Kaur et al Asian Journal of Dental and Health Sciences. 2021; 1(1):24-32 [24] AJDHS.COM Available online at ajdhs.com Asian Journal of Dental and Health Sciences Open Access to Pharmaceutical and Medical Research Copyright © 2021 The Author(s): This is an open-access article distributed under the terms of the CC BY-NC 4.0 which permits unrestricted use, distribution, and reproduction in any medium for non-commercial use provided the original author and source are credited Development and Evaluation of Aceclofenac Liposomes Gurleen Kaur* , Zaquiyya Naaz, Kapil Kumar , Deepak Teotia Department of Pharmaceutics, Global Institute of Pharmaceutical Education and Research, Kashipur- 244713, Uttarakhand, India Article Info: _________________________________________ Article History: Received 17 Nov 2021 Reviewed 11 Dec 2021 Accepted 18 Dec 2021 Published 25 Dec 2021 _________________________________________ Cite this article as: Kaur G, Naaz Z, Kumar K, Teotia D, Development and Evaluation of Aceclofenac Liposomes, Asian Journal of Dental and Health Sciences. 2021; 1(1):24-32 DOI: http://dx.doi.org/10.22270/ajdhs.v1i1.8 Abstract ___________________________________________________________________________________________________________________ This review gives concise information about the application of dendrimers as drug delivery carrier in the field of drug delivery. Due to their unique architecture these have improved physical and chemical properties. Due to their terminal groups these show high solubility, miscibility and reactivity. Dendrimers have well defined size, shape, molecular weight and monodispersity. These properties make the dendrimers a suitable carrier in drug delivery application. Dendrimers are unimolecular miceller in nature and due to this enhances the solubility of poorly soluble drugs. Their compatibility with DNA, heparin and polyanions make them more versatile. Dendrimers, also referred as modern day polymers, they offer much more good properties than the conventional polymers. Due to their multivalent and mono disperse character dendrimers have stimulated wide interest in the field of chemistry biology, drug delivery, gene therapy and chemotherapy. Self-assembly produces a faster means of generating nanoscopic functional and structural systems. But their actual utility in drug delivery can be assessed only after deep understanding of factors affecting their properties and their behaviour in vivo. Keywords: Dendrimers, Drug targeting, nanoscale carriers. *Address for Correspondence: Gurleen Kaur, Department of Pharmaceutics, Global institute of pharmaceutical and research, Kashipur - 244713, Uttarakhand, India ORCID ID: https://orcid.org/0000-0001-7753-6880 Introduction: The Liposomes are specifically targeted drug delivery system which helps to carry the drug at a specific site and shows its therapeutic effect. Over the past few decades, liposomes have received widespread attention as a carrier system for therapeutically active compounds, due to having a specialized characteristic such as the capability to incorporate both hydrophilic as well as in hydrophobic drugs, low toxicity, good compatibility, lack of immune system activation and targeted delivery of a bioactive compound to the site of action.1 Liposomes are colloidal vesicular structures composed of one and more than one lipid bilayer surrounding an equal number of aqueous compartments. Generally, liposomes are simple, small microscopic vesicle structures that incorporate both the type of drug either it is hydrophilic and lipophilic.2 The main aim of any drug delivery system is to minimize toxicity and increase its effectiveness, safety, target specificity, and target ability at a particular site. The liposomes are so formed to targeting and site-specific delivery of a drug, to increase the circulation, time of drug and release slowly for the extended action of a drug, drug protective from degradative enzymes. The liposomes are directly delivered to the drug at a targeted