Folate-conjugated liposome as effective Drug delivery system for Quercetin

Thi Dac Ngan Thai¹^,², Nguyen Tuong Vi Le¹^,², Van Chinh Nguyen¹, Tong Hung Quach¹^,²,

Cuu Khoa Nguyen¹^,^2*

¹Department of Materials and Pharmaceutical Chemistry, Institute of Applied Materials Science,

Vietnam Academy of Science and Technology, 01 TL29 District 12, Ho Chi Minh City 700000, Vietnam.

²Graduate University of Science and Technology, Vietnam Academy of Science and Technology,

Hanoi 100000, Vietnam.

*Corresponding Author E-mail: nckhoavnn@yahoo.com

ABSTRACT:

Active ingredient (AI), particularly quercetin (Q), has been known as types of nature-derived chemotherapic agents in cancer treatment. However, the advantages of this agent concerning antineoplastic activity were restricted by its poor water solubility. Therefore, the encapsulation of AI in nano-mediated drug delivery is expected to create diverse effects and can sufficiently increase their therapeutic outcomes. The aim of this present study was to effectively prepare folate-conjugated liposome (L-F) that can enhance the delivery of Q. L-F containing Q (Q-L-F) was successfully prepared by thin film technique, using tween 80-ethylenediamine-acid folic as the surface-modified moiety. Physicochemical parameters, including morphology, particles size, zeta potential, drug encapsulation efficiency and release profiles were investigated. In addition, in vitro cytotoxicity of the prepared formulation was evaluated against NCI-H460 cell line. Results showed that the prepared Q-L-F had a mean size of about 166.8 nm with low polydispersity index (below 0.5) and high encapsulation efficiency (96.6%). The release profile showed a sustained release of Q up to 48 h. Moreover, Q-L-F liposomal system was proposed to have the enhanced toxicity effect toward cancerous cells with expressed folate receptors due to the targeting of folic acid conjugated. In support for this, cell proliferation using SRB assay on NCI-H460 cells demonstrated that Q-L-F exhibited higher cytotoxicity than quercetin loaded conventional liposome (Q-L). For the purpose of researching, the data could serve as proof for the potential of L-F as a sustained delivery system for Q in anti-cancer therapy.

KEYWORDS: Biomaterial, Liposome, Folate-conjugated liposome, Drug delivery systems, Quercetin.

INTRODUCTION:

Quercetin, a natural flavonoid present in many edible fruits and vegetables, has been receiving growing attention due to its immense benefits to the human health. Besides the renowned antioxidant effect, quercetin has been found to be anti-carcinogenic, antiviral, antibacterial, anti-inflammatory, etc., and thus need proper and in-depths studies to exploit all the potential benefits of this natural-based drug¹^,²^,³^,⁴. In accordance to the anti-carcinogenic effect of quercetin, promising ideas about the applications of this drug in chemotherapy has also emerged. Ideally, quercetin after injected into circulation will be able to reach the areas with cancerous cells and interact with those cells, thereby eliminating the threats produced by tumours⁵. However, quercetin is poorly soluble, especially in aqueous media like the human serum, the characteristic which derived from the chemical structure of this drug itself. Therefore, it is a quite challenging process to effectively deliver quercetin in human’s circulation because of its poor solubility, instability under physiological conditions, low bioavailability-high accumulation in the spleen and liver, thereby reducing its applicability when used in its normal form⁶^,⁷^,⁸^,⁹^,¹⁰. As the chemical structure of a substance is the main feature to its functions, chemically changing quercetin’s innate structure without alter its function will be less likely to be possible. In the process of solving this problem, drug delivery systems seems to be more rational.

To overcome these issues, folate-conjugated liposome has attracted much attention as an effective carrier for drug delivery. While conventional nanoparticles are designed to reach the tumour site passively by EPR (enhanced permeability retention) effect, those with modified moieties aim to target cancer cells by specific bonding, allows the highly selective release of drug-carrier systems¹¹^,¹². According to the expanding research area on tumour specific receptors, some receptors present uniquely in cancerous cells, while they are absent in their normal counterpart. Chief among the unique receptor is the folate receptor. Folate molecule binds with high affinity to folate receptor and ingests cell interior through endocytic mechanism¹³^,¹⁴. Therefore, the application of folate-conjugated liposomes can reduce side effects by avoid normal growing cells and only release cargo at the targeted site.

