 ml 7 ml 7 ml 5. Methanol - - - 3 ml 3 ml 3 ml 6 Chloroform 5 ml 5 ml 5 ml - - - 7. Phosphate buffer pH 6.8 50 ml 50 ml 50 ml 50 ml 50 ml 50 ml 3.1.5. Evaluation of liposomes22 Determination of percentage drug entrapment efficiency Drug entrapment efficiency was calculated by using centrifugation method. 10 ml of liposome suspension was taken and centrifuged at 15,000 rpm for 20 mins. The supernatant liquid was collected and suitably diluted. Then the absorbance was taken at 233 nm with the help of UV double beam spectrophotometer using pH 6.8 as a blank. Morphology analysis The prepared Pregabalin liposomes for all the formulations were viewed under for observing the vesicle formation and discreteness of dispersed vesicles. A slide was prepared by placing a drop of liposome dispersion on a glass slide and cover slip was placed over it and this slide was viewed under optical microscope at 40X magnification. Photographs were taken to prepared slides using digital camera33. In vitro drug release study: Apparatus: USP TYPE II (Paddle) RPM : 50 Temperature: 37 ºC ± 0.5 ºC Time : 30 min. interval Up to 8 hrs The in vitro release for all the formulated Pregabalin liposomes were carried out for 8 hours in phosphate buffer p H 6.8. The studies were carried in USP dissolution apparatus II (Paddle) at 37ºC ± 0.5ºC and 50 rpm speed. 900 ml of phosphate buffer p H 6.8 was used as a dissolution medium. Equivalent to 100 mg of Pregabalin liposome was taken in a dissolution jar contains dissolution medium and the paddle was rotated at 50 rpm. 1 ml of samples were withdrawn at every 30 min. upto 480 mins and make upto 10 ml with pH 6.8 and analyzed for Pregabalin content at 233 nm with pH 6.8 as blank using double beam UV double beam spectrophotometer79. Particle size determination23 The particle size determination is done by using Malven particle size analyzer. Groups of particles are dispersed in a liquid medium and measured as they are circulated between the flow cell, which is placed in the measurement unit, and a dispersion bath in the sampler. The dispersion bath incorporates a stirrer and an ultrasonic sonicator. A pump delivers the dispersed suspension to the flow cell. The pump is specially designed to ensure both liquid medium and the particles are circulated. It can be controlled from a PC. Organic solvents can be used as dispersion media. Stability studies24, 25: The behavior of the liposome to retain the drug was studied by storing the liposome at two different temperature conditions, i.e., 4ºC (refrigerator RF), 25°C±2ºC for a period of 1 month. The liposomal preparations were kept in sealed World Journal of Biology Pharmacy and Health Sciences, 2025, 21(03), 161-174 167 vials. At 30th day the samples were analyzed for the drug content following the same method described in % drug encapsulation efficiency and in vitro drug release. And also the liposomes were studied for their morphology. 4. Results and discussion The research study was aimed to formulate Pregabalin liposomes to sustain the action of drug for over the period of 8 hours. The liposomes were prepared by physical dispersion method and ether injection method. Soya lecithin and cholesterol were used for encapsulating the drug and also to release the drug in sustained manner. Chloroform, ether and methanol were used as a solvent. Phosphate buffer pH 6.8 was used as a hydration medium for loading the drug. Preformulation studies such like solubility analysis, melting point and FT – IR studies were carryout before the formulations. After formulation, the liposomes were evaluated for various parameters like percentage drug entrapment efficiency, microscopic analysis, particle size analysis, in vitro drug release studies and stability study. 