Nanotechnology in Bladder Cancer: Diagnosis and Treatment

cance s Re iew Nano echnology in Bladde Cance : Diagnosis and T ea men Mahmood Ba ani 1, Seyedeh Ma yam Hosseinikhah 2, Abbas Rahda 3,* , Leila Fa houdi 2, Rabia A shad 4, Magali Cucchia ini 5,* and Sadanand Pandey 6,7,*   Ci a ion: Ba ani, M.; Hosseinikhah, S.M.; Rahda , A.; Fa houdi, L.; A shad, R.; Cucchia ini, M.; Pandey, S. Nano echnology in Bladde Cance : Diagnosis and T ea men . Cance s 2021,13, 2214. h ps://doi.o g/ 10.3390/cance s13092214 Academic Edi o : Lukas Lusua di Recei ed: 31 Ma ch 2021 Accep ed: 4 May 2021 Published: 5 May 2021 Publishe ’s No e: MDPI s ays neu al wi h ega d o ju isdic ional claims in published maps and ins i u ional a il- ia ions. Copy igh : © 2021 by he au ho s. Licensee MDPI, Basel, Swi ze land. This a icle is an open access a icle dis ibu ed unde he e ms and condi ions o he C ea i e Commons A ibu ion (CC BY) license (h ps:// c ea i ecommons.o g/licenses/by/ 4.0/). 1Depa men o Chemis y, Shahid Bahona Uni e si y o Ke man, Ke man 76169-14111, I an; [email p o ec ed] 2Nano echnology Resea ch Cen e , Pha maceu ical Technology Ins i u e, Mashhad Uni e si y o Medical Sciences, Mashhad 91886-17871, I an; [email p o ec ed] (S.M.H.); [email p o ec ed] (L.F.) 3Depa men o Physics, Facul y o Science, Uni e si y o Zabol, Zabol 98613-35856, I an 4 Depa men o Pha macy, Quaid-I-Azam Uni e si y, Islamabad 45320, Pakis an; [email p o ec ed] 5Cen e o Expe imen al O hopaedics, Saa land Uni e si y Medical Cen e , 66421 Hombu g/Saa , Ge many 6Depa men o Chemis y, College o Na u al Science, Yeungnam Uni e si y, 280 Daehak-Ro, Gyeongsan 38541, Ko ea 7Pa icula e Ma e Resea ch Cen e , Resea ch Ins i u e o Indus ial Science & Technology (RIST), 187-12, Geumho- o, Gwangyang-si 57801, Ko ea *Co espondence: [email p o ec ed] (A.R.); [email p o ec ed] (M.C.); [email p o ec ed] o [email p o ec ed] (S.P.) Simple Summa y: Bladde cance (BC) is he ou h mos common cance among men and he en h mos common cance among women. Since he o e all p ognosis o BC has no changed in he las 30 yea s, he e is a compelling medical need o de elop new diagnos ic and he apeu ic app oaches. Nano echnology has been ex ensi ely de eloped o cance managemen , including cance diagnosis, de ec ion, and ea men . Se e al nanopa icles (NP) can be used in in i o cance diagnos ics, in i o imaging enhancemen , and d ug loading echniques. In his e iew, we examine he cu en s a e o nano echnology in he diagnosis and ea men o bladde cance . We in es iga ed he unc ion o me al NPs, polyme ic NPs, liposomes, and exosomes accompanied he apeu ic agen s o BC he apy, and hen ocused on he po en ial o nano echnology o imp o e con en ional app oaches in sensing. Abs ac : Bladde cance (BC) is he second mos common cance o he u ina y ac in men and he ou h mos common cance in women, and i s incidence ises wi h age. The e a e many con en ional me hods o diagnosis and ea men o BC. The e a e some cu en bioma ke s and clinical es s o he diagnosis and ea men o BC. Fo example, adio he apy combined wi h chemo he apy and su gical, bu esidual umo cells mos ly cause umo ecu ence. In addi ion, chemo he apy a e ansu e h al esec ion causes high side e ec s, and lack o selec i i y, and low sensi i i y in sensing. The e o e, i is essen ial o imp o e new p ocedu es o he diagnosis and ea men o BC. Nano echnology has ecen ly spa ked an in e es in a a ie y o a eas, including medicine, chemis y, physics, and biology. Nanopa icles (NP) ha e been used in umo he apies as app op ia e ools o enhancing d ug deli e y e icacy and enabling he apeu ic pe o mance. I is no ewo hy, nanoma e ial could be educed he limi a ion o con en ional cance diagnosis and ea men s. Since, he majo disad an ages o he apeu ic d ugs a e hei insolubili y in an aqueous sol en , o ins ance, pacli axel (PTX) is one o he impo an he apeu ic agen s u ilized o ea ing BC, due o i s abili y o p e en cance cell g ow h. Howe e , i s majo p oblem is he poo solubili y, which has con i med o be a challenge when imp o ing s able o mula ions o BC ea men . In o de o educe his challenge, an i-cance d ugs can be loaded in o NPs ha can imp o e wa e solubili y. In ou e iew, we s a e se e al nanosys em, which can e ec i e and use ul o he diagnosis, ea men o BC. We in es iga e he unc ion o me al NPs, polyme ic NPs, liposomes, and exosomes accompanied he apeu ic agen s o BC The apy, and hen ocused on he po en ial o nano echnology o imp o e con en ional app oaches in sensing. Cance s 2021,13, 2214. h ps://doi.o g/10.3390/cance s13092214 h ps://www.mdpi.com/jou nal/cance s Cance s 2021,13, 2214 2 o 29 Keywo ds: bladde cance ; nanopa icles; he apy; diagnosis 1. In oduc ion The bladde is an essen ial o gan o he u ina y sys em ha ing majo oles in em- po a y u ine s o age ia immense olded in e nal linings, as well as i s expulsion due o bladde muscula u e con ac ions and elaxa ions [ 1 , 2 ]. The bladde is ea u ed wi h he apex a he uppe loca ion, main body, iangula -shaped pos e io undus, as well as neck o con e gence o undus [ 3 ]. BC is he common cance o he u ina y ac , o igina ing inside he linings o he bladde consis ing o u o helial cells [ 4 ]. U o helial cells a e he sou ce o connec ion be ween he kidney and bladde [ 5 ]. BC is he ou h mos common cance in men and en h in women causing 15,000 dea hs annually in he USA [ 6 ]. BC includes a a ie y o o ms, depending on how i de elops in he bladde ’s speci ic cells, i.e., u o helial