In this study, folate-conjugated liposome (L-F) using tween 80-ethylenediamine-acid folic (Tween 80-EDA-FA) has been developed for an effective delivery of quercetin (Q). Conjugation of Tween 80-EDA-FA was carried out in two steps. First, EDA was conjugated with FA (EDA-FA), Tween 80 was conjugated with 4-nitrophenyl chloroformate (Tween 80-NPC), and in second step, EDA-FA was conjugated to Tween 80-NPC. Different analytical tools, included fourier transform infrared (FTIR) and proton nuclear magnetic resonance (¹H-NMR) for chemical structure, scanning electron microscopy (SEM) for particles imaging, and dynamic light scattering (DLS) for size distribution and surface charge were employed. Further, encapsulation efficiency and drug release profiles and in vitro cytotoxicity of these systems were investigated. To all intents and purposes, the aim of this study is to create an effective folate-conjugated liposome for a controlled Q delivery.

MATERIALS AND METHODS:

Materials:

Lecithin was purchased from TCI chemicals (Japan). Cholesterol was obtained from Acros Organic (USA). Quercetin, SRB (Sulforhodamine B), folic acid, ethylenediamine, Tween 80, hexadecyltrimethylammonium bromide (CTAB) and trichloro-acetic acid were purchased from Merck (Germany). Maltodextrin was purchased from Zhucheng Dongxiao Biotechnology (China). NCI-H460 was obtained from ATCC (USA).

Synthesis of Tween 80-EDA-FA:

Briefly, FA was reacted with an excess amount of EDA in 50mL NaOH, and then adjusted to neutral pH. The excess EDA was removed under vacuum to obtain EDA-FA. For the synthesis of Tween 80-EDA-FA, Tween 80 (1mmol) and NPC (3.6mmol) were stirred for 6 h at room temperature. Finally, EDA-FA (1mmol) was added to react for 12 h. Sephadex column was used to obtain Tween 80-EDA-FA. The structure of Tween 80-EDA-FA was conﬁrmed by FTIR spectroscopy (Perkin Elmer) and ¹H-NMR spectra (Bruker Avance 500).

Preparation of Q-L-F:

The thin film hydration method was used to prepare Q-L-F¹⁵. Briefly, lecithin (1.4mmol), cholesterol (0.26 mmol), CTAB (0.025mmol) and quercetin (0,007 mmol) were first dissolved in a mixture of chloroform and methanol (3:1, v/v). This lipid mixture was then placed onto the rotary evaporator under vacuum at 45°C for 2 h to remove all solvents. Tween 80(5%, v/v) in deionized water was added to the dried lipid film for 2 h at 60°C to form the liposomes, and homogenized by sonication for 30min. After that, 5mL of Tween 80-EDA-FA (13.4 ppm) was added and stirred at 60℃ in dark for 2 h¹⁶. Finally, maltodextrin (0.23mmol) was added and filtered via a 0.22µm membrane before storing at 4℃. Besides, Q-L was also prepared as Q-L-F without the addition of Tween 80-EDA-FA.

Characterizations:

The morphology of Q-L-F was observed by SEM (FE SEM Hitachi S4800).

The particle size and surface charge of liposomal formulations were determined by DLS (SZ-100, Horiba).

Encapsulation efficiency (EE)

The Q encapsulation was determined by a UV-Vis at 370 nm. The free Q in the supernatant was defined as the total amount of not encapsulation Q and was removed by centrifugation at 3500rpm for 30min. Total Q concentration was assumed as the added active substance. EE of Q was expressed as the percent of the trapped drug. The %EE equation as follows: %EE = ((total Q – free Q) x 100%)/total Q.

In vitro drug release study:

Q release from Q-L, Q-L-F and free Q into PBS (pH 7.4) was monitored by a dialysis method (MWCO 3500 Da). Each sample (1mL) were first taken into dialysis bags and placed in 20ml of PBS at 37°C under gentle stirring. Dialysis medium (1mL) was withdrawn at given time points and replaced with 1mL fresh PBS. The withdrawn dialysis medium of each samples were assayed for Q content by measuring absorbance at 370nm.