4.1. Preformulation studies 4.1.1. Solubility The drug should be dissolve in solvents and also dissolution medium so the solubility analysis for the drug was important. The solubility of raw drug was determined by dissolving in distilled water, methanol and phosphate buffer pH 6.8. The drug was found to be freely soluble in water, soluble in methanol and phosphate buffer p H 6.8. Melting point The melting point was confirmed the Pregabalin present in raw material of drug. It was found to be 224ºC within the specification range. So it confirmed Pregabalin present in raw material of drug. 4.1.2. Drug – excipients interaction studies: The FT – IR studies of pure Pregabalin, cholesterol, soya lecithin and Pregabalin+ cholesterol + soya lecithin were conduct to study the interaction between the drug and excipients. IR spectral analysis showed that the fundamental peaks and patterns of the spectra were similar both in pure drug and combination containing drug and highest proportion of excipients. This indicated that there was no chemical interaction between Pregabalin and the other excipients used in the formulations. The spectral data are presented in Table No. 2 and spectral peaks were presented graphically in Figure No. 1 – 2. 4.1.3. FT – IR spectrum of pure pregabalin Figure 1 FT – IR Spectrum of pure Pregabalin World Journal of Biology Pharmacy and Health Sciences, 2025, 21(03), 161-174 168 Table 2 FT – IR Spectrum of pure Pregabalin Wave length (cm-1) Functional group 3372 N-H stretching 1582 Amino N-H bending 1466 CH3 bending alkanes 1057 C-N Stretching 957 Alkene C-H bending FT – IR SPECTRUM OF COMBINATION OF Pregabalin, CHOLESTEROL AND SOYA LECITHIN Figure 2 FT – IR SPECTRUM OF COMBINATION OF Pregabalin, CHOLESTEROL AND SOYA LECITHIN Table 3 FT – IR Spectrum of combination of Pregabalin + cholesterol + soya lecithin Wave length (cm-1) Functional group 3372 N-H stretching 1582 Amino N-H bending 1466 CH3 bending alkanes 1057 C-N Stretching 957 Alkene C-H bending Table 4 FT – IR Spectrum of pure Pregabalin, cholesterol, soya lecithin and combination of Pregabalin + cholesterol + soya lecithin Functional group N-H stretching (cm-1) Amino N-H bending (cm-1) CH3 bending alkanes (cm-1) C-N Stretching (cm-1) Alkene C-H bending (cm-1) Drug 3372 1582 1466 1057 957 Cholesterol 3421 - 1466 1057 955 Soya lecithin 3379 1620 1464 1104 864 Combination of drug + cholesterol + soya lecithin 3372 1582 1466 1057 957 World Journal of Biology Pharmacy and Health Sciences, 2025, 21(03), 161-174 169 4.1.4. Evaluaton of metormin HCL liposomes Percentage drug entrapment efficiency The percentage drug entrapment efficiency of liposomes were prepared by physical dispersion method and ether injection method. The formulations was formulated by varying the cholesterol – soya lecithin ratio. It was found to be that percentage drug entrapment efficiency of formulations F 1, F 2 and F 3 were 86.60 %, 79.90 % and 73.10 % respectively and formulations F 4, F 5 and F 6 were 30.47%, 39.58% and 39.69% respectively. The results may adjudge physical dispersion method have better drug entrapment efficiency than ether injection method. Morphology analysis The morphology characters of liposomes were analyzed by optical microscopy (Olympus Opto System, India) and the images were taken using digital camera. The formulation F 1, F 2, F 3, F4, F 5 and F 6 microscopic images were showed in Figure No. 3.-8 Prepared liposomes F 1 to F 6 shows well identified morphology characters. Figure 3 Microscopic image (45 X) of F 1 formulation Figure 4 Microscopic image (45 X) of F 2 formulation Figure 5 Microscopic image (45x) of F 3 formulation Particle size analysis The particle size analysis was carried out by particle size analyzer for all the prepared liposome formulations. The particle size for all the formulated liposomes were found be in the range of 30.617 µm to 0.031µm and graphically showed. The particle size data showed that when the concentration of soya lecithin was increased the particle size was decreased for all the formulations of Pregabalin liposomes in prepared by both methods. The particle size of Pregabalin liposomes of F 3 and F 6 were found to be lower when compared with other formulations this may be due to higher concentration of soya lecithin. Figure 6 Microscopic image (45x) of F 4 formulation Figure 7 Microscopic image (45x) of F 5 formulation Figure 8 Microscopic image (45x) of F 6 formulation