Sustainable Technology for the Fabrication of Liposomal Phases

Liposomes are self-assembled lipid vesicles capable of encapsulating both hydrophili c and hydrophobic therapeutics, making them versatile platf orms in drug deliv ery and biomedical technology . In this stud y , the limitations of the classical thin-film hydration

were criticall y e v aluated, and a sustainable, systematicall y optimized strategy was established for generating defined liposomal lamellar phases. Hy dration conditions wer e optimized, and 4 mL of buffer per 10 mg of lipid w as determined to be optimal f or effecti ve r eh ydr ation and improved statistical r eliability of vesicle measur ements. A refined probe-sonication protocol (20% amplitude, 5 s ON/55 s OF F pulse) enabled controlled transformation of multivesicular v esi cles into stable multilamellar and unilam ellar ves icles at net ON-tim es of 90 s and 185 s, respectively , without overheating or contamination. In addition, a Python-based machine-learning t ool was de veloped for vesicle size charact erization. Collectively , these optimizations provided a r eproducible and sustainable framework f or preparing liposomes across different lamellar phases.

BF , CM, SEM, MVV , ML V , UL V 1. Introduction A liposome is an artificially synthesized, self-assembled lipid v esicle composed of a single bila yer or multiple concentri c bilay ers enclosing an aqueous core. Alec B angham first introduced the field of liposomology and char acteri zed liposom e structure in the mid-1960s. Since then, liposomes ha ve been extensi vely studied as one of the simplest biomimetic sy st ems, resembling miniature cells without the complexities of nuclei or cytoplasm (Sessa and W eissmann, 1968; Bangham et al., 1974; A dler and Schiemann, 1985; Bibi et al ., 2011; Aranda-Lar a et al., 2020; Andra et al. , 2022) . T h e s i z e o f l i p o s o m e s r a n g e s f r o m ~ 2 0 n m t o s e v e r a l µ m , w h i l e t h e l i p i d b i l a y e r thickness is typically 3–5 nm (Liu et al., 2022). Phosphatid ylcholine (PC) lipids constitute ~40–50% of cell membranes (van der V een et al., 2017). In this study , dim yristoylphosphatidylcholine (DMPC), a commonly used lipid with a low main phase transition temperature (T c ≈ 23 °C), is employed for liposome pr epar ation (Drabik et al., 2020). Based on lamellarity , liposomes are classified as unilamellar vesicles (UL V s), m ultilam ellar vesicles (M L V s), and multivesicular vesic les (M VV s) ( Giuliano et al., 2021). UL V s ma y be further categorized as SUV s ( 1 µ m ) , a l t h o u g h s u c h s i z e d i s t i n c t i o n s a r e n o t t h e f o c u s o f t h e p r e s e n t s t u d y ( N s a i r a t et al., 2023 ). Liposomes are widel y explor ed in drug deli v ery as they encapsulat e hydrophilic drugs within the aqueous core and h ydrophobic drugs within the lipid bila y er , thereby .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted May 13, 2026. ; https://doi.org/10.64898/2026.05.09.724055doi: bioRxiv preprint enhancing stability , biocompatibility , and membrane penetr ation (Gomez and Hosseinidoust, 2020; Umbarkar et al., 2 021; Mehta et al., 2023). They interact with cells through endocytosis, e xocytosis, and lipid exchange, and serv e as vaccine ad ju v ants and targeted nanocarriers for proteins, nu cleic acids, imaging agents, and cancer therapeutics (W ang et al., 2019; Zhang and Sun , 2021; Fult on and Najahi-Missaoui, 2023). V arious macroscale methods—such as thin-film h yd ration, extrusion, re v erse