In vitro cytotoxicity study:

The cytotoxic effects of Q-L and Q-L-F against NCI-H460 cells were evaluated by using SRB assay. Approximately 7000 cells (NCI-H460) per well were seeded in the 96-well plates at 37°C overnight and were then treated with Q-L and Q-L-F at 5µg/mL for 2 days.

Figure 2.FT-IR spectra of Tween80-EDA-FA.

CPT (Camptothecin) was used as a positive control and non-treated cells were used as control. Total protein of treated cells was fixed with 50% trichloroacetic acid and stained with 0.2% SRB. The results were read at two wavelengths (492 nm for soluble dye and 620nm for cells) using an ELISA reader. The percentage of growth inhibition (I%) was calculated according to below formula¹⁷: %I = (1- OD_t/OD_c) x 100. OD_c and OD_t were the optical density value of the control sample and the test sample, respectively.

RESULTS AND DISCUSSION:

Synthesis of Tween 80-EDA-FA:

Successful synthesis of Tween 80-EDA-FA was confirmed by ¹H-NMR and FT-IR as show in figure 1, 2. Tween 80-EDA-FA has typical chemical shift protons ¹H-NMR (500 MHz, DMSO-d6) 0.83-0.86 (3H, C1'), 1.23 (14H, C2'), 1.51 (2H, C3'), 1.91-1, 95 (10H, C4', C23), 2.22-2.26 (10H, C4', C24), 6.63-6.65 (6H, C15, C19), 7.59-7.61 (6H, C16, C18). In addition, as shown in the FT-IR spectra of Tween 80-EDA-FA (figure 2), the absorption band of EDA-FA at 3298 cm^-1 (–NH–), and Tween 80 at 2927cm^-1 (CH₃–CH₂–) and 1608 cm^-1 (–CH=CH–) were clearly identified, which proved that Tween 80-EDA-FA was successfully conjugated.

Characterization of Q-L-F:

The mean diameter of Q-L-F was approximately 170.97 ± 3.66 nm with quite narrow size distribution (0.486 ± 0.034) and negative surface charge (-15.30±2.30mV) (figure 3). Compared to other studies with similar interests, the average hydrodynamic size of Q-L-F liposome were consistent with other systems: the range for the size of an average lecithin-based liposome is about 100- 500 nm¹⁸^,¹⁹^,²⁰. According to the previous statement, sizes of lecithin-based liposomes can be varied with the changes of lipid, surfactants, or additives components. Achieving such size of 170 nm in this study (with the folic acid-Tween 80 outer shell included) set up a promising technique and formulation to produce liposome with appropriate sizes for biomedical purposes. Moreover, mean diameter obtained from SEM were in the range of 100–300nm and was close to the size measured by DLS. In fact, nanoparticles with size ranged between these two values are considered as having the most potent for tumor targeting purpose. SEM image also showed that Q-L-F exhibited a uniform spherical appearance and no aggregation. Regarding the physical properties, the liposomal system also experienced appropriate zeta potential, which can facilitate the process of preventing aggregation that commonly observed in nanoparticles due to weak innate forces²¹^,²²^,²³^,²⁴. Q-L-F possesses net negative zeta potential which can overcome those weak forces between the individual particles and prevent those from aggregating.

The EE of Q-L-F (96.6±1.2%) was found to be higher than Q-L (85.4 ± 1.5%). This marked disparity in EE can be explained on the basis of liposomal leakage protection of Tween 80-EDA-FA as a coating agent for liposome modification²⁵^,²⁶^,²⁷^,²⁸^,²⁹. Regarding the result of increased loading capacity of liposomal systems with and without modifiers (Tween 80- EDA-folic acid), it can be stated that the incorporation of these moieties to the liposome significantly increase the encapsulation ability of liposome. This proved that Tween-EDA-FA can be used for both purposes, that is to target tumor using FA, and to increase the loading drug-having the commercial and efficient aspects of a drug delivery system.