ca cinoma, squamous cell ca cinoma, and e y a e adenoca cinoma in mucus-sec e ing cells o he bladde [ 7 ]. As a as symp oms o BC a e conce ned, i is o en accompanied by hema u ia, equen pain ul u ina ion, and pel ic pain [ 8 ]. The mechanism besides he p opaga ion o BC lies in he esis an mu a ions in he umo sup- p esso genes [ 9 ]. Howe e , o he isk ac o s include smoking, old age, ch onic bladde in lamma ion, inhe i ance his o y, and exposu e o ce ain chemicals and dyes [ 4 , 10 – 13 ]. As a as a gene alized diagnosis o he BC is conce ned, i can be ini ially de e mined h ough cys oscopy by inse ing a cys oscope ube ha ing a lens in o he u e h a o exam- ine s uc u al changes. Cys oscopy can be modi ied ia a aching a specialized ool o collec ing a sample o biopsy de e mina ion. Fu he mo e, he u ine sample can also be op ed o collec o analysis o he cance cells p esence [ 14 , 15 ]. A e he con i ma ion o he p esence o aces o cance cells in u ine con i ming BC. Fu he p eceded diagnos ics can also be pe o med o examine he se e i y, and i includes CT scan, magne ic esonance imaging (MRI), posi on emission omog aphy (PET), bone scan, and ches X- ay. The diag- nosis o BC is challenging owing o he as exis ence o gaps accompanying o e - es ing, o e -diagnosis, o e - ea men , non-speci ici y, and he e ogeneous na u e o malignan BC cells [ 16 ]. Howe e , ea men is a ied acco ding o he ype and se e i y based on low g ade (benign) and high g ade (me as a ic) BC [ 8 , 17 ]. The e o e, he op ed me hods o BC he apy include su ge y o emo ing cance cells di ec ly ia ansu e h al esec ion o bladde umo (TURBT), cys ec omy, neobladde econs uc ion, and ileal condui [18,19]. Chemo he apy o BC includes he use o combina o ial chemo he apeu ic agen s in he o m o gemci abine and cispla in (GC), cispla in, me ho exa e, and inblas ine (CMV), and gemci abine and pacli axel (GP) di ec ly in he bladde ia in a esical chemo he apy o sys emic chemo he apy [ 17 , 20 , 21 ]. Radia ion he apy uses beams o powe ul ene gy, such as X- ays and p o ons, o des oy he cance cells [ 22 ]. Immuno he apy is he mos used ea men p o ocol o BC ia in a esical and in a enous me hods [ 23 ]. In a esical immuno he apy can be done using he bacillus Calme e-Gue in (BCG) accine o media e he immune sys em eac ion ha di ec s ge m- igh ing cells o he bladde [ 24 ]. Howe e , in- a enous immuno he apy can be pe o med h ough a ious immuno he apy d ugs [ 25 ]. Ne e heless, all hese modali ies me hods a e associa ed wi h an inc eased economic bu den, non-pa ien compliance, and need o a ge ed deli e y, and mos impo an ly wi h he in ol emen o i ulen ac o s in umo supp esso genes [ 26 ]. Chemo he a- peu ics agen s u ilized in chemo he apy a e speci ically associa ed wi h damaging cells in he bone ma ow, in es inal mucosal linings and hai ollicles, as well as de eloping se e e in ec ion and a igue due o he deple ion o leukocy es and ed blood cells [ 27 ]. In e ms o a gene alized diagnosis o BC, i can be de e mined i s by cys oscopy, which in ol es inse ing a cys oscope ube wi h a lens in o he u e h a o in es iga e s uc u al changes [ 28 ]. Cys oscopy can be modi ied ia a aching specialized ool o collec ing sam- ple o biopsy de e mina ion [ 29 , 30 ]. Fu he mo e, u ine sample can also be op ed o collec o analysis o he cance cells p esence. A e he con i ma ion o he p esence o aces o Cance s 2021,13, 2214 3 o 29 cance cells in u ine con i ming BC, u he p eceded diagnos ics can also be pe o med o examine he se e i y and i includes CT scan, magne ic esonance imaging (MRI), posi on emission omog aphy (PET), bone scan and ches X- ay [ 31 – 33 ]. The diagnosis o BC is challenging owing o he as exis ence o gaps accompanying o e - es ing, o e -diagnosis, o e - ea men , non-speci ici y, and he e ogeneous na u e o malignan BC cells [ 34 ]. To o e come he limi a ions o con en ional diagnos ic me hods o BC, a signi ican numbe o nano echnology-based bioassays a e highly encou aged [ 35 ]. In his ega d, luo escen cys oscopy has been de eloped using 5 -aminole ulinic acid (5-ALA) phos osensi ize ollowed by in a esical adminis a ion, and ha e he capabili y o p o icien abso p ion o cance cells by showing in ense ed colo compa ed o su ounding no mal issues [ 36 ]. Simila ly, ligand media ed app oach-based nano-senso s a e also o g ea in e es as hey can be de eloped by conjuga ing BC speci ied amino acid-based ligand PLZ4. PLZ4 unc- ionalized nanomicelles p e e en ially enhance he up ake o only cance cells unde he mechanis ic o pho odynamic diagnosis [ 37 ]. Role o gold nanopa icles (GNP) in he diagnosis o BCs by inducing plasmon esonance is i eplaceable and esul ing in colo s o isible di e ence o be obse ed by naked eyes [ 38 ]. Ca ionic GNPs can be u ilized wi h biocompa ible anionic hyalu onic acid (HA) o p oduce isible blue colo change [ 39 ]. Ul a-small pa icles o i on oxide (USPIO) anging om 30 o 50 nm in diame e ha e he capabili y o up ake by e iculoendo helial sys em o ul a-sensi i e diagnosis [40]. Nano echnology ad en s he ield o an i-cance modali ies ia imp o ing he d ug loading by deco a ing he su ace o nanopa icles wi h a ge ed ecep o ligands, highly exp essed on umo su aces [ 41 – 48 ]. Nanoma e ials bypass he side e ec s o con en- ional he apy by imp o ing he speci ici y and pha macokine ics o an i-cance d ugs [ 49 – 52 ]. An i-cance NPs o ea ing BC a e GNPs and hey modi ied he acids and p o ein molecules o acili a ing he apid killing o