evaporation, ethanol injection, electroformation, freeze-drying, and double emulsion— are widel y used for liposome pr eparation (Šturm and PoklarUlrih, 2021). How ever , these approaches provide limited control over v esicle size and morphology (Danaei et al., 2018 ), whereas biological sy stems r equir e highl y d efined geometries for specialized func tion (Choi et al., 2023). Moreover , precise control over c argo release remains a critical ch allenge, as sy st emic use of liposomes is often limited by rapid clear ance, instability , and unintended drug r elease (Na gayasu et al., 1999; Kim and Jeong, 2021). The article is structured as follows. First, the methodology employed for lipos ome preparation is descr ibed in detail. Next , the main res ults are presented, including the influence of uniform lipid thin-film formation on v esicle morphology , the o p t i m i z a t i o n o f r e h y d r a t i o n b u f f e r v o l u m e , a n d t h e c a l i b r a t i o n o f p u l s e d p r o b e s o n i c a t i o n f o r t h e f o r m a t i o n o f d i s t i n c t l i p o s o m a l p h a s e s . F i n a l l y , t h e f i n d i n g s a r e summarized, and a concluding perspecti ve is provided outlining ke y challenges and pot ential future dir ections for ad vancing liposome-based systems. 2. Methods 2.1 Liposome Preparation by Thin-Film Hyd ration Different methods can be employed to prepare various liposomal phases. In the present study , the thin-film h ydration t echnique (Bangham method) was employed f or lipid vesicle prepar ation. Dim yristoylphosphatidylcholine (DMPC) was select ed due to its low main phase transition t emperatur e (T c = 23 ° C) (Drabik et al., 2020) . Lipids were dissol ved in chloroform, follow ed by solvent evapor ation to form a thin lipid film. Th e dried film was h ydrated with HEPES buffer (Lu and Qi, 2021; Lombardo and Kiselev , 2022) and mechanically agitat ed to gener ate multivesicular vesicles (MVV s). Subsequent probe sonication was performed to downsize MVV s into multilamellar vesicles (ML V s) and u nilamellar v esicles (UL V s). V esicle morphology and phase identification wer e carried out using bright-field microscopy (BF), confocal microscopy (CM), and scanning electron microscopy (SEM) (Fig. S1 in S1 of Supplementary Information (SI)) (R obson et al., 2018). These imaging techniques enabled quantification of vesi cle number , size distribution, lamellarity , and structur al characteristics (Lujan et al ., 2019). 2.2 Pr eparation of Lipid Thin Film: Influence of Film Uniformity on V esicle Morphology Thin lipid films w er e pr epared by either air drying or rotary e vaporation to evaluate their effect iv eness in producing uniform films. .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted May 13, 2026. ; https://doi.org/10.64898/2026.05.09.724055doi: bioRxiv preprint F or film preparation, 10 mg of DMPC was d issol v ed in 1 mL chloroform t o ensure complet e solubilization. When fluorescence microscop y w as perf ormed, 18:1 Liss Rhod PE w as added at a concentration of 1 µM during this st ep (Shohda et al., 2015). Other lipid-compatible fluorescent dyes wer e selected based on spectral compatibility and lipid composition. Glassw are cleaning procedur es and detailed air-drying protocols are provided in S2 and S3 of SI, respecti vel y (Has and Sunthar , 2020). 