In vitro drug release profile:

Q release from Q-L, Q-L-F, and free Q are showed in figure 4. It is clear that Q-L and Q-L-F showed slightly similar Q release profiles, while free Q showed a quick release profile. Free Q showed about 69% release for a period of first 4 h, whereas only 34.6% and 21.7% of Q were released from Q-L and Q-L-F, respectively. For the stage of 22 – 38 h, the speed of Q releasing from Q-L continuously increasing while Q-L-F almost remaining. After 38 h, the release rate of Q in Q-L and Q-L-F exhibited sustained release which the accumulated releases were 61.8% and 40.8%, respectively. Directly compared to other studies, Q-L-F system showed a much slower and sustained releasing profile that is different by many reasons, most important of which can be the modification with Tween 80-folic acid polymer as a modifier. One study with conventional liposome and PEG modified liposome loading with quercetin demonstrated that the drug was release to over 50% content before the first 12 hours³⁰. Other study on folate-liposome load with a hydrophobic drug also suggested a release of 50% after first 24 hours³¹. These two reports were put in comparison with the Q-L-F system to indicate that the formulation used in this study has a significant effect on the release of loaded drug, and can be considered as beneficial for the purpose of drug delivery.

Figure 4. In vitro drug release profile of Q, Q-L and Q-L-F.

Cytotoxicity assay:

SRB assays were performed to evaluate the inhibition of growth of lung cancer cells of the synthesized Tween 80-EDA-FA conjugate for folate-conjugated liposome-based drug delivery systems. As shown in figure 5, folate-conjugated liposome (Q-L-F) showed well inhibition capability compared with conventional liposome (Q-L). The percentage inhibition of growth of lung cancer cells NCI-H460 of Q-L-F was significantly high (29.56%) in comparison with Q-L (8.08%). Folate-conjugated liposome had shown a marginally improved inhibitory potential as compared to untagged delivery system. Therefore, Q-L-F was more potent to improve effectiveness and efficiency of quercetin. According to researches of the same interests, the effects of folic acid on the viability of cells can be confirmed in this study, where the uptake of systems conjugated with folic acid were significantly improved and produced a higher toxicity compare to bare systems (without folic acid)³²^,³³. Based on the theory of enhanced targeting due to the folic acid- folate receptor bonding, the results partly proved that folic acid can produce the targeting ability instead of passively targeting observed in conventional liposome. The overall effect of folic acid as surface modifier on liposomal system can be confirmed in this study and suggested a new direction for developing new strategies for drug delivery systems.

Figure 5. Inhibition of growth of NCI-H460 cells exposed to 5 µg/mL of Q-L, Q-L-F, and CPT.

CONCLUSIONS:

In this study, folate-conjugated liposome based on Tween 80-EDA-FA was successfully synthesized for Q delivery. The physical properties of Q-L-F were spherical shapes, particle size around 170.97 nm, and surface charge around -15.30mV, which would be suitable in vivo drug delivery. The EE calculations, as well as a sustained release profile of around 40% after 48 h, suggest that the prepared folate-conjugated liposome is a promising candidate for a stable delivery system for Q. Moreover, Q-L-F had a higher percentage of cytotoxicity to cancer cells than Q-L. Therefore, the results clearly show that this Q-L-F has potential and is suitable for developing a drug delivery system to improve the therapeutic effect of Q.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENTS:

This research is funded by the Department of Science and Technology of Ho Chi Minh city (DOST) under decision number 1301/QĐ-SKHCN and contract number 123/2019/HĐ-QPTKHCN.

REFERENCES:

1. Bachmetov, L, Gal‐Tanamy, M, Shapira, A, et al. Suppression of hepatitis C virus by the flavonoid quercetin is mediated by inhibition of NS3 protease activity. Journal of viral hepatitis. 2012 Aug 16; 19(2):e81-88.doi: 10.1111/j.1365-2893.2011.01507.x

2. Thapa, M, Kim, Y, Desper, J, et al. Synthesis and antiviral activity of substituted quercetins. Bioorganic & medicinal chemistry letters. 2012 Jan 1; 22(1):353-356.doi: 10.1016/j.bmcl.2011.10.119

3. Mohanty, S, Pal, A, Si, SC. Flavonoid as Nutraceuticals: A Therapeutic approach to Rheumatoid Arthritis. Research Journal of Pharmacy and Technology. 2020 Apr 9; 13(2):991-998.doi: 10.5958/0974-360X.2020.00184.5

4. Saha, S, Roy, A, Bahadur, S, et al. Fabrication and in-vitro evaluation of liposomal quercetin and its optimization. Research Journal of Pharmacy and Technology. 2018 Jun 12; 11(1):61-64.doi: 10.5958/0974-360X.2018.00012.4