cance cells [ 53 ]. GNPs help in p o iding s abil- i y, as well as a s ong a ini y o a achmen o ligands a ge ing BC [ 54 ]. Mos e ec i ely u ilized nanoca ie s in he ea men o BC include, polyme ic nanopa icles because hei syn hesis is easy and cos -e ec i e, p o ides supe io iabili y and biodeg ada ion [ 55 ]. Polyme ic nanopa icles u ilized in he ea men o BC a e a ailable in a wide ange o na - u al and syn he ic polyme s, cons i u ing mac omolecules poly (lac ide-coglycolide), poly (lac ic acid), poly (cap olac one), and chi osan [ 55 – 62 ]. Simila ly, he lipid-based nanopa i- cles a e also impa ing hei ole in ea ing BC by adap ing o mula ion s a egic o ms o solid lipid nanopa icles (SLNs) and nanos uc u ed lipid ca ie s (NLC) composed o phospholipids [ 63 ]. Lipid-based nanopa icles a e highly ad an ageous in encapsula ing an i-cance d ugs and hei si e-speci ic loading ia o e coming hei solubili y issues [ 64 ]. Mo eo e , p o ein nanopa icles help in acili a ing he a ge ed and con olled elease o o al deli e y using p o eins such as albumin, gela in, gliadin, and legumin [65]. 2. Diagnosis o Bladde Cance 2.1. Cu en Diagnosis App oaches o Bladde Cance Cu en ly, di e en app oaches such as cys oscopy, biopsy, u ina y cy ology, and imaging p ocedu es a e used o de ec BC [ 66 ]. A hin, na ow ube (cys oscope) is placed h ough he u e h a o pe o m cys oscopy. The cys oscope has a lens ha helps o inspec inside o he u e h a and bladde o signs o disease [ 67 ]. U ine cy ology is a p ocess in which a sample o u ine is examined unde a mic oscope o moni o o cance p ob- lems [ 68 ]. Finally, imaging examina ions such as compu e ized omog aphy (CT) u og am, magne ic esonance imaging (MRI), posi on emission omog aphy (PET), bone scan, and ches X- ay can help wi h ea ly diagnosis and in luence ca e decisions [69]. T adi ional me hods ha e limi ed sensi i i y and capaci y o p o ide accu a e and speci ic in o ma ion on he condi ion, and hey ely g ea ly on he size shi o lymph nodes and he p esence o ana omic anomalies, which is ypically he p ima y c i e ia used o de e mine he diagnosis [ 29 , 70 ]. Nanopa icles (NP) wi h a nanome ic scale a e no el ma e ials ha we e i s used o de elop scanning p obe mic oscopy and disco e molecula s uc u es, and since been used in a a ie y o diagnos ic applica ions [71]. Cance s 2021,13, 2214 4 o 29 2.2. Cu en Bioma ke s and Tes s in Bladde Cance Diagnosis The iden i ica ion o bioma ke s in u ine, issue, and blood has been sugges ed as impo an componen s o p ecision medicine o add ess exis ing sho comings in he diag- nosis, ea men , and ollow-up o BC. These mode n molecula es s can aid in de ec ing disease ea lie , isk-s a i ying pa ien s, imp o ing oncological ou come p edic ion, and op imizing a ge he apies. We looked a he exis ing s a e o he a and he success o he mos p omising and accessible bioma ke s. BTA s a ® , BTA TRAK ® , NMP22, and U oVysionTM a e he only u ina y diagnosis sys ems app o ed by he Food and D ug Ad- minis a ion (FDA) o BC diagnosis and ollow-up. Meanwhile, ImmunoCy TM/uCy +TM is only app o ed o BC ollow-up [ 72 ]. Telome ase, nuclea ma ix p o ein 22 (NMP22), cy oke a in 19, su i in, hyalu onidase (HAase), apolipop o ein A1 (ApoA1), miRNA-21, and galec in-1 p o ein, on he o he hand, a e e ec i e bioma ke s in he diagnosis o BC [73,74]. Despi e some p omising indings, pa icula ly in e ms o imp o ed sensi i i y, when compa ed o u ina y cy ology in he diagnos ic en i onmen . These molecules ha e ye o be inco po a ed in o ou ine clinical p ac ice, owing o he di icul y in deciding he equi ed scena io o use and he lack o high-quali y p ospec i e ials. Tha esul ing in a low le el o p oo . To de ine he posi ion o hese p omising bioma ke s, mo e p ospec i e esea ch and b oad in e na ional collabo a ions a e needed [ 75 ]. Nano echnology has been ex ensi ely de eloped o cance managemen , including cance diagnosis, iden i ica ion, and ea men . Nanopa icles o cance ea men ha e a ple ho a o po en ial uses, bu mos a e s ill in he p eclinical s age. GNPs, o example, would be used in cance sc eening in i o s udies, in i o imaging imp o emen , and as po en ial d ug loading s a egies. Aside om ha , di e en p epa a ion me hods al e pa icle sizes, shapes, and s uc u es, educing hei clinical u ili y [76–79]. 2.3. Nanoma e ials o Imaging App oach Cu en imaging me hods, such as compu ed omog aphy and magne ic esonance imaging (MRI), ely hea ily on he size shi o lymph nodes o he p esence o ana omic anomalies as he p ima y c i e ia o diagnosis [ 80 ]. Nanopla o m o con as agen s has shown some p omising esul s in imaging app oaches o add ess his p oblem [81]. BC, as p e iously men ioned, has he highes ecu ence a e o all cance s, owing in pa o insu icien ansu e h al esec ion. The ailu e o cys oscopes o iden i y in isible lesions du ing he esec ion p ocess esul s in inadequa e esec ion [ 82 ]. Da is e al e al- ua ed he abili y o he endoscope app oach and su ace-enhanced Raman NPs o de ec bladde issue as cance ous o no mal (Figu e 1) [ 83 ]. A e opically adminis a ion o NPs o human bladde issue samples, bo h issue pe meabili y-based (passi e) a ge ing and an ibody-based (ac i e) a ge ing was assayed. The ecei e ope a ing cha ac e is ic egion unde he cu e (ROC AUC), o mul iplexed molecula imaging o CD47 and Ca bonic Anhyd ase 9 umo p o eins was 0.93. (0.75, 1.00). Fu he mo e, wi h a ROC AUC o 0.93, passi ely a ge ed NPs allowed issue classi ica ion. As