2.2.1 Air-Drying Method Thin films w ere f ormed by e vaporating chloroform under ambient conditions in different containers to ev aluate the effect of geometry on film uniformity and vesi cle morphology . Air drying in an Eppendorf tube led to uneven sol vent e vaporation due to its cylindrical shape, sharp edges, and limited surface ar ea, causing lipid accumulation at the base and formation of thicker r egions or sediment-like aggr egates, which produced heterogeneous vesicle size distribution upon hydr ation (Fig. S2(a) in SI). In contrast, a conca v e w atch glass pro vided a larger evaporation surface and yield ed comparati vel y more unif orm vesicle distribution after hydration, despit e slightly thicker lipid deposition t oward the center (Fig. S2(b) in SI). Overall, container geometry influenced evaporation rat e, film thickness, and lipid distribution, and air drying frequently resulted in incomplete solvent r emoval and film heterogeneity (Hadian et al ., 2014). 2.2.2 R otary E vapor ation Method F or rotary ev aporation, the lipid–ch loroform solution was transferred to a round- bottom flask and e vaporated under r educed pressu re at temperatures above T c u s i n g a two-step protocol. Initially , sol vent removal w as performed at 41 °C and 480 mbar with rotation at 60–70 rpm for 15 min t o form a thin lipid film. Subsequently , the temperature was lowered to 36 °C and the pressur e r educed t o 56 mbar while maintaining 70 rpm for an additional 10 min t o enhance drying under higher vacuum (Zhu et al., 2013). Alternati vely , residual chloroform was eliminated by brief vacuum desiccation (650 mmHg for 1 min) followed by overnight vacuum. Controlled rotation ensur ed uniform lipid spr eading along th e flask surface, and reduced pressure promot ed efficient sol vent removal. Upon reh ydration, these films produced vesicles with homogeneous spatial di stribution and narrow size dispersion (Fig. S2(c)), outperf orming air-dried films (Fig. S2(a), (b)) by yielding thinner , more uniform films with minimal residual solv ent contamination, ther eby providing superior control over film quality and v esicle homogeneity . .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted May 13, 2026. ; https://doi.org/10.64898/2026.05.09.724055doi: bioRxiv preprint 3. Results and Discussion: 3. 1. O p t i m i za t i o n of Re h yd r a t i on B uf f e r V o l um e T he li pid fi lm s pr ep a red b y a i r d ry ing or r otary e v apor ati on w er e h y d rat ed i n aqueous bu ff er , su ch as p h osphat e- buff er ed sali ne or HEP ES bu ff er ( L u and Qi , 20 21; L ombar do and K i s ele v , 20 2 2) . In t hi s stu d y , HEPE S b uff er , a wi d el y used bi ologi c al buf f er th at closel y mi m ic s p h y s i ologi cal conditi ons (Nadd af D ezful i et al., 201 4 ) , w as em plo y e d (composi t i on det a i led i n S4, SI). Aft er r e h y dr ati on, s amp les w er e i n cu b at ed at 37 °C f or 1 h a n d vo r texe d fo r 1 m i n pr i o r to br i g ht - f i e l d i m a g i ng . Di r e c t vi s ua li z a t i o n wi th cont rol led di l uti on w as adopt ed t o a v o i d spat ial h et er ogenei t y as s o c ia t ed wi t h m a n ual s m e a r in g a n d t o e n s u r e r e p r o d u c ib l e v e s ic l e d is t r ib u t io n . T o c a l i b r a t e v e s i c l e d e n s i t y a n d m i n i m i z e o v e r l a p , t h e r e h y d r a t i o n v o l u m e w a s s y s t e m a t i c a l l y v a r i e d ( 0 . 5 , 2 , 3 , 3 . 5 , 4 , a n d 5 m L f o r 1 0 m g l i p i d ) . V e s i c l e a r e a s w e r e e xtr act ed fr om m i cros cop y i m a g es (F i g. 1 ( a )), and t he p r obabi li t y di str ibution, P( A ), w as d e t e r m i n e d ( F i g . 