× About the Journal Scope of the Journal SPER Publications Editorial Board Abstracting and Indexing Articles in Press Current Issue Archives Submit Article Author Guidelines Advertise Join as Reviewer Contact Editorial Policies and Peer Review Process Journal Policies Publishing Ethics
[An Official Publication of ISF College of Pharmacy, Moga]

Original Article
Year : 2019   |  Volume : 11   |  Issue : 2   |  Page : 63-68  

Preparation and evaluation of liposomes of diclofenac sodium using round-bottom flask method

Sankha Bhattacharya, Omprakash Sahu, Shatrughna Chaudhary, Puja Banik, Nandita Bhowmik, Varsha Shodhi

Correspondence Address:Department of Pharmaceutics, Indo Soviet Friendship College of Pharmacy, Moga, Punjab, India

Source of Support: Nil, Conflict of Interest: None Declared

DOI: 10.4103/2231-4040.197331


Aim: The goal of the research was to prepare liposome containing diclofenac sodium in the round-bottom flask method. Materials: The liposomes consisting of phosphatidylcholine and cholesterol have been tested to be appropriate components which give amphiphilic nature and flexibility. Method: Liposomes had been organized through the use of rotator evaporator (HEIDOLPH G3, Germany) for 30 min at 50 rpm and at 40–60°C temperature. The different types of instruments used for this type of operation were sonicator (LABMAN Scientific Instrument), UV spectrophotometer, Delsa C Particle Analyzer, Motic microscope, and Fourier-transform infrared spectroscopy. Result: The prepared liposomes had been analyzed for particle size, zeta potential, entrapment efficiency, and vesicle morphology. From the outcomes of this research it was found that Batch-C (8:2) was found to be the most optimized formulation batch. Conclusion: liposomal formulation of diclofenac confirmed a proper entrapment efficiency and better stability profile. Thus, it concluded that Batch-C was very promising carrier for liposomal delivery and showing a new possibilities applications for diclofenac sodium.

Keywords: Liposomes, diclofenac sodium, phosphatidylcholine, entrapment, lipid, cholesterol, stearyl amine, particle analyzer

How to cite this article:
Bhattacharya S, Sahu O, Chaudhary S, Banik P, Bhowmik N, Shodhi V. Preparation and evaluation of liposomes of diclofenac sodium using round-bottom flask method. Pharmaspire 2019;11(2):63-68.


Diclofenac is a nonsteroidal anti-inflammatory drug (NSAID) with analgesic, anti-inflammatory, and antipyretic activities.[1] It is a monocarboxylic acid consisting of phenylacetic acid having a (2,6-dichlorophenyl) amino group at position II.[2] Diclofenac indicates its activities through inhibiting cyclooxygenase (COX)-2 enzyme with increased efficiency than it does (COX)-1,[3] which are enzyme responsible for producing prostaglandins. Like different NSAIDs,[4] it is regularly used as first line remedy for acute and chronic pain and inflammation from a range of causes.[5] Diclofenac significantly binds to plasma albumin. It has a brief half-life in plasma (1–2 h) and solely 50% of the drug reaches the systemic circulation. Diclofenac undergoes oxidative metabolism to hydroxyl metabolites as properly as conjugation to glucuronic acid, sulfate, and taurine.[6] The major metabolite is 4´-hydroxy diclofenac which is generated using CYP2C9.[7] Overdose of diclofenac can reason lethargy, drowsiness, nausea, vomiting, and epigastric ache and gastrointestinal bleeding.[8] Lipos means “fat” and Soma means “body,” the title liposome is derived from these two Greek words. Liposome had been first produced in England in 1961 by means of Alec D. Bangham. In liposomes, the phospholipids blended with water and at once shaped a sphere due to the fact one stop of each molecule is hydrophilic and one quit is hydrophobic.[9] Liposomes are in the structure of vesicles that might also be bilayer or multilayers. The polar molecules in liposomes permit to encapsulated[10] the polar molecules. Amphiphilic and lipophilic molecules are solubilized inside the phospholipid or in accordance to their affinity closer to the phospholipid. Liposomes can be formulated as an aerosol, suspension, or in a semisolid structure such as a cream, gel, or dry powder.[11] They can be administered topically or parenterally. Liposomes usually recognized as foreign particles and endocytosed through mobile phone of MPS (mononuclear phagocytic system). Liposomes can enhance the therapeutic recreation and safety of drugs in drug delivery system using supply them at the target site of action for extended duration of time.[12]
As a delivery system liposomes are turning into extra beneficial in drug administration to human body due to distinct advantages: (1) Controlled hydration, protein stabilization, (2) reduce exposure of sensitive tissues to toxic drugs, (3) carry both water and oily soluble payloads, (4) ideal specific gravity and possibility of producing then exclusive size ranges, (5) flexible for systemic and non-systemic administration, (6) non-toxicity, (7) target ability, (8) biodegradability, and (9) biocompatibility.