site of action and provide maximum therapeutic efficacy and help to prevent the drug from any degradation and protect the body from any inappropriate and adverse drug reaction.3,4 Liposomes provide a wide range of attention, it provides a targeted carrier for many drugs such as anticancer, anti- depressant, anti-asthmatic, anti-fungal and also helps to deliver the drug at a targeted site. Due to their phospholipid bilayer structure, liposomes can easily cross the drug from the blood- brain barrier (BBB) in the case of the hydrophilic nature of anti-depressant drugs.5,6 The structure of phospholipid is amphipathic in nature, due to this it helps to incorporate water-soluble drug as well as a lipid-soluble drug as well as lipid-soluble drug. The tail of lipophilic is repelled by the water and the head of hydrophilic is repelled by the lipid.7 Liposomes play a major role in the pharmaceutical industry, cosmetic and dermatologist and carry both hydrophilic and lipophilic drugs and entrap the drug by liposomes and target the drug at a specific-organs. Due to their structure, liposomes also help to prevent the drug from oxidation. Liposome helps to penetrate the dermatological preparation into the deeper skin.8 The objective of this delivery system is to target the drug at specific site during the time period of treatment as to produce the stable, efficacious and safe delivery system of Aceclofenac to overcome complications related to oral route by formulating the liposomes of Aceclofenac for topical use. Aceclofenac used in the pain induced by rheumatoid arthritis and osteoporosis, which reduce the level of PGE2 in synovial fluid & suppresses the production from blood polymorphonuclear leukocytes (mononuclear leukocytes). This delivery system is used in Open Access Research Article DOI: http://dx.doi.org/10.22270/ajdhs.v1i1.8 http://jddtonline.info/ http://dx.doi.org/10.22270/ajdhs.v1i1.8 https://orcid.org/0000-0001-7753-6880 https://orcid.org/0000-0001-7753-6880 https://orcid.org/0000-0003-0481-7109 Kaur et al Asian Journal of Dental and Health Sciences. 2021; 1(1):24-32 [25] AJDHS.COM study because of their specialized characteristics and helps to incorporate the Aceclofenac efficiently and used as topically and ultimately reduce the oral side effects of Aceclofenac.9 Figure 1: Liposome10 Materials and Method: Materials Aceclofenac was received as a gift sample from S. P Pharma, Chandigarh India. Lecithin, Methanol, Cholesterol, Mannitol, and Chloroform were taken from the Global Institute of pharmaceutical education and research institute Kashipur Laboratory. All other materials and chemicals used were of either pharmaceutical or analytical grade. Method: Preparation of topical drug-loaded Aceclofenac liposomes was prepared by the thin-film hydration method. In this method, 3gm of mannitol powder and cholesterol in 3%, 2%, 4%, and 5% were placed in 250ml RBF and held at a temper e f - d f ed eed f f -30 min in a rotatory evaporator.11 Aceclofenac (50mg) and lecithin with a ratio of 0.1:1, 0.1:2, 0.1:3 and 0.1:4 was dissolved in methanol and chloroform in the ratio of 1:4 v/v and add 0.5ml aliquot of the above organic solution were introduced in RBF containing mannitol and cholesterol at 37⸰C. After drying a second aliquot (0.5ml) of the solution was added and then dried, a thin film is formed on the surface of RBF and placed in a desiccator overnight and the sieved with 100 mesh. The Aceclofenac-loaded liposomes were prepared and mentioned as f1, f2, f3, and f4.12 Composition of different formulation: Formulation code Drug (mg) Chloroform (ml) Methanol(ml) Lecithin(gm) Mannitol(gm) Cholesterol (%) F1 50 4 1 1 3 3% F2 50 4 1 2 3 2% F3 50 4 1 3 3 4% F4 50 4 1 4 3 5% Figure 2: Lipid Film Hydration Method13 Pre-formulation studies Pre-formulation study related to drug is necessary to develop the effective and safe dosage form. It is the first step to form any dosage form. It is also helping to shows the compatibility between excipients and drug and also finds out the physical and chemical characteristic. Pre-formulation study is necessary to develop the: -  Safe and effective use of drug.  