1 ( b ) ) , a p p r o x i m a t i n g v e s i c l e s a s c i r c u l a r ( A = π r ² ) . A c u t o f f a r e a ( A c = 50 µ m ² ; r c ≈ 4 µ m ) w a s u s e d t o d i s t i n g u i s h o v e r l a p p i n g ( A > A c ) fr om non-o verlapp i ng ( A ≤ A c ) v es icles. T he o v e rlap fr a cti on decr eas ed w i t h i n cr e asi n g v olu me and plat eaue d a t 4 m L (F ig . 1 ( c ) ) , w h e r e a s v e s i cl e c o u n t s in it ia l l y i n c r e a s e d ( 2 m L ) d u e t o r e d u c e d o v e rlap and subs equent l y d ecl i ned at hi g he r v olum es be cau s e of di lu tion ( Fi g. 1 ( d ) ) . A t 5 mL, v es i c le d ens i ty bec ame i nsu ffi cien t f or rel i able i m agi ng . A cc or di n gl y , 4 m L w a s i d enti f ied as th e opt i mal r eh y d r ati o n v ol ume, balanc i ng h igh v e s i c le co un t wi t h mi n i mal o v e rlap f or r obust bri ght- fi eld visua lizat i on and qu anti t ati v e anal y sis (Zhu et al., 2013). F i g u r e 1 : A f t e r r e h y d r a t i o n o f a 1 0 m g D M P C t h i n f i l m w i t h b u f f e r ( 0 . 5 – 5 m L) , s a m p l e s w e r e i n c u b a t e d a t 3 7 ° C f o r 1 h , v or t e x e d f o r 1 m in , a n d i m a ge d by b r i gh t - fi e ld m ic r os c op y . ( a ) V e s i c l e d i s t r i bu t i on s a t d i f f e ren t b u ff e r v ol um e s . ( b) Pro bability d is tri b ut io n of ves i c l e area, P (A). A cutoff area (A c = 5 0 µ m ²) d i s tingui s h e s overlapped (A > A c ) from no n - o v e r la pp e d ( A < A c ) v esicles (grey ) . (c) Pe rcent ag e of ove rl app ed and non-o v erla pp e d ve sicles ve rsu s bu ff e r v o lume, sho w ing re d uced o v erla p wi th in cre a s i ng v olu m e a nd sa tu r ation at 4 mL. (d) T o t al v esicle co u n t a s a fun c t i o n of bu ff e r v o lume. Error ba rs deno t e s tand ard deviation fro m 20 imag e s. .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted May 13, 2026. ; https://doi.org/10.64898/2026.05.09.724055doi: bioRxiv preprint F i gure 2: Schem a tic repres e n t atio n o f th e p u l s ed prob e sonic a t ion, s h ow i ng amp li t ud e , A w ith n u mber of cycle s. (a) shows the pulse d probe sonica ti o n c a rried o ut f o r t ot al 9 cy cles (1 0 se co nd ON an d 50 sec o nd OF F p e r cy cle ) f or 30% a m plit u de, whi l e (b) t h e same f or tot al 37 cyc l e s ( 5 second ON and 55 s econd OF F p e r cycle ) f or 2 0 % amp l it ud e. 3 . 2 . Optimi z ati on of Pr obe Soni catio n t o Obtai n Di ff er ent l i posomal Ph a s es A f t er r eh y d r ati on an d v ort e xi ng (1 m i n), MVV s w er e f ormed (Fi g. 1 ( a ); Fig. S4( a) in S7, SI ) . V esicle downs i z i ng w as perf o rmed t o ob t ai n ML V s and UL V s. Among a v ai l able m ethods—ho mogeni zation, sonicat i o n, and e x t r u s ion (Lombar do and K i sele v , 2022 )— soni cat i on i s p r ef e rr ed f or eff icient s ize contr ol vi a acou stic d i s ruption of li p i d aggr eg at es ( M au luc ci et al., 2 005 ) . B ath s on i cat i on (90 min; S5, SI ) pr ed omi nan tl y yi el d ed ML V s w i th r e si du al MVV s (Fi g. S3 , S5, SI), consist ent wi th li mit ati ons i n pow er and freq u ency c o ntr ol ( d e F r e i tas et al., 201 9; Morr isse y , 20 01 ). P robe sonicati o n (130 W , 20 kHz, 6 m m prob e) w as the r ef or e em plo y ed . C onti n uous soni cat i on at 30 % am pli tu de f or 3 0 s (Sha rma et al., 202 3 ; Zen g et al . , 2 023) i n duc ed h e at i ng and m etalli c c on tami n ati on (F i g. S4(b) i n S7, SI ) . I c e- bath co oli ng ( Lo mbar do and Ki sel e v , 2022 ) ris k ed ph as e perturbati o n d ue t o thermal c y cling. T o min i mi z e t her m al and cont ami nat i on ef f ec t s, p ulsed pr ot ocols w er e opti m i zed . A t 3 0% ampli t ude (10 s ON / 5 0 s O FF) , UL V s appe ar ed at net O N-t i mes ≥6 0 s and w er e e vident at 90 s (Fi g . 