compa ed o s anda d bladde u o helium, passi ely a ge ed nanopa icles pene a ed i e- ime deepe and a ached o umo issue a 3.3- ime highe concen a ions in cance . Indica ing ha he e is an imp o ed su ace pe mea ion and e aining unc ion in human BC. Quan um do s (QD) a e luo escen nanopa icles wi h supe io op ical p ope ies compa ed o o ganic dyes, bu in i o use o hem is cons ained by he po en ial o sys emic oxici y. Topical adminis a ion o a ge ed nanopa icles is appealing, because i allows o minimal exposu e and dose educ ion [ 84 ]. Pan e al. success ully indica ed ha opical (i.e., in a esical) adminis a ion o QD-conjuga ed an i-CD47 esul ed in e ec i e ex i o endoscopic imaging o human BC. In addi ion, hey in es iga ed he biodis ibu ion and oxici y o ee QD, and an i-CD47-QD in mice in i o [ 85 ]. An i- CD47-QD biodis ibu ion in i o was measu ed using induc i ely coupled plasma mass spec ome y. While some mice showed ex a esical biodis ibu ion o QD, indica ing a ou e o sys emic exposu e unde some condi ions, he e was no subs an ial agg ega ion Cance s 2021,13, 2214 5 o 29 o QD ou side o he bladde on a e age. Up o 7 days a e injec ion, he e we e no signs o high oxici y. In a esical adminis e ing o a ge ed NPs can minimize sys emic exposu e, bu o clinical use, NPs wi h well-es ablished bio - sa e y cha ac e is ics should be used o educe long- e m con amina ion in si ua ions o d ug oxici y. Figu e 1. The use o su ace-enhanced Raman sca e ing nanopa icle (NPs) o mul iplexed imaging o bladde cance issue [83]. The use o op ical imaging echnology in conjunc ion wi h cance -speci ic molecula imaging agen s may be an e ec i e solu ion o p omo ing cance de ec ion and enabling image-guided su gical [ 86 ]. The mos common endoscopic ea men o BC is whi e ligh cys oscopy, which has a low de ec ion abili y. De eloping op ical imaging inno a ions ha e a lo o p omise o be e diagnos ic p ecision, bu he e a e no any imaging agen s o molecula speci ici y ye [ 87 ]. Pan e al demons a ed s ong de ec ion o BC wi h clinical-g ade luo escence imaging app oaches, con ocal endomic oscopy, and blue ligh cys oscopy in esh su gically ex ac ed human bladde s using luo escen ly labeled CD47 an ibody (an i-CD47) as a molecula diagnos ic agen [ 88 ]. The speci ici y and sensi i i y o CD47- a ge ed imaging wi h blue ligh cys oscopy we e 82.9% and 90.5%, espec i ely. Based on he esul s o his s udy, CD47- a ge ed molecula imaging could help wi h BC diagnosis and esec ion p ecision. Despi e nume ous esec ions and long- e m chemo and immuno he apy, mos pa ien s wi h non-muscle in asi e BC unde go ecu ence o wo sening, necessi a ing cys ec omy and a poo p ognosis. Un inished esec ion and eimplan a ion o cance cells a e po en ial causes, which could be a oided wi h be e esec ion and adju an ea men [ 89 ]. Liang e al p oduced a guided d ug o u o helial ca cinoma iden i ica ion, luo escence-guided esec ion, and deep-pene a ing adju an pho odynamic he apy (UC) [ 90 ]. The me hod was based on up-con e sion nanopa icles (UCNP), which can hold a pho osensi ize and con e deep-pene a ing nea -in a ed ligh in o high-ene gy isible ligh , which is needed o umo in e p e a ion as well as he de elopmen o eac i e oxygen species in he pho osensi ize . The mean pho oluminescence o cells in he a ge ed g oup was 5 o 8 imes highe han in con ol g oups as a esul o he labeling, makes i o quick de ec ion o posi i e cells wi h low backg ound au o- luo escence. QDs a e commonly used in biomedical ields as luo escen labels. Yuan e al de el- oped quan um do (QD) luo escen p obes ha we e linked o a monoclonal an ibody agains p os a e s em cell an igen (PSCA) (QD-PSCA). The a ge ed imaging o QD-PSCA p obes in EJ human bladde u o helial cance cells was in es iga ed using his nanopla - o m. They also looked in o he e icacy o non-in asi e umo - a ge ed imaging using hese Cance s 2021,13, 2214 6 o 29 QDs in i o condi ions. By chemical co alen linking, QDs wi h an emission wa eleng h o 605 nm (QD605) was co alen ly linked wi h PSCA o p oduce QD605-PSCA luo escen p obes. An ul a iole spec opho ome e and a luo escence spec opho ome e we e used o es and de e mine he op ical p ope ies o he p obes coupled and uncoupled wi h PSCA. To iden i y and in e p e imaging o he p obes o EJ cells, di ec immune- luo escen labeling was used. The indings showed ha QD605-PSCA p obes main ained QD605’s luo escen p ope ies, as well as he PSCA p o ein’s immunogenici y. The p obes we e able o iden i y he PSCA p o ein exp essed in BC cells wi h high speci ici y, and he luo escence was s able and long las ing. QD-PSCA luo escen p obes may be help ul o p ecise a ge ed labeling and imaging in bladde u o helial cance cells, acco ding o his epo . The p obes also ha e good op ical du abili y, making hem sui able o non-in asi e a ge ed imaging, ea ly de ec ion, and a ge ed in i o umo he apy es ing. 2.4. Nanoma e ials in Biosenso De elopmen Resea che s iden i ied p omising u ina y bioma ke s o he de ec ion o BC as a esul o hei e o s o educe he cos s o BC diagnosis and imp o e pa ien s’ quali y o li e by emo ing unnecessa ily in asi e diagnos ic es s [ 91 – 97 ]. The po en ial bioma ke o ea ly diagnosis o BC is elome ase, nuclea ma ix p o ein 22 (NMP22), cy oke a in 19, su i in, hyalu onidase (HAase), apolipop o ein A1 (ApoA1), miRNA-21, galec in-1 p o ein, e c. [ 98 ]. Biosenso s o iden i ying u ina y bioma ke s ha e been es ablished in andem wi h hei disco e y, and hey can gi e low de ec ion limi s, a la ge linea esponse ange, high s abili y, and high accu acy [99]. 