{"paper_id":"06e7a65c-c619-48e5-8650-60f0f95fa46e","body_text":"Sustainable T echnology for the F abrication of Liposomal Phases \nA ysh w arya  Ra v ik u m ar 1 , S w athi  S h a n m ug am 1  a nd  A n i r ba n  P o ll e y 1/i2  \n1 S cho ol of C hem ica l  and Bio tec h nology ,  SA S T RA  Un i v ers i ty ,  T rich y -T an jo r e  Road, Than ja v u r , 6 1 3401, T a mil Nadu , \nIn dia \n*Co rr es p ond ing autho r . E -m ail: an i r b an . p o ll ey@ gm ai l . c o m  \n \nAbstract: \nLiposomes are self-assembled lipid vesicles capable of encapsulating both hydrophili c \nand hydrophobic therapeutics, making them versatile platf orms in drug deliv ery and \nbiomedical technology . In this stud y , the limitations of the classical thin-film hydration \nmethod were criticall y e v aluated, and a sustainable, systematicall y optimized strategy \nwas established for generating defined liposomal lamellar phases. Hy dration conditions  \nwer e optimized, and 4 mL of buffer per 10 mg of lipid w as determined to be optimal f or \neffecti ve r eh ydr ation and improved statistical r eliability of vesicle measur ements. A \nrefined probe-sonication protocol (20% amplitude, 5 s ON/55 s OF F pulse) enabled \ncontrolled transformation of multivesicular v esi cles into stable multilamellar and \nunilam ellar ves icles at net ON-tim es of 90 s and 185 s, respectively , without overheating \nor contamination. In addition, a Python-based machine-learning t ool was de veloped for \nvesicle size charact erization. Collectively , these optimizations provided a r eproducible \nand sustainable framework f or preparing liposomes across different lamellar phases.  \nKeywords: BF , CM, SEM, MVV , ML V , UL V \n1. Introduction \nA liposome is an artificially synthesized, self-assembled lipid v esicle composed of \na single bila yer or multiple concentri c bilay ers enclosing an aqueous core. Alec B angham \nfirst introduced the field of liposomology and char acteri zed liposom e structure in the \nmid-1960s. Since then, liposomes ha ve been extensi vely studied as one of the simplest \nbiomimetic sy st ems, resembling miniature cells without the complexities of nuclei or \ncytoplasm (Sessa and W eissmann, 1968; Bangham et al., 1974; A dler and Schiemann, \n1985; Bibi et al ., 2011; Aranda-Lar a et al., 2020; Andra et al. , 2022) . \nT 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  \nthickness is typically 3–5 nm (Liu et al., 2022). Phosphatid ylcholine (PC) lipids \nconstitute ~40–50%  of cell membranes (van der V een et al., 2017). In this study , \ndim yristoylphosphatidylcholine (DMPC), a commonly used lipid with a low main phase \ntransition temperature (T c ≈ 23 °C), is employed for liposome pr epar ation (Drabik et al., \n2020). Based on lamellarity , liposomes are classified as unilamellar vesicles (UL V s), \nm ultilam ellar  vesicles (M L V s), and multivesicular vesic les (M VV s) ( Giuliano et al., 2021). \nUL V s ma y be further categorized as SUV s (<100 nm), L UV s (100–1000 nm), and GUV s \n( > 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  \net al., 2023 ). \nLiposomes are widel y explor ed in drug deli v ery as they encapsulat e hydrophilic \ndrugs within the aqueous core and  h ydrophobic drugs within the lipid bila y er , thereby \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted May 13, 2026. ; https://doi.org/10.64898/2026.05.09.724055doi: bioRxiv preprint \n\nenhancing stability , biocompatibility , and membrane penetr ation (Gomez and \nHosseinidoust, 2020; Umbarkar et al., 2 021; Mehta et al., 2023). They interact with cells \nthrough endocytosis, e xocytosis, and lipid exchange, and serv e as vaccine ad ju v ants and \ntargeted nanocarriers for proteins, nu cleic acids, imaging agents, and cancer \ntherapeutics (W ang et al., 2019; Zhang and Sun , 2021; Fult on and Najahi-Missaoui, \n2023).  \nV arious macroscale methods—such as thin-film h yd ration, extrusion, re v erse \nevaporation, ethanol injection, electroformation, freeze-drying, and double emulsion—\nare widel y used for liposome pr eparation (Šturm and PoklarUlrih, 2021). How ever , \nthese approaches provide limited control over v esicle size and morphology (Danaei et \nal., 2018 ), whereas biological sy stems r equir e highl y d efined geometries for specialized \nfunc tion (Choi et al., 2023). Moreover , precise control over c argo release remains a \ncritical ch allenge, as sy st emic use of liposomes is often limited by rapid clear ance, \ninstability , and unintended drug r elease (Na gayasu et al., 1999; Kim and Jeong, 2021). \nThe article is structured as follows. First, the methodology employed for \nlipos ome preparation is descr ibed in detail. Next , the main res ults are presented, \nincluding the influence of uniform lipid thin-film formation on v esicle morphology , the \no 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  \ns 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  \nsummarized, and a concluding perspecti ve is provided outlining ke y challenges and \npot ential future dir ections for ad vancing liposome-based systems.  \n2. Methods \n2.1 Liposome Preparation by Thin-Film Hyd ration \nDifferent methods can be employed to prepare various liposomal phases. In the \npresent study , the thin-film h ydration t echnique (Bangham method) was employed f or \nlipid vesicle prepar ation. Dim yristoylphosphatidylcholine (DMPC) was select ed due to \nits low main phase transition t emperatur e (T\nc  = 23 ° C)  (Drabik et al., 2020) . \nLipids were dissol ved in chloroform, follow ed by solvent evapor ation to form  a \nthin lipid  film. Th e dried film was h ydrated with HEPES buffer (Lu and Qi, 2021; \nLombardo and Kiselev , 2022) and mechanically agitat ed to gener ate multivesicular \nvesicles (MVV s). Subsequent probe sonication was performed to downsize MVV s into \nmultilamellar vesicles (ML V s) and u nilamellar v esicles (UL V s). \nV esicle morphology and phase identification wer e carried out using bright-field \nmicroscopy (BF), confocal microscopy (CM), and scanning electron microscopy (SEM) \n(Fig. S1 in S1 of Supplementary Information (SI)) (R obson et al., 2018). These imaging \ntechniques enabled  quantification of vesi cle number , size distribution, lamellarity , and \nstructur al characteristics (Lujan et al ., 2019). \n2.2 Pr eparation of Lipid Thin Film: Influence of Film Uniformity on V esicle Morphology \nThin lipid films w er e pr epared by either air drying or rotary e vaporation to \nevaluate their effect iv eness in producing uniform films. \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted May 13, 2026. ; https://doi.org/10.64898/2026.05.09.724055doi: bioRxiv preprint \n\nF or film preparation, 10 mg of DMPC was d issol v ed in 1 mL chloroform t o ensure \ncomplet e solubilization. When fluorescence microscop y w as perf ormed, 18:1 Liss Rhod \nPE w as added at a concentration of 1 µM during this st ep (Shohda et al., 2015). Other \nlipid-compatible fluorescent dyes wer e selected based on spectral compatibility and \nlipid composition. Glassw are cleaning procedur es and detailed air-drying protocols are \nprovided in S2 and S3 of SI, respecti vel y (Has and Sunthar , 2020). \n2.2.1 Air-Drying Method \nThin films w ere f ormed by e vaporating chloroform under ambient conditions in \ndifferent containers to ev aluate the effect of geometry on film uniformity and vesi cle \nmorphology . Air drying in an Eppendorf tube led to uneven sol vent e vaporation due to \nits cylindrical shape, sharp edges, and limited surface ar ea, causing lipid accumulation at \nthe base and formation of thicker r egions or sediment-like aggr egates, which produced \nheterogeneous vesicle size distribution upon hydr ation (Fig. S2(a) in SI). In contrast, a \nconca v e w atch glass pro vided a larger evaporation surface and yield ed comparati vel y \nmore unif orm vesicle distribution after hydration, despit e slightly thicker lipid \ndeposition t oward the center (Fig. S2(b) in SI). Overall, container geometry influenced \nevaporation rat e, film thickness, and lipid  distribution, and air drying frequently \nresulted in incomplete solvent r emoval and film heterogeneity (Hadian et al ., 2014). \n2.2.2 R otary E vapor ation Method \nF or rotary ev aporation, the lipid–ch loroform solution was transferred to a round-\nbottom flask and e vaporated under r educed pressu re at temperatures above T\nc  u s i n g  a  \ntwo-step protocol. Initially , sol vent removal w as performed at 41 °C and 480 mbar with \nrotation at 60–70 rpm for 15 min t o form a thin lipid film. Subsequently , the \ntemperature was lowered to 36 °C and the pressur e r educed t o 56 mbar while \nmaintaining 70 rpm for an additional 10 min t o enhance drying under higher vacuum \n(Zhu et al., 2013). Alternati vely , residual chloroform was eliminated by brief vacuum \ndesiccation (650 mmHg for 1 min) followed by overnight vacuum. Controlled rotation \nensur ed uniform lipid spr eading along th e flask surface, and reduced pressure \npromot ed efficient sol vent removal. Upon reh ydration, these films produced  vesicles \nwith homogeneous spatial di stribution and narrow size dispersion (Fig. S2(c)), \noutperf orming air-dried films (Fig. S2(a), (b)) by yielding thinner , more uniform films \nwith minimal residual solv ent contamination, ther eby providing superior control over \nfilm quality and v esicle homogeneity . \n \n \n \n \n \n \n \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted May 13, 2026. ; https://doi.org/10.64898/2026.05.09.724055doi: bioRxiv preprint \n\n3. Results and Discussion:  \n3. 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  \nT 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  \nbu 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 \nand  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 \nclosel 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  \n(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 \n1 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  \ncont 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 \ns 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 .  \nT 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  \ns 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  \ne 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 \nd 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\nc  =  \n50  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  \n( 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  \na 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  \no 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 \n5  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  \ni 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  \no 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).  \n \nF 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 ,  \nv 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)  \nPro bability  d is tri b ut io n  of ves i c l e  area, P (A). A cutoff area (A\nc  = 5 0 µ m ²) d i s tingui s h e s overlapped (A > A c ) from  no n -\no 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 \nre 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  \nba rs  deno t e  s tand ard  deviation fro m  20  imag e s. \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted May 13, 2026. ; https://doi.org/10.64898/2026.05.09.724055doi: bioRxiv preprint \n\n \nF 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  \nthe 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, \nwhi 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.  \n3 . 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   \nA 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,  \nSI ) . 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 \nm ethods—ho mogeni zation, sonicat i o n, and e x t r u s ion (Lombar do and K i sele v , 2022 )—\nsoni 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 \naggr 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 \nyi 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  \nand  freq u ency  c o ntr ol ( d e  F r e i tas et al., 201 9;  Morr isse y ,  20 01 ). \nP 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 \nsoni 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 \nh 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 \nand  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  \nt 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  \n(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  \n(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 . \nA  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  \ns 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  \n(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.  \nA 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 ;  \nFig. 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  \ngeom 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 \nyi 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 \n(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  < 1.5 µm ). \nP r o b a b i l i t y  d i s t r i b u t i o n s  ( F i g .  S 5  i n  S 8 , S I )  c o n f i r m e d  M V V  >  M L V  >  U L V , c o n s i s t e n t  w i t h  \nr epr esen tativ e i mages  ( Fi g.  S4  i n S7, SI) .  \nC 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 \nph 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  \nm 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  \nm 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 , \nw 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  \np rodu c t i on of d i sti nct l i p os o mal phase s ( S10  in  SI) .  \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted May 13, 2026. ; https://doi.org/10.64898/2026.05.09.724055doi: bioRxiv preprint \n\nThese findings show that sonication amplitude and duty cycle govern vesicle \nmorphology: high amplitude yields rapid UL V formation but risks thermal damage, \nwhereas low-amplitude pulsed sonication achieves the same outcome more safely over \nlonger times. Therefore, tuning sonication parameters enables controlled and reliable \nproduction of the desired vesicle type  ( S10  in SI) . \n \nF 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 \nsoni 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  \ndet 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  \n(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  \nan 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  \n(gre e n ), M LV  ( gray),  and ULV (blue) p h a se s.  \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted May 13, 2026. ; https://doi.org/10.64898/2026.05.09.724055doi: bioRxiv preprint \n\n \n \n \nF 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 \npro 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) \nre 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 \nsqua 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. \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted May 13, 2026. ; https://doi.org/10.64898/2026.05.09.724055doi: bioRxiv preprint \n\n \n \n4. Conclusion \nA sy st emat ic an d optim iz ed w orkflow f or l iposome p rep ar ation  i s establi s hed , \nint 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 \ndown 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  \ninf 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  \npr 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  \nglas 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  \nfilm 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), \nf 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 \nmbar , 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 \nopt 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 \nmain 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 \nv 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)  \npr 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. \nIn 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 %  \nampl itude f or 30 s  ind uces  he atin g and  m e t alli c con tamin ation. Pul sed soni cation  at  \n30% 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 \neff 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, \ncorr 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 , \nr 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  \nfr 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 , \nr 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  \nduring gold sput t ering.  Qu antit ati v e r obustn es s is enh an ced using a m achin e le arning –\nbased 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  \nr 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  \nr 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 .  \n \n5. Conflict of Interest: \n 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 .  \n6. Acknowledgements: \n 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  \n(ANRF),  Dep ar t ment of Sci enc e  and T ech nology , Go v ernmen t of India [ SER B-\nSR G /2022 /00148 9] an d  T .R.  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