2 (a); Fi g. S6 i n S9, SI) , thou gh h eati ng r emai n ed si gn i f i c ant . A low er ampli t ude (2 0% ) wi t h 5 s O N / 55 s OFF w a s then s y st em ati c all y v aried ( 5– 1 85 s net ON-t i me; F i g. 2(b) ) . M L V s e merged at 90 s ( 18t h c y cl e) and U L V s at 160– 18 5 s (32n d– 37t h c y c l es ). Th e ext ende d OFF pe r iod l i m i t ed h e ati ng an d pr obe e r osi on. A ppr o xi mat el y 20 i mag es pe r c y c l e w er e anal yz ed us i ng a cu st om Pytho n w orkflow (S6 ; Fig. S7 i n S9, SI). V esicle ar e a (A) and rad i us (R) w er e e xtr act ed a s sum i ng circ ular geom etry (A = π R²; R = √(A/π ) ) . G a u s sian fi t s t o P( R ) e x c l ud i ng ra r e lon g-tai l e v ents yi el d ed m ean r ad i i w ith e rr or ( Fig . 5c– d ) . The e v olu ti on of m ean r ad ius w i th ON-t i me (Fi g . 5e ) clas si fied ph as e s a s M VV ( R > 4 µm), M L V ( R ≈ 1.5– 4 µm ) , and UL V ( R M L V > U L V , c o n s i s t e n t w i t h r epr esen tativ e i mages ( Fi g. S4 i n S7, SI) . C onfocal m i c roscop y (F i g. 4 (a – d) ; S1.2 i n SI) r e v eal ed lamell ar stru ctu re at 90 s (ML V ph a s e) , whi le S E M a t 18 5 s ( F i g. 4 ( e– f ) ; S 1 . 3 i n S I ) sh o we d s phe r i c a l U L V s wi t h s l i g ht m embr ane und ulati on . O v e r all, s o n i cati on am pli tu de and du ty c y cl e go v er n v esicle m or p hology: hi gh er ampli t ude acc eler at es UL V f ormati o n b ut i ncrea ses t h ermal r i sk , w h er eas low- ampli t ude pu lsed ope r ati on en ables contr olled , c on tami n ati on- mi nimi zed p rodu c t i on of d i sti nct l i p os o mal phase s ( S10 in SI) . .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted May 13, 2026. ; https://doi.org/10.64898/2026.05.09.724055doi: bioRxiv preprint These findings show that sonication amplitude and duty cycle govern vesicle morphology: high amplitude yields rapid UL V formation but risks thermal damage, whereas low-amplitude pulsed sonication achieves the same outcome more safely over longer times. Therefore, tuning sonication parameters enables controlled and reliable production of the desired vesicle type ( S10 in SI) . F i gure 3 : Chara ct e ri z a t ion of ve sicles. (a) Brig h t -field i m a ge a t a n ON-t i m e of 1 8 5 s ( 3 7t h c yc l e) duri ng p u lsed p r o be soni c ati o n a t 2 0 % a m plitude. (b ) V esicle i den t i f ic ati o n usi n g a c u s t om Pytho n-b ased machi ne learni ng w o rkflo w (c ont o ur det e ctio n). (c ) His t og ram of ves i c l e ra dii ( R ). (d) No rm a liz ed p robabili ty d is trib u t io n , P ( R ), fitt ed w ith a G a ussian f u nct ion (e x clu di n g ra re long -t ai l e ve n ts) t o o b t ain the m ean ra di us wit h error . A ppr oximat ely 2 0 images ( ~40 0 0 ves i c l e s) were an alyz e d per time poin t. (e ) E voluti o n of me a n ve s i cle ra dius wi th ON-time at 3 0 % and 2 0 % ampl i t ud e s, h ighl i g h t ing MVV (gre e n ), M LV ( gray), and ULV (blue) p h a se s. .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted May 13, 2026. ; https://doi.org/10.64898/2026.05.09.724055doi: bioRxiv preprint F i gure 4: C on f oc a l an d SEM chara ct e ri z a t ion of l iposomes . (a,b ) C o nf o c a l imag es a t t he 37th and 18t h c ycles o f pulsed pro be son i c ati o n (20% a m p li tu de; 5 s ON / 55 s OF F). ( c , d) Three - dim e n sion al Z -s t a ck a nd m a g n ifi ed view (do tt ed squ are) re v eali ng lamella r s truct ure in dic a t ive o f th e M LV p ha s e. (e) S EM imag e at th e 37th cy cle. ( f ) M a gni fied v i e w (b lack dott ed squa re ) sho w i n g sp h e ri c a l ULV s wi th sli g h t m embran e un d u la tion. .