2.4.1. Gold Nanopa icle Su i in is one o he bioma ke s used o diagnose BC [ 100 ]. Using he ELISA ap- p oach and gold nanopa icle (GNP) co alen ly a ached o su i in an ibody, Jazaye i e al desc ibed eac i e an ibodies o su i in p o ein as a bioma ke o de ine he BC phase [ 38 ]. The plasma and u ine concen a ions o su i in inc eased d ama ically in he T3 and T4 phases o he disease (high g ades), acco ding o ELISA da a, as compa ed o heal hy people. Su i in p o ein was also ound in he u ine samples o pa ien s o all classes using c oss-linked GNPs (low and high g ades). Ano he u ina y bioma ke o BC has been iden i ied as hyalu onidase (HAase) [ 101 ]. Nossie e al. de eloped a simple colo ime ic GNPs assay o de ec ing u ina y HAase ac i i y quickly and accu a ely [ 102 ]. The o ma ion o gold s uc u es and a ed o blue colo change came om cha ge in e ac ion be ween ca ionic CTAB-co e ed gold NPs and polyanionic hyalu onic acid (HA). In addi ion, poly-ca ionic chi osan was used o collec all nega i ely cha ged compounds in u ine. In a s udy o 40 bladde ca cinoma pa ien s, 11 benign bladde lesions pa ien s, and 15 heal hy people, he de eloped GNP echnique was compa ed o zymog aphy o apid iden i ica ion o u ina y HAase ac i i y. The assay sensi i i y was 82.5% s. 65% o zymog aphy, and he speci ici y o bo h es s was 96.1%. The HAase ac i i y was measu ed using he A530/A620 abso p ion a io o he eac ed GNP solu ion. The highes cu -o achie ed abou 93.5 U/ng p o ein wi h 81% speci ici y and 90% sensi i i y. 2.4.2. G aphene Peng e al de eloped a magne ic g aphene oxide (GO)-linked o P ussian blue (PB) (PMGO) as a pe oxidase-mimicking nanozyme wi h g ea oxidizabili y o 3,3 0 ,5,5 0 - e ame h ylbenzidine (TMB). Fo colo ime ic immunosensing o apolipop o ein A1 (ApoA1) as a bioma ke o BC, his p obe p o ides conside able abso p ion capaci y [ 103 ]. Immunosen- so biochip was unc ionalized wi h ApoA1 an ibody (AbApoA1) and PMGO. In he exis ence o sel -linkable PMGO, he linea de ec ion ange was signi ican ly expanded ( om 0.05 o 100 ng/mL) as compa ed o he g oup wi hou signal enhancemen ( om 1 o 100 ng/mL). The immunosenso e alua ion o ApoA1 in he u ine o BC pa ien s and heal hy people was s ongly associa ed wi h enzyme-linked immunoso ben assay Cance s 2021,13, 2214 7 o 29 measu emen s; howe e , ApoA1 concen a ions in high-g ade BC pa ien s we e ma kedly la ge han in low-g ade BC pa ien s. In medical and biological science, molecula beacon (MB)-senso s ha e a high impac on p ope diagnosis. The syn hesis o bo h quenche and luo ogen in nucleic acid p obes, would inc ease he s ain o o ganic syn hesis wo k and c ea e di icul ies in p ecisely egula ing he ela i e dis ance be ween quenche and luo ogen, possibly leading o alse-posi i e and alse-nega i e ou comes [ 104 ]. Ou e al. published a single labeled MB (FAM-MB, wi h ca boxy luo escein as luo ogen and no quenche ) o de ec elome ase ac i i y using GO [ 104 ]. They design an easy, esponsi e, and selec i e sys em using a label- ee beacon (AIE-MB) based on accumula ion emission luo ogen (silole-R), o u he simpli y his design, namely label- ee s a egy. The AIE-MB media ed comb-like DNA s uc u e esul s in highe agg ega ion o silole-R and, hus, hea y luo escence emission when elome ase is added. They can iden i y elome ase wi h g ea e sensi i i y as a esul o his, and show how i can be used o diagnose BC. Duan e al used a sys em o analyze miRNA-21 and elome ase by a nicking enzyme- assis ed signal ampli ica ion and GO [ 105 ]. The use o a DNA molecula beacon p obe o p e en he o ma ion o G-quad uplexes boos s elome ase ac i i y. The implemen a ion o GO g ea ly educes backg ound noise. This bina y assay can di e en ia e be ween u ine om BC pa ien s, cys i is pa ien s, and heal hy people, acco ding o es s on 258 u ine samples. E en ually, his echnique has a lo o p omise when i comes o disc imina ing be ween muscle-in asi e and non-muscle-in asi e BCs. 2.4.3. Ino ganic Nanopa icles Galec in-1 p o ein has ecen ly been iden i ied as a use ul u ina y bioma ke o BC di- agnosis and p ognosis [ 106 ]. Shaikh e al. p oposed a esponsi e and p ecise impedime ic immunosenso o de ec ing Galec in-1 p o ein in clinical u ine samples quan i a i ely and wi hou he use o labels. In o al, nine gold in e digi a ed mic oelec odes (3 × 3 a ay) make up he immunosenso [ 106 ]. They used Galec in-1/Al2O3 nanop obes (Galec in-1 an ibody bonded o alumina NPs) ha can be speci ically apping on he mic oelec ode su ace using posi i e dielec opho esis (p-DEP) o achie e highe sensi i i ies. The median no malized impedance di e ence du ing immunosensing o 22 cance pa ien s and 26 no mal pa ien s is 27% and 10%, espec i ely, acco ding o he clinical es s, wi h a cu -o poin o 19.5% abo e which he speci ici y and sensi i i y o BC diagnosis we e 80% and 82%, espec i ely. The enhancemen o de ec ion p ecision and speci ici y has been hinde ed by high backg ound noise caused by con aminan s and o he analy es in biological mix u es. Wang e al de eloped an ul alow backg ound bio-chip based on ime-ga ed luminescen p obes assis ed by pho onic c ys als (PC) o high speci ici y and sensi i i y iden i ica ion o BC- ela ed miRNA bioma ke s in u ine samples [ 107 ]. Au o- luo escence can be e icien ly elimina ed when combined wi h he luminescence-enhanced capabili y o PCs and he ime- ga ed