Types of liposomes


Niosomes are self-assembly of non-ionic amphiphiles in aggregate with different lipidic surfactants in aqueous medium.[13] Niosomes and liposomes have main distinction of chemical stability and relatively low cost. Both are at the risk of aggregation, fusion, and encapsulated drug leakage.
Recently Proniosomes are showing many advantages over Noisome i.e., minimization of physical instability issues such as aggregation, fusion, and encapsulated drug leakage. They additionally furnish effortless transportation, distribution, storage, and dosage.


Unmodified liposomes are up to 105 times much less deformable than transfersomes.[14] Transfersomes are having particle size of around 200-300nm with a capability of passing through stratum corneum. From the different research findings, it is confirmed that, Niosomes andliposomes having nearly equal entrapment efficiency.


Ethosomes composed of phospholipids (soy phosphatidylcholine), ethanol, and water. They are multilamellar vesicles (MLVs) and recognized for permeability enhancer.[15] Ethosomes have higher molecule entrapment efficiency of more than a few lipophilicities, for example, acyclovir, minoxidil, and testosterone.


Proliposomes are free-flowing particles that form liposomes on contact with water.[16] They are water-soluble porous material as a carrier; consequently, drug and phospholipid are deposit in microporous shape of carrier material, consequently keep the free-flowing characteristics. Proliposomes can be stored in dry sterilized state, if wanted they dissolved with water to form isotonic multilamellar liposomes.

Advantages of liposomes

  • Liposomes are biocompatible, biodegradable, non-toxic, and non-immunogenic.
  • Suitable delivery of hydrophobic, amphipathic, and hydrophilic drugs.
  • Protect the drug from exterior environmental by means of encapsulation.
  • Increased stability, minimize toxicity, enhance the therapeutic activity.
  • Reduce exposure of toxic drugs to sensitive tissues.

Disadvantages of liposomes

  • Production value is high.
  • Leakage and fusion of encapsulated drug.
  • Short half-life.

Classification of liposomes

Based on structure

  1. Unilamellar vesicles
    • Small unilamellar vesicles (SUVs): Size ranges from 20 to 40 nm.
    • Medium unilamellar vesicles: Size ranges from 40 to 80 nm.
    • Large unilamellar vesicles: Size ranges from 100 to 1000 nm.
  2. Oligolamellar vesicles (OLVs)
    • OLVs are made up of 2–10 bilayer of lipids surrounding a large inside volume.
  3. MLVs
    • They have a number of bilayers. They have onion like shape and spherical bilayer of MLV enclosing a massive wide variety of SUV

Based on technique of preparation

  1. REV: Reverse-phase evaporation method.
  2. DRV: Dehydration rehydration method.
  3. VET: Vesicle prepared through extrusion techniques.

Stages of liposome preparation

  1. Drying down lipids from organic solvent.
  2. Dispersion of lipid in aqueous media.
  3. Purifying the liposomes.
  4. Analyzing the ultimate product.