Compatibility study of drug with different excipients.  To find the release kinetics.14 Physical appearance of drug is also observed, its color, odour, taste. Organoleptic properties: -  Color: - Powder of white crystalline  Odour - Odourless  Taste: - tasteless  Solubility: - Water insoluble, in acetone soluble freely and solubilise in alcohol. Angle of repose It is done to check the flow property of powder. It is range from 0 to 90 degree. In this method, glass funnel is used.15 Tan Ɵ – h/r Whereas, Ɵ =Angle of repose h- heap height r- Heap Radius Kaur et al Asian Journal of Dental and Health Sciences. 2021; 1(1):24-32 [26] AJDHS.COM Relation b/w flowability and angle of repose Angle of repose Flowability <20 Excellence 20 to 30 good 30 to 40 Passable >40 Very poorly Bulk density It depends on size of particle, its shape and adhering tendency. For this, a powder mass is taken in 10ml measuring cylinder. And then, filling was done; cylinders dropped at the surface from one inch height in 2sec interval. The bulk density determination calculated by: - Pb = M/Vb Whereas, Pb- Bulk dens. M- Powder wt. Tapped density In this, sample is taken in measuring cylinder and tapped and then calculated by following method. Pt = M/Vt Whereas, Pt = Tapped density M= powder weight Vt = Tapped density Carr’s Compressibility Index: This method is used for determining weight uniformity. ’ I dex = B de i y – Tapped density/Tapped density ×100 Car’s compressibility Index Percentage Compressibility Description of flow 5 to15 Excellence 12 to 16 good 18 to 21 Fairly 23 to 28 Poorly 28 to 35 Poorly 35 to 38 very poorly Greater than 40 Extreme poorly Solubility analysis The solubility of Aceclofenac was done by using a different solvent. In this method, an amount of solvent taken in a test- tube after that drug also added in it and left overnight for the complete solubilization. On the next, solution was sonicated for some time after that a small amount of solution is pipette out around 0.1ml and further dilutions were prepared by using this. After a several dilutions were prepared, absorbance was determined with different concentration by using UV spectrophotometer with the blank solution. By using the calibration curve, amount of dissolve drug was calculated.16 Determination of melting point In this method, the melting point of Aceclofenac was determined to check the purity of drug. At which temperature, a substance starts melts known as melting point. It is carried out by M.P apparatus, in this drug filled in capillary and one ended of the capillary sealed with the help of flame and attached with thermometer. Note the time at which drug starts melts. Moisture content Determination Formulation was allowed to content of moisture study by Infra-red moisture balance by placing liposomes for 10 minutes in 105⸰C. Calibration curve preparation: - Calibration curve of Aceclofenac prepared in PO43- buffer 6.8. Calibration curve preparation in phosphate buffer 6.8 In this method, 10mg of Aceclofenac powder were dissolved in 10ml of PO43- buffer having 6.8 PH to produce 1000ug/ml. After that several dilutions were prepared by using the above solution having 0.5ml, 1ml, 2ml, 3ml, 4ml was taken and diluted with phosphate buffer 6.8 up to 100ml. The prepared dilutions were analysed by UV- spectrophotometer at 273nm. 17 Compatibility studies of drug- excipients: FTIR study FTIR studies were done for determining compatibility b/w drug and excipients. This study was performed by using the saturated potassium bromide. Drug sample were prepared with KBr pellets i.e., 2mg sample in 200mg KBr with a hydrostatic force for 5.2N cm-2 for 3 minutes. Development of Aceclofenac liposomes by thin film hydration tech. In this, a specified lipid &drug amount were dissolved with chloroform in an RBF. After that, evaporation of solvents takes ce d ce he hi fi by ed ci g he e e. T ce’ solvent was removed by using vacuum by storing the flask overnight and then film was hydrated with phosphate buffer 6.8.18 RESULT: Flow property of Aceclofenac powder S.NO Properties of powder F1 F2 F3 F4 1 Angle of repose 31.5±0.02 31.2±0.10 30.1±0.07 31.4±0.04 2 Bulk density(gm/ml) 0.65±0.05 0.66±0.05 0.68±0.03 0.62±0.01 3 Tapped density 0.72±0.09 0.74±0.11 0.78±0.07 0.73±0.05 4 ’ i dex 9.76±0.06 9.75±0.08 9.72±0.06 9.8±0.06 Kaur et al Asian Journal of Dental and Health Sciences. 