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted May 13, 2026. ; https://doi.org/10.64898/2026.05.09.724055doi: bioRxiv preprint 4. Conclusion A sy st emat ic an d optim iz ed w orkflow f or l iposome p rep ar ation i s establi s hed , int egrati ng thin -film f or m ation, contr olled d r y in g, cal ibr at ed r e h y dr at ion, v esi c l e down s i zin g, and ad v an ced ima ge a nal y si s (F ig. S8 in SI). Cont ainer ge ometry m ark edl y inf luenc es film uni f ormity and v esi cle m orphology: s h a r p- edg ed E ppend or f tu bes pr oduce h et er ogeneous film s and br oad v esi c l e di stribu tions, w he re as cu rv ed w at ch glas s es y ield com parat i v el y unif o rm f ilms; th e most hom ogeneous and com plet el y dr ied film s ar e obtain ed b y ro tary e v apo r ati on at 41 °C and 4 80 mbar f or 15 min ( 60 –70 rpm), f ollow ed b y 36 ° C an d 56 m bar f or 10 m in (70 rpm) and o v e rnig ht de s ic c at ion at 866 .6 mbar , w hil e ai r d ry ing oft en r esult s in s t i c k y , i n complet el y d ri ed f ilms. R eh y dr at ion i s opt imized at 4 mL HEP ES buff er per 1 0 mg l ipid , min im iz ing v e si c le o v e rlap w hil e main tainin g suff ic ien t conc ent rat ion; low er v olum es inc re ase o v e rlap , w her ea s h i gh er v olum es r ed u ce v e si cle den sit y . F or MV V d ownsiz ing, b ath s o nic ation ( u p t o 90 mi n) pr edomi nantl y yi elds ML V s withou t cont aminat ion b ut requ ir es p rolon ged pr oc es s in g. In con tr as t, p rob e s on i c at ion enab l es r apid s iz e r ed uction; con tinuo us o perat ion at 3 0 % ampl itude f or 30 s ind uces he atin g and m e t alli c con tamin ation. Pul sed soni cation at 30% ampl itude (10 s ON/5 0 s O FF , 9 c y cl es ) r ed uc es cont aminat ion bu t ret ains the rmal eff ects, w her ea s fu r t he r o ptim iz a t ion t o 20% amplit ude (5 s ON /55 s OFF ) f or 37 cy cle s, corr espond ing t o net O N tim es of 9 0 s an d 185 s, r epr odu c ib l y ge n e rat es ML V s and UL V s , r es p ect i v e l y , withou t det ectable he ati ng o r pr obe-der i v ed p arti cles ; r esid ual tit anium fr agment s a r e r emo v ed b y cent r i fu g ation at 10 ,000 × g f or 3 mi n. F or SEM pr epar ation , r emo v al of e x c es s buf f er com ponent s , in c lu ding su cr ose, i s essent ial t o p re v ent c h arging during gold sput t ering. Qu antit ati v e r obustn es s is enh an ced using a m achin e le arning – based P ython image a n al y s i s w o r k f low . Collecti v el y , th e opt imiz ed pr ot ocol pr o vides a r epro du cible, con taminat ion- mini m ized , and th er m all y cont rolled str at egy f or pr e ci s e r egul ation o f liposome ph ase beh a v i or , v esicl e s i ze d istr ibuti on, and str uctur al int egrity . 5. Conflict of Interest: Th e au thors de c l ar e that the r e ar e n o confl ic t s o f int er e st . 6. Acknowledgements: A.P . a cknow ledg es th e s u pport un d er A nusandh an N at ional R e s e ar ch F ound ation (ANRF), Dep ar t ment of Sci enc e and T ech nology , Go v ernmen t of India [ SER B- SR G /2022 /00148 9] an d T .R. R ajag opalan r es ear ch fu nd, S A S TRA Uni v er s ity , In dia . 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