luminescence o long-li e ime luminescence pla o ms. Thus, de ec ion sensi i i y would be d ama ically imp o ed. Taking ad an age o hese ad an ages, a de ec ion limi o 26.3 M was ob ained. Mo eo e , he biochip pe o ms well in de ec ing u ina y miRNA, wi h s ong eco e ies. The p oduced biochip has ul alow backg ound and luminescence enhancemen capabili ies, making i an excellen ool o de ec ing BC- ela ed miRNA in u ine. 2.4.4. Ex acellula Vesicles (EV) Ex acellula esicles (EV), which include mic o esicles and exosomes, can be ound in he human body, and he concen a ion o exosomes and hei ela ed bioma ke s such as nucleic acids and p o eins can help diagnosis p ocedu e [ 108 ]. Liang e al c ea ed an op imized double- il a ion mic o luidic sys em ha sepa a ed and pu i ied EVs (200 nm) om u ine be o e quan i ying hem using a mic ochip ELISA (Figu e 2) [ 109 ]. The amoun o u ina y EVs was ma kedly la ge in pa ien s wi h BC (n= 16) han in sa e con ols (n= 8) Cance s 2021,13, 2214 8 o 29 acco ding o he obse a ions. This op imized EV double- il a ion sys em had a sensi i i y o 81.3% and a speci ici y o 90%, acco ding o ecei e ope a ing cha ac e is ic (ROC) analysis (16 BC pa ien s and 8 heal hy con ols). As a esul , his in eg a ed echnology has a lo o po en ial o be used in combina ion wi h u ina y cy ology and cys oscopy in clinics and a poin -o -ca e (POC) se ings o enhance clinical diagnosis o BC. Figu e 2. Sepa a ion and iden i ica ion o EVs om u ine using a mic o luidic sys em wi h in eg a ed double- il a ion. ( A ) Illus a ion o a mic o luidic sys em wi h double il a ion. ( B ) Schema ic o a comple ed double- il a ion p ocess. ( C ) Desc ip ion o an on-chip di ec ELISA o EV iden i ica ion. ( D ) A sma phone ha is used, o pho og aph he ELISA esul , and hen anspo ed o a lap op o da a p ocessing using ImageJ [109]. 3. Applica ions o Nanoma e ials in T ea men o BC As men ioned ea lie , BC is conside ed one o he mos ecu en u ogeni al cance s in he wo ld [ 110 ]. BC is an epi helial ca cinoma ha unusual cells in he epi helial lining mul iply wi hou any con ol. The mos equen his ological class o BC is ansi ional cell ca cinoma (TCC), also named u o helial cell ca cinoma (UCC) [ 41 ]. Se e al me hods a e gene ally used o ea men s o pa ien s who su e ed om BC, including chemo he apy, su ge y o umo , adio he apy, immune he apy, s em cell ans o ma ion, and adical cys ec omy. Howe e , as we know, hese me hods will ha e many side e ec s such as es ic ed bioa ailabili y, oxici y, non-selec i i y, as clea ance, and limi a ion in me as asis. The e o e, esea che s a e s udying o ind newe ea men me hods wi h ewe side e ec s and mo e he apeu ic e ec s [ 111 ]. Gene ally, BC is a e y he e ogeneous and complex disease wi h a ious biological sub ypes. So, i has many challenges in g ading, classi ica ion. App oxima ely, 70% o pa ien s who su e ed om bladde UCC show a supe icial ca cinoma named non-muscle-in asi e BC (NMIBC), while he o he pa ien s (30%) p og ess a muscle-in asi e ca cinoma (MIBC) bea ing he dange o he me as a ic sp ead o he umo [ 112 ]. The mos common ea men o BC is su ge y, bu he indings ha e been shown ha in app oxima ely 80% o pa ien s who unde wen su ge y, he umo ecu ed a e 5 yea s. The e o e, chemo he apy is s ill conside ed he main and impo an ea men in he inhibi ion o umo ecu ence and p og ession. Al hough chemo he apeu ic agen s, such as axanes, cispla in, gemci abine, e c., acili a e p olong- e m su i al in many BC pa ien s, he g ea ecu ence a e o he umo and se ious ad e se ou comes o he apeu ic agen s emain o BC he apy. The e o e, mo e e ec i e and no el managemen is necessa y o inc ease he quali y and quan i y o li e o he BC pa ien s [112]. Nowadays, nano echnology has been d ama ically helped us o diagnose and ea a wide a ie y o cance s like ha BC. Recen ly, se e al nanopa icles (NP) such as polyme ic NPs, lipid NPs, me allic NPs ha e been used o help BC he apy. Va ious o ms o NPs Cance s 2021,13, 2214 9 o 29 can inc ease he solubili y o d ugs wi h weakly soluble, and mul i- unc ional NPs ha e accep able esul s agains enal, bladde , p os a e cance . NPs a e also u ilized as a d ug deli e y sys em (DD) o inc ease e ec s and in e ac ions be ween d ugs and he u o he- lium. Fu he mo e, nano echnology can also associa e wi h o he mode n echnologies o ad ance enhance e ec i ely [ 113 ]. In his e iew, we in end o ocus on nano echnologies wi h po en ial and use ul applica ions in BC ea men , also we will in es iga e se e al nanoma e ials as nanoca ie s o educe side e ec s and enhance he e ec o chemo he apy d ugs o imp o e ea men o BC. 3.1. Some Impo an The apeu ic Agen s o T ea men o BC Recen ly, se e al e o s ha e been done o dec ease he de imen al e ec s o he apeu- ic agen s du ing he cance ea men p ocedu e o emo e he side e ec s on he closed cells and issues. Addi ionally, se e al me hods ha e been ound o enhance d ug e icacy in he umo , de eloping mode n DDS and a ge ing sys ems [ 114 ]. Chemo he apeu ic agen s ha e classi ied in o se e al g oups in ol e cy o oxic and cy os a ic agen s which ha e shown accep able e ec s alone o in combina ion wi h o he cance he apies. Fo example, I ino ecan (a semisyn he ic de i a i e o he plan alkaloid) and doxo ubicin (DOX) ( he mos popula an h acycline) a e chemo he apeu ic agen s in ol e opoiso- me ase inhibi o s, which used o many cance s. They ha e some impo an side e ec s such as neu openia and ca dio oxici y, espec i ely. O he