Therapeutic application of liposome:

Liposome provides an optimal therapeutic efficacy and safety in contrast to present formulations. Some of the essential therapeutic applications of liposome in drug delivery include:

  1. Site avoidance delivery.
  2. Site specific targeting.
  3. Intracellular drug delivery.
  4. Sustained release drug delivery.
  5. Intraperitoneal administration.
  6. Immunological adjuvants in vaccine.

The liposome drug delivery systems possess significant role in drug formulation to enhance the therapeutics, other novel applications of liposome are

  • Protection toward enzymatic degradation: Liposomes are no longer prone to enzymatic degradation so the entrapped drug is accordingly protected.
  • Drug targeting: The want to drug targeting is to avoid the drug distribution to minimize the drug waste.
  • Topical drug delivery: Increase the permeability of pores and skin for a number of entrapped drugs.
  • Respiratory drug delivery: Liposomal aerosols are used.
  • Liposome in tumor therapy: Long-term therapy of anticancer drug causes various toxic side effects. (Liposome is used having low dose).
  • Liposome as carrier of drug in oral treatment: Steroid used for arthritis can be included into massive MLVs.
  • Alteration in blood glucose levels in diabetic animals used to be acquired through oral administration of liposome encapsulated insulin. Figure 1 describes about basic structure of liposomes.


Diclofenac sodium (DS) used to be bought from Alka Pharmaceuticals, Hyderabad, lecithin soy 30% used to be bought from HiMedia Laboratories. Ltd., Mumbai, cholesterol was once supplied using Central Drug House Pvt. Ltd., New Delhi, methanol (purchased from SD Fine Chem Limited, Mumbai), chloroform used to be bought from Rankem, Thane, Maharashtra, and phosphate buffer

Preparation of liposome

The RBF technique was once used to put together DS -loaded liposome.[17] In this approach soy lecithin, cholesterol used to be dissolved in chloroform in exclusive molar ratio soy lecithin & cholesterol was once taken respectively in one of a kind ratio (6:4, 7:3, and 8:2) in round-bottom flask (RBF). Respective quantity of chloroform and methanol used to be taken &, brought into the RBF to dissolve the lipids. In any other beaker, DS used to be dissolved in phosphate buffer (pH = 7.4). Solvent combination was once evaporated through the usage of rotatory evaporator (HEIDOLPH G3, Germany) for 30 min at 50 rpm and at 40–60°C temperature. Dry thin film was formed on facet wall of RBF when evaporation was once completed. Then, rehydration of thin film was once carried out with drug and phosphate buffer solution. After hydration, the answer was once sonicated (LABMAN Scientific Instrument, Chennai) for 5 min at 30°C temperature [Table 1].

Characterization of liposome

Morphology of liposome

Shape & and morphology of liposome have been observed under Motic microscope at appropriate magnification. Shape of liposome particles had been observed spherical. The liposomes had been stained the usage of sulforhodamine B solution (sulforhodamine was once organized with the aid of the use of 0.5% solution of acetic acid and water).

Particle size and zeta potential of liposome

Delsa C particle analyzer was once used to measure particle size of liposome. Then, cuvette used to be crammed through sample carefully and there should not be formation of bubbles. The cuvette used to be positioned into the instrument through opening the lid of zetasizer and sample was once analyzed. Zeta potential of DS -loaded liposome was once observed through the use of Beckman Coulter & zeta potential had been measured for three batches and values have been determined −15.68 (batch A), −16.29 (batch B), and −13.68 (batch C).

Standard curve of DS

The dilution was once organized from stock solution of DS (100 µg/mL) to get concentration of 5, 10, 15, 20 &, and 25 µg/mL, respectively. Absorbance of DS was once measured at 276 nm in ultraviolet–visible spectrophotometer. The calibration curve used to be plotted between absorbance verses concentration [Figure 2].