5. Choubey, A, Bajpai, P, Jain, S. Nanomedicines based Drug delivery system and their significant role in Herbal Formulations: A Review. Research Journal of Pharmacy and Technology. 2020 Oct 28; 13(10):5034-5039.doi: 10.5958/0974-360X.2020.00881.1

6. Patra, M, Mukherjee, R, Banik, M, et al. Calcium phosphate-quercetin nanocomposite (CPQN): A multi-functional nanoparticle having pH indicating, highly fluorescent and anti-oxidant properties. Colloids and Surfaces B: Biointerfaces. 2017 Jun 1; 154:63-73.doi: 10.1016/j.colsurfb.2017.03.018

7. Le, NTT, Nguyen, DTD, Nguyen, NH, et al. Methoxy polyethylene glycol–cholesterol modified soy lecithin liposomes for poorly water‐soluble anticancer drug delivery. Journal of Applied Polymer Science. 2021 Sep 22; 138(7):49858.doi: 10.1002/app.49858

8. Le, NTT, Vu, MT, Nguyen, NH, et al. Preparation and in vitro evaluation of PEGylated liposomes as effective nanocarrier for delivery of oxaliplatin. Journal of Materials Research. 2021 Jan 14; 36:1-12.doi: 10.1557/s43578-021-00105-9

9. Dai Hai, N. 5-fluorouracil encapsulated CS-mPEG nanogels for controlling drug release. Vietnam Journal of Chemistry. 2017 Aug 31; 55(4):446.doi: 10.15625/2525-2321.2017-00489

10. Hamzah, H, Hertiani, T, Pratiwi, SUT, et al. Efficacy of Quercetin against Polymicrobial Biofilm on Catheters. Research Journal of Pharmacy and Technology. 2020 13(11):5277-5282.doi: 10.5958/0974-360X.2020.00923.3

11. Chauhan, SB, Gupta, V. Recent advances in liposome. Research Journal of Pharmacy and Technology. 2020 Apr 30; 13(4):2053-2058.doi: 10.5958/0974-360X.2020.00369.8

12. Yadav, C, Asif, M, Akhtar, J, et al. An outline on Curcumin: Its pro-liposome and Liposome Formulations. Research Journal of Pharmacy and Technology. 2020 Dec; 13(12):6276-6283.doi: 10.5958/0974-360X.2020.01092.6

13. Scaranti, M, Cojocaru, E, Banerjee, S, et al. Exploiting the folate receptor α in oncology. Nature Reviews Clinical Oncology. 2020 Jun; 17(6):349-359.doi: 10.1038/s41571-020-0339-5

14. Rana, A, Bhatnagar, S. Advancements in folate receptor targeting for anti-cancer therapy: A small molecule-drug conjugate approach. Bioorganic Chemistry. 2021 Jul; 112:104946-104946.doi: 10.1016/j.bioorg.2021.104946

15. Ruozi, B, Tosi, G, Forni, F, et al. Ketorolac tromethamine liposomes: encapsulation and release studies. Journal of liposome research. 2005 Dec 9; 15(3-4):175-185.doi: 10.1080/08982100500364214

16. Liang, X, Fan, J, Zhao, Y, et al. A targeted drug delivery system based on folic acid-functionalized upconversion luminescent nanoparticles. Journal of biomaterials applications. 2017 Mar 28; 31(9):1247-1256.doi: 10.1177/0885328217701289

17. Nguyen, M-NT, Ho-Huynh, T-D. Selective cytotoxicity of a Vietnamese traditional formula, Nam Dia long, against MCF-7 cells by synergistic effects. BMC complementary and alternative medicine. 2016 Jul 16; 16(1):1-10.doi: 10.1186/s12906-016-1212-z

18. Kumar, D, Sakhare, N, Das, S, et al. Development of technetium-99m labeled ultrafine gold nanobioconjugates for targeted imaging of folate receptor positive cancers. Nuclear Medicine Biology. 2021 Feb; 93:1-10.doi: 10.1016/j.nucmedbio.2020.11.001

19. Mutalik, S, Salian, SR, Avadhani, K, et al. Liposome encapsulated soy lecithin and cholesterol can efficiently replace chicken egg yolk in human semen cryopreservation medium. Systems biology in reproductive medicine. 2014 Jun; 60(3):183-188.doi: 10.3109/19396368.2014.902521