chemo he apy d ugs such as oxalipla in, ca bopla in, cispla in, melphalan, and cyclophosphamide a e also conside ed alkyla ing agen s. Thei consump ion is usually associa ed wi h he ollowing side e ec s: gas oin es inal oxici y, ca dio ascula oxici y, neph o oxici y, and hema ologic oxici y. The o he chemo he apeu ic d ugs, such as inc is ine, inblas ine, PTX, and doce axel, a e g ea ly sui able and use ul agains a b oad ange o cance s; howe e , hese he apeu ic agen s a e also showing pa icula es ic ions such as hei oxici y, side e ec s, expensi e, e c., he majo and common side e ec s o hese an i-cance agen s a e dec eased blood cells, hai loss, and immunosupp ession. The eason o hese side e ec s is ha hey also a ge no mal cells, which a e apidly di iding in he body. Consequen ly, nanoma e ials a e a p omising d ug deli e y ca ie , and used in he apeu ic p ocedu es designed o sol e some o hese side e ec s [114]. Role o Phy ochemical Agen s in BC The apy Phy ochemicals a e conside ed plan -de i ed ma e ials ha ha e a g ea e ec on se e al diseases and can be use ul o he inhibi ion o p og essi e diseases. Fu he mo e, phy ochemicals ha e a conside able ole as na u al d ugs in down egula ing he mul iple d ug esis ance (MDR) in di e en ypes o ca cinoma pa icula ly in BC [ 115 ]. In BC, cu cumin (CUR) plays a majo ole, as i can p e en cell p oli e a ion and dec ease ela ed eac ions ha lead o me as asis. These mechanisms a e p obably accompanied by down- egula ing be a-ca enin exp ession and e e sing he epi helial-mesenchymal ansi ion (EMT) p ocess. Apigenin is ano he na u al p oduc capable o enhance amoun s o eac i e oxygen species (ROS), and can dec ease he le el o glu a hione (GSH) in BC. On he o he hand, Res e a ol capable ep esses cell p oli e a ion and agi a e apop osis ia signal ansduce and ac i a o o ansc ip ion 3 (STAT3) pa hways and de elopmen o umo in a xenog a model in BC. The e o e, phy ochemical compounds could con ol he MDR p ocess o UCC and inc ease he e ec o an i-cance d ugs [115]. Phy ochemicals ha e a wide olume o dis ibu ion, and caused accumula ion in se e al o gans. Mo eo e , he easibili y o he expansion o esis ance h ough many pa h- ways is he main challenge o he e ec i e applica ion o phy omedicines in cance he apy. To con ol he challenge associa ed wi h con en ional he apies, using nanoma e ials a e being pe o med [116]. In a ecen s udy, Jung Cho and cowo ke s in es iga ed se e al phy ochemical agen s such as CUR, capsaicin, que ce in, es e a ol, and hei combina ion wi h gemci abine. They ha e c ea ed a gemci abine esis ance u o helial cell ca cinoma (UCC) cell line Cance s 2021,13, 2214 16 o 29 Figu e 5. Deli e y o he apeu ic agen loaded in micelle. Many esea che s applied polyme ic micelles in o de o ea men o BC, o in- s ance, Zhou and e al. designed a a ge ed DDS wi h po en ials o in a esical ins illed chemo he apic agen s in o de o ea men o supe icial BC [ 135 ]. They used amphiphilic di-block copolyme poly ( ε -cap olac one)-b-poly (e hylene oxide) (PCL-b-PEO), which polye hylene oxide (PEO) is conside ed as a hyd ophilic block and poly-cap olac one (PCL) as a hyd ophobic block. Fi s , cyclic a ginine-glycine-aspa ic acid-D-phenylalanine-lysine C (RGD K) pep ide and lo escen label was conjuga ed wi h he copolyme and o me ly accumula ed in o micelles. C (RGD K) pep ide showed high a ini y o α β 3 in eg in, which o e exp ess on he su ace o T24 cell lines, when DOX was loaded in o he sys em. As a esul o he con ocal analysis, i was disco e ed ha encapsula ing DOX h ough he c (RGD K) pep ide would aid micelles in a ge ing umo cells wi h s ong an i-p oli e a ion o umo bladde cells [135]. In ano he simila s udy, ano he a ge ed d ug deli e y sys em was in es iga ed in o de o in a esical ins illed an i-cance agen . In his s udy, bo h olic acid (FA) and luo escein iso hiocyanna e (FITC) was deco a ed o PCL-b-PEO-NH 2 copolyme s uc u e. The cellula up ake mechanism o T-24 cells applying con ocal demons a ed ha PCL-b-PEO-FA has mo e up ake, due o o e exp ession o FA ecep o on he su ace o ca cinoma cells. In i o , he adjus ed wi h FA has much lowe cell iabili y wi h DOX loaded in micelles han wi hou a ge ed micelles, acco ding o he cell cy o oxici y expe imen . Addi ionally, his s uc u e can educe ad e se e ec o DOX ia ecep o media ed endocy osis mechanism [135]. Zhong and e al. designed a sui able me hod in o de o de elopmen a polyme ic micelle s uc u e by using DOX as chemo he apy d ug which is co alen ly conjuga ed o i-me hyl-chi osan (TMC) wi h beclin-1 siRNA (Si-Beclin1/DOX-TMC) [ 149 ]. The si- beclin1/DOX-TMC micelle displayed mo e cy o oxic e ec o bo h d ug-sensi i e BIU-87 and d ug- esis an BIU-87/ADR cell lines. This o mula ion showed s ong capaci y o au ophagy in BC cells. In ac , DOX may cause inducing p o ec ing au ophagy in his ype o cell line. I is no ewo hy ha , he equipped si-beclin1/DOX-TMC micelle p e- en s he beclin-1 p o ein exp ession o p e en p o ec i e au ophagy o BIU-87/ADR cells, bu simul aneously inducing apop osis mechanism. In ac , hey can demons a e a new s a egy o o e come chemo he apy esis ance by using nanoma e ial. Since ha , egula ing he apop osis and au ophagy a e combined by sui able design and p epa a- Cance s 2021,13, 2214 17 o 29 ion o mul i- unc ional biocompa ible nanoca ie s o siRNA and d ug co-deli e y. So, codeli e y o DOX and becline-1 SiRNA desc ibed syne gis ic e ec o supp ess d ug esis- an BC. In i o in