Entrapment efficiency

Entrapment efficiency of liposomal formulation was once got using centrifugation method.[18] Here, 10 ml liposomal suspension was once poured into a centrifugation tube and centrifuged at 1000 rpm for 10 min. The clear supernatant used to be similarly used for the determination of free drug through the use of UV–visible spectrophotometer at 276 nm examined underneath the UV–visible spectroscopy. The entrapment efficiency was once calculated the use of the following formula:

Entrapment effectivity percent = (Ct−Cf)/Ct × 100
Where, Ct the concentration of total drug and Cf is the concentration of entrapped drug.


Fourier-transform infrared (FTIR) spectroscopy

FTIR spectroscopy of drug (DS), cholesterol, soy lecithin, physical mixture,[19] and drug-loaded liposome has been obtained by means of conformist using KBr disc/pallet method (Agilent Cary 630 FTIR), the 1:150 ratio of KBr & beaten powder pattern used to be blended collectively and compressed the use of Jasco MP-1 minihydraulic compactor. The spectroscopical research was once carried out from 4000 cm−1 to 400 cm−1with a resolution of 420 scans for the prepared anhydrous KBr pallets of drug and specific formulations. The FTIR data was found to be in-between the wavelengths of 4000 cm−1 and 400 cm−1.



Formulation was developed using RBF method and the formulation is discussed in Table 2. The formulation and its excipients were first analyzed using FTIR. The FTIR spectra of DS used to be displaying characteristics peaks at 3161.34 cm−1, 166.97 cm−1, and 1223.46 cm−1, signifying the presence of strong ammonium stretching, amide N-H & C=O stretch, O-H & and C-O stretch; cholesterol indicates significant stretching at 3161.65 cm-1 indicating C=O stretch; the place else soy lecithin indicates characteristics peaks at 3677.64 cm−1, 3174.25 cm−1, 2993.95 cm−1, 1851.24 cm−1, 1772.77 cm−1, and 1212.71 cm−1 indicating amide N-HC=O stretch; physical mixture indicates characteristics peaks at 3645.21 cm−1 and 3039.10 cm-acid monomeric C=O stretch; ester C=O & C-O strong stretch; liposome except drug shows characteristic peaks at 3710.96 cm−1, 2881.43 cm−1, and 1795.35 cm−1 indicating amide N-H & C-O stretch , alkyl C-H stretch, acid chloride C=O stretch; drug-loaded liposome show characteristics peaks at 3655.06 cm−1, 3161.65 cm−1, and 1784.60 cm−1 indicating amide N-H & and C-O stretch, ammonium ion stretch. As in contrast DS liposome without drug & drug-loaded liposome indicates narrow peaks in distinctive wavenumbers indicating drug loading and amorphous nature of formulations. IR characteristics confirm encapsulation of DS inside of the liposome.
Pre-formulation research of DS received with the aid of maximum absorption of UV spectroscopy and it used to be discovered to be at 276 nm and the approach prepared using Beer’s law with linearity plot. Liposomes prepared with lipids such as soy lecithin and cholesterol had been utilized to furnish the encapsulation and rigidity of associated DS. The liposomes formulation was once prepared by way of round-bottom flask method. The physical traits had been studied to determination of vesicle morphology, size distribution, and entrapment efficiency used to be found to be within the desirable limits [Table 2].
The liposomes had been found as desirable sphericity with smooth surface. The particles had been discrete and separate with no agglomeration or aggregation. The particle size of the liposome used to be determined the usage of microscopy studies [Figure 3].

Size distribution studies

The liposomal dispersions have been characterized for size distribution the usage of dynamic scattering light technology (particle size analyzer). Higher the positive or negative zeta potential value [Figure 5], larger will be its colloidal stability. The greater zeta potential fee was once observed for diclofenac coated liposomal formulation batch-C (8:2). These show good kinetic stability of the liposomes. Therefore, the diclofenac encapsulated liposomes show better stability [Figure 6].




Liposomal formulation of diclofenac allowed a massive enchantment of its therapeutic effectiveness in terms of drug permeation. Furthermore, from the learn about it used to be confirmed that liposomal formulation of diclofenac confirmed a proper entrapment efficiency and better stability profile. Thus, it concluded that formulation is a very promising carrier for liposomal delivery and growing a new possibilities application of diclofenac.