20. Yokota, D, Moraes, M, Pinho, S. Characterization of lyophilized liposomes produced with non-purified soy lecithin: a case study of casein hydrolysate microencapsulation. Brazilian Journal of Chemical Engineering. 2012 Jun; 29(2):325-335.doi: 10.1590/S0104-66322012000200013

21. Zhang, W. Nanoparticle aggregation: principles and modeling. Nanomaterial. 2014 811:19-43.doi: 10.1007/978-94-017-8739-0_2

22. Kumar, PR, Lakshmi, AV. An overview on nanobased drug delivery system. Research Journal of Pharmacy and Technology. 2020 Oct; 13(10):4996-5003.doi: 10.5958/0974-360X.2020.00875.6

23. Thomas, JA, Abraham, S, Raju, SP, et al. Formulation and Evaluation of Anti-Arthritis Drug Loaded Stealth Liposomes. Research Journal of Pharmacy and Technology. 2021 14(3):1313-1318.doi: 10.5958/0974-360X.2021.00233.X

24. Joseph, AE, Abraham, S, Raju, SP, et al. Formulation and Evaluation of levofloxacin stealth liposome using different polymers. Research Journal of Pharmacy and Technology. 2021 14(3):1493-1498.doi: 10.5958/0974-360X.2021.00265.1

25. Le, NTT, Nguyen, CK, Nguyen, DH. Carboplatin delivery system based on poly (ethylene glycol) methyl ether–cholesterol modified soy lecithin liposomes. Advances in Natural Sciences: Nanoscience and Nanotechnology. 2020 Nov 30; 11(4):045016.doi: 10.1088/2043-6254/abcaf7

26. Gao, W. Preparation and evaluation of folate receptor mediated targeting liposomes. Liposome-Based Drug Delivery Systems. 2021 Jul 1; doi: 10.1007/978-3-662-49231-4_12-1:167-178.doi: 10.1007/978-3-662-49231-4_12-1

27. Li, Z, Xiong, X, Peng, S, et al. Novel folated pluronic F127 modified liposomes for delivery of curcumin: preparation, release, and cytotoxicity. Journal of microencapsulation. 2020 Feb 17; 37(3):220-229.doi: 10.1080/02652048.2020.1720030

28. Nikam, NR, Patil, PR, Vakhariya, R, et al. Liposomes: A Novel Drug Delivery System: An Overview. Asian Journal of Pharmaceutical Research. 2020 Jun 3; 10(1):23-28.doi: 10.5958/2231-5691.2020.00005.2

29. Mulla, TS, Thorat, MS, Rayate, Y, et al. Liposome as a drug carrier. Asian Journal of Research in Pharmaceutical Science. 2019 Jul 3; 9(2):141-147.doi: 10.5958/2231-5659.2019.00021.3

30. Gang, W, Jie, WJ, Ping, ZL, et al. Liposomal quercetin: evaluating drug delivery in vitro and biodistribution in vivo. Expert opinion on drug delivery. 2012 Jun; 9(6):599-613.doi: 10.1517/17425247.2012.679926

31. Gazzano, E, Rolando, B, Chegaev, K, et al. Folate-targeted liposomal nitrooxy-doxorubicin: An effective tool against P-glycoprotein-positive and folate receptor-positive tumors. Journal of controlled release. 2018 Jan 28; 270:37-52.doi: 10.1016/j.jconrel.2017.11.042

32. Karpuz, M, Silindir-Gunay, M, Kursunel, MA, et al. Design and in vitro evaluation of folate-targeted, co-drug encapsulated theranostic liposomes for non-small cell lung cancer. Journal of Drug Delivery Science Technology. 2020 Jun; 57:101707.doi: 10.1016/j.jddst.2020.101707

33. Tan, S, Wang, G. Lung cancer targeted therapy: folate and transferrin dual targeted, glutathione responsive nanocarriers for the delivery of cisplatin. Biomedicine Pharmacotherapy. 2018 Jun; 102:55-63.doi: 10.1016/j.biopha.2018.03.046

Received on 21.08.2021 Modified on 02.10.2021

Research J. Pharm. and Tech. 2022; 15(6):2741-2746.

DOI: 10.52711/0974-360X.2022.00459