2021; 1(1):24-32 [27] AJDHS.COM Organoleptic properties of Aceclofenac S.NO Properties Results 1. Physical appearance White crystalline powder 2. Odor Odorless 3. Taste Tasteless 4. Solubility Practically Insoluble in water, freely soluble in acetone, soluble in alcohol (95%) 5. Melting point 149-153◦C 6 Moisture content 0.6% Solubility: - The solubility study of Aceclofenac performed and result observed in the form of calibration curve. Calibration of Aceclofenac in methanol S.no Concentration(µg/ml) Absorbance(nm) 1. 2 0.049±0.02 2. 4 0.086±0.04 3. 6 0.0139±0.012 4. 8 0.198±0.010 5. 10 0.246±0.14 6. 12 0.298±0.016 Calibration Curve of Aceclofenac in Methanol Compatibility study: - Compatibility study was done to determine the interaction of drug with excipients. The peak of Aceclofenac and peak of Aceclofenac and mannitol is almost similar. So, the sample of Aceclofenac properties matched with the standard value.13 FTIR Spectra of API Aceclofenac FTIR Spectra of Aceclofenac and Mannitol FTIR Spectra Interpretation FUNCTIONAL GROUP (wave number cm) O-H C-H C=O NH2 P=O S-OR Aceclofenac 3399.78 3317.18 2941.64 613.08 1788.78 1718.70 1577.92 1247.99 1146.76 844.85 881.81 Aceclofenac +Mannitol 3276.24 2833.83 3028.41 2994.23 1770.71 1718.70 1579.75 1145.75 896.00 848.71 Calibration curve of aceclofenac in methanol y = 2x - 2 R2 = 1 0 2 4 6 8 10 12 14 16 0 1 2 3 4 5 6 7 8 9 concentration A b s o rb a n c e Kaur et al Asian Journal of Dental and Health Sciences. 2021; 1(1):24-32 [28] AJDHS.COM Characterization of liposomes of Aceclofenac Batch code Yield (%) Entrapment Efficiency (%) F1 56.37±0.003 95±0.05 F2 60.23±0.06 85±0.03 F3 65.46±0.04 89.45±0.07 F4 62.45±0.07 79.15±0.02 Comparison of Entrapment Efficiency of Different Liposomes of Aceclofenac In vitro cumulative percent drug release profile of Aceclofenac of batch F1 to F4 Time(hour) F1 F2 F3 F4 0 0 0 0 0 1 37±0.007 34±0.06 27±0.05 18±0.02 2 48±0.002 37±0.07 32±0.003 22±0.019 3 51±0.03 42±0.05 35±0.01 24±0.002 4 64±0.014 52±0.02 41±0.012 27±0.005 5 67±0.020 58±0.04 46±0.008 46±0.09 Percentage of Drug Released From Liposomes of Aceclofenac of Batch F1 to F4 Release kinetic study Kinetic study of formulation F1 Time (hour) Square root of time Log time Cumulative percent drug release Log Cumulative percent drug release % ARA Log cumulative % drug remaining 1 1 0 37 1.56 63 1.79 2 1.4 0.30 48 1.68 52 1.71 3 1.7 0.47 51 1.70 49 1.69 4 2 0.60 64 1.80 36 1.55 5 2.2 0.69 67 1.82 33 1.51 0 50 100 150 200 F1 F2 F3 F4 % E n tr ap m en t Ef fi ci en cy Batch code % Drug Entrapment Efficiency of liposomes of aceclofenac of Batch F1 to F4 yield % Entrapment Efficiency 0 10 20 30 40 50 60 70 80 0 1 2 3 4 5 6 C U M U LA TI V E % D R U G R EL EA SE TIME IN HOURS IN VITRO CUMULATIVE % DRUG RELEASE OF LIPOSOMES OF ACECLOFENAC OF BATCH F1 TO F4 F1 F2 F3 F4 Kaur et al Asian Journal of Dental and Health Sciences. 2021; 1(1):24-32 [29] AJDHS.COM Kinetic study of formulation F2 Time (hour) Square root of time Log time Cumulative percent drug release Log Cumulative percent drug release % ARA Log cumulative % drug remaining 1 1 0 34 1.53 66 1.81 2 1.4 0.30 37 1.56 63 1.79 3 1.7 0.47 42 1.62 58 1.76 4 2 0.60 52 1.71 48 1.68 5 2.2 0.69 58 1.76 42 1.62 Kinetic study of formulation F3 Time (hour) Square root of time Log time Cumulative percent drug release Log Cumulative percent drug release % ARA Log cumulative % drug remaining 1 1 0 27 1.43 73 1.86 2 1.4 0.30 32 1.50 68 1.83 3 1.7 0.47 35 1.54 65 1.81 4 2 0.60 41 1.61 59 1.77 5 2.2 0.69 46 1.66 