  1. Futaki N, Yoshikawa K, Hamasaka Y, Arai I, Higuchi S, Iizuka H, et al. NS398, a novel non-steroidal anti-inflammatory drug with potent analgesic and antipyretic effects, which causes minimal stomach lesions. Gen Pharmacol 1993;24:105-10.
  2. Griswold DE, Adams JL. Constitutive cyclooxygenase (COX-1) and inducible cyclooxygenase (COX-2): Rationale for selective inhibition and progress to date. Med Res Rev 1996;16:181-206.
  3. Malhotra SD, Rana DA, Patel VJ. Comparison of analgesic, anti-inflammatory and anti-pyretic efficacy of diclofenac, paracetamol and their combination in experimental animals. Int J Basic Clin Pharm 2013;2:458-65.
  4. Small RJ. Diclofenac sodium. Clin Pharm 1989;8:545-58.
  5. Vane J, Bakhle Y, Botting RJ. Cyclooxygenases 1 and 2. Annu Rev Pharmacol Toxicol 1998;38:97-120.
  6. Gan J, Ma S, Zhang DJ. Non-cytochrome P450-mediated bioactivation and its toxicological relevance. Drug Metab Rev 2016;48:473-501.
  7. Si D, Wang Y, Zhou YH, Guo Y, Wang J, Zhou H, et al. Mechanism of CYP2C9 inhibition by flavones and flavonols. Drug Metab Dispos 2009;37:629-34.
  8. Smolinske SC, Hall AH, Vandenberg SA, Spoerke DG, McBride PV. Toxic effects of nonsteroidal anti-inflammatory drugs in overdose. Drug Saf 1990;5:252-74.
  9. Swami H, Kataria MK, Bilandi A, Kour P, Bala SJ. Liposome: An art for drug delivery. Int J Pharm Sci Lett 2015;5:523-30.
  10. Ostro MJ, Cullis PR. Use of liposomes as injectable-drug delivery systems. Am J Hosp Pharm 1989;46:1576-88.
  11. ÇağdaŞ M, Sezer AD, Bucak S. Liposomes as Potential Drug Carrier Systems for Drug Delivery. London: Intech; 2014. p. 1-100.
  12. Shi J, Xiao Z, Vilos C, Votruba A, Langer RS, Farokhzad OC. Lipid-polymer Hybrid Particles. Google Patents; 2017.
  13. Uchegbu IF, Vyas SP. Non-ionic surfactant based vesicles (niosomes) in drug delivery. Int J Pharm 1998;172:33-70.
  14. Oidu B. Uptake of Liposomes Into Bacterial Cells. South Africa: Nelson Mandela Metropolitan University; 2013.
  15. Cosco D, Celia C, Cilurzo F, Trapasso E, Paolino DJ. Colloidal carriers for the enhanced delivery through the skin. Expert Opin Drug Deliv 2008;5:737-55.
  16. Song KH, Chung SJ, Shim CK. Preparation and evaluation of proliposomes containing salmon calcitonin. J Control Release 2002;84:27-37.
  17. Chandu VP, Arunachalam A, Jeganath S, Yamini K, Tharangini K, Chaitanya GJ. Niosomes: A novel drug delivery system. Int J Nov Trends Pharm Sci 2012;2:25-31.
  18. Agarwal R, Katare O, Vyas SJ. Preparation and in vitro evaluation of liposomal/ niosomal delivery systems for antipsoriatic drug dithranol. Int J Pharm 2001;228:43-52.
  19. Jithan A, Swathi M. Research paper development of topical diclofenac sodium liposomal gel for better antiinflammatory activity. Int J Pharm Sci Nanotech 2010;3:986-93.


Contact SPER Publications

SPER Publications and Solutions Pvt. Ltd.

#730, Tower B, i-Thum IT Park,
Sector 62, Noida,
Uttar Pradesh 201301 [Delhi-NCR] India
Phone: +91-930-190-7999 / +91-120-410-0035