54 1.73 Kinetic study of formulation F4 Time (hour) Square root of time Log time Cumulative percent drug release Log Cumulative percent drug release % ARA Log cumulative % drug remaining 1 1 0 18 1.25 82 1.91 2 1.4 0.30 22 1.34 78 1.89 3 1.7 0.47 24 1.38 76 1.88 4 2 0.60 27 1.43 73 1.86 5 2.2 0.69 46 1.66 54 1.73 Drug release kinetic with model fitting Formulation code R2 n value Best fit model Mechanism of release Zero order First order Higuchi matrix F1 0.987 0.9875 0.9676 0.4407 First order Non-Fickian diffusion F2 0.9811 0.888 0.9703 0.3836 First order Non-Fickian diffusion F3 0.9886 0.9301 0.8807 0.3923 First order Non-Fickian diffusion F4 0.9298 0.9341 0.8929 0.5859 Zero order Fickian diffusion Kinetic release model of zero order release 0 10 20 30 40 50 60 70 80 0 1 2 3 4 5 6 % C U M U LA TI V E D R U G R EL EA SE TIME(hr) F1 F2 F3 F4 Kaur et al Asian Journal of Dental and Health Sciences. 2021; 1(1):24-32 [30] AJDHS.COM Kinetic release model of first order release Kinetic release model of Higuchi release Kinetic release model of Korsmeyer peppas release DISCUSSION: Preformulating studies: Preformulating study of Aceclofenac was done by placing the following test: -i.e., melting point, solubility, flow property was done according to I.P. Physical appearance: - The organoleptic properties of liposome were observed by physical and visual method, properties were matched with the standard drug and the prepared Aceclofenac liposomes were sticky in appearance. Solubility: - Aceclofenac is freely soluble in acetone, soluble in alcohol (95%) and practically insoluble in water. Aceclofenac liposome solubility matched with the standard drug. Melting point: - The melting point of Aceclofenac liposomes was found to be 150◦C and the standard range is 149-153◦C. Flow property of Aceclofenac  Angle of repose: - It was found to be 31.5, it indicates the powder is passable.  Bulk density: - It was found to be 0.65 having good flow property.  Tapped density: - It was found to be 0.72. Preparation of calibration curve The calibration curve of Aceclofenac was plotted in phosphate buffer having pH 6.8 and the graph was plotted between concentration (x-axis) and absorbance (y-axis). The results of calibration curve of Aceclofenac were shown in Figure 16. Table 10 shows the absorbance of Aceclofenac standard 0 20 40 60 80 100 0 1 2 3 4 5 6 LO G % A R A TIME(hr) f1 f2 f3 f4 0 20 40 60 80 0 0.5 1 1.5 2 2.5 % C U M U LA TI V E D R U G R EL EA SE SQUARE ROOT OF TIME F1 F2 F3 F4 0 0.5 1 1.5 2 0 0.2 0.4 0.6 0.8 LO G % C R TIME (hr) F1 F2 F3 F4 Kaur et al Asian Journal of Dental and Health Sciences. 2021; 1(1):24-32 [31] AJDHS.COM solution containing 10-50 µg/ml of drug in phosphate buffer pH 6.8. In vitro drug release studies of liposome In vitro- drug release study was carried out in USP XIII dissolution test apparatus type II. In this, a temperature was set at 37◦C ±5◦C and set at 50rpm. phosphate buffer of 1000 ml and set for 12 hours. Release of drug at different time interval has been analyzed by UV spectrophotometer at 274 nm. Compatibility study Compatibility study of drug and excipient was done by FTIR method. The peak of alone Aceclofenac and peak of Aceclofenac with excipients was almost same but a little different due to presence of excipients. There was no appearance or disappearance of peaks found in the drug-lipid mixture which confirms the absence of any chemical interaction between the drug and excipients. Release kinetics: - Drug release kinetic model are used to illustrate the drug release mechanism. For this various model are used like zero order, first order, Higuchi, korsmeyer peppas model to obtain the value of R2 and n-value for the determination of best fit model. R2 value was compared for all the formulation which shows the best fit model and by noticing n value which is from korsmeyer peppas model. Release mechanism was described by an equation.19 Mt/M∞ = n Followed by standard release mechanism N value Release mechanism 0.5 Fickian diffusion 0.5