es iga ion was ca ied ou in BIU-87/ADR xenog a BC models and si-beclin1/DOX-TMC nanomicelles showing signi ican ly supp essed umo g ow h [ 149 ]. Pan e al de eloped nanosized micelles loaded wi h PTX and in es iga ed an i- umo e ec agains BC [ 136 ]. S uc u e o micelle- o med elodend ime s was syn hesized ia conjuga ion o 8 uni s o cholic acid a one end o PEG and a BC-speci ic a ge ing pep ide named PLZ4 (cance speci ic ligand) a he o he end. I should be no ed ha , cys eine (Cys) was p esen ed be ween he cholic acid and PEG when p epa ed disul ide c oss-linked PLZ4 nanomicelles (DC-PNM). Expe imen al esul s and an i- umo e ec we e con i med in mice wi h immune sys em ine iciency, ca ying pa ien -de i ed BC xenog a s (PDX). A e IV adminis a ion, DC-PNM de ini ely a ge ed he BC PDXs. Fu he mo e, DC-PNM loaded wi h PTX can emo e cispla in esis ance, and inc ease he median su i al om ee PTX compa ed o mice ca ying PDXs. As esul , DC-PNM exhibi ed s able in he sodium dodecyl sul a e (SDS) solu ion and p ecisely a ge ed he BC xenog a s. Thus, he sys em enhanced he e icacy o PTX as an an i-cance d ug [136]. In ano he s udy, he combina ion o micelles and PLZ4 was e alua ed o s udding o imaging and ea men in BC cases. PTX, as an an i-cance , we e loaded o deco a ed micelles. The a ge ing micelles accumula ed in cy oplasm o 5637 human cells and T24 cell line. The esul showed enhancemen o d ug deli e y in a ge si e. Addi ionally, educe PTX oxici y and inc ease o e all su i al [150]. 3.2.3. Applica ions o Polyme ic NPs o T ea men o BC Polyme ic based NP sys em a e he mos used among he NPs ha can be p epa ed om se e al polyme s, including na u al o syn he ic ma e ials composed o mac o- molecules such as poly (lac ide-coglycolide) (PLGA), poly(lac ic acid) (PLA), poly(e- cap olac one)(PCL), poly(alkyl cyanoac yla es), and chi osan. Due o physicochemical s uc u es o polyme ic NPs, we in ended o in es iga e solid NPs, polyme ic micelles, dend ime sys em [ 151 ]. Ad an ages o polyme ic NPs ha e a ac ed in e es o e he pas yea s. Fo example: he sui able size a ound 100–300 nm, o e en less han 100nm; a e able o con ol elease; less expensi e; p o ec he d ug agains en i onmen al eac ions [ 152 ]. Howe e , polyme ic NPs a e oxic o pa ien s, o example, PLA ha e poo oughness, deg ada ion speed slow o chi osan ha e poo s eng h, low wa e -solubili y [ 153 ]. So, i is essen ial o imp o e hei biocompa ibili y and educe oxici y in o de o biomedical usage. As we know, a e esec ion o he umo in o de o ea men o pa ien s who su e om BC, chemo he apy is conside ed as a s anda d way o p e en ing he de elopmen o he disease in he pa ien s. Howe e , he high a e o exc e ion o chemo he apeu ic d ugs is a majo challenge in hei e ec i eness. I is no ewo hy ha polyme s wi h mucoadhe- si e ea u es can imp o e he e ec i eness and pene abili y o chemo he apeu ic d ugs. The e o e, i could be help o inc ease e en ion ime o d ug deli e y in he pos -su ge y ea men o BC pa ien s. In a ecen esea ch, scien is s designed a p omising d ug deli e y sys em by chi osan as a posi i ely cha ged polyme ic NP sys em in o de o inc ease he adhesion and pe meabili y o p od ug gambogic acid wi hin he bladde wall. They used a educ ion sensi i e ca ie o anspo he eac i e oxygen species (ROS) ac i a ed p od ug o gambogic acid in o de o ea men o o ho opic BC. Fu he mo e, hey examined he a ious le el o glu a hione (GSH) and ROS be ween no mal and cance cells, he dual esponsi e NP ca ie can selec i ely deli e he d ug in o BC cells. As esul , he sys em can signi ican ly p e en he g ow h o BC cells in an o ho opic supe icial BC model wi hou leading o des uc ion o no mal cells [137]. In ano he s udy, Li e al. designed luo ina ed chi osan nanosys em in o de o inc ease ansmucosal deli e y o sonosensi ize -a ached ca alase o sonodynamic (SDT) BC he apy pos -in a esical ins illa ion. The SDT nano-pla o m which a ansmucosal O2-sel p oduc ion is designed in o de o ha e mo e e icien SDT agains BC [ 154 ]. In he s udy, chi osan deco a ed wi h lou (FCS) is made as a mo e e ec i e sa e ansmucosal Cance s 2021,13, 2214 18 o 29 deli e y nanoca ie o accumula e wi h meso- e a (4-ca boxyphenyl) po phine-bonded ca alase (CAT-TCPP). The enginee ed CAT-TCPP/FCS NPs a e in a esical ins illa ion in o he bladde showed e y good in a umo al, and also ansmucosal pene a ion. Fu he mo e, i could e icien ly p oduce hypoxia in umo mic oen i onmen by he ca alase-ca alyzed O2 gene a ion om cance cells endogenous H 2 O 2 o u he de elop he he apeu ic e icacy o SDT o abla e o ho opic BC cells unde ul asound. In ano he wo k, Zhu e al p epa ed DOX and IR780@PEG-PCL-SS NPs in o de o c ea e a chemo pho o he mal he apy. In pho o he mal he apy, ligh abso p ion makes pho osensi ize s gene a e hea . I can cause all o a ge cells o unde go nec osis and apop- osis. Thus, his me hod can p omo e he apeu ic e icacy. In his s udy, he o mula ion o NP which con ain DOX and IR780 a e sensi i e o p esence o GSH in mic o-en i onmen al o BC cells and nea -in a ed lase i adia ion. The e o e, he NP can elease d ugs unde nea -in a ed lase i adia ion (IR) in he p esence o GSH, which enable he con olled he elease o d ugs. The esul o he in i o s udy demons a ed ha signi ican e icacy and sui able size o umo ob ained a e 21 day o ea men [ 140 ]. In ano he esea ch, poly (e hylene glycol)-b-(poly(e hylene diamine l-glu ama e)-g-poly( ε -benzyoxyca bonyl-l- lysine)- -poly(l-lysine)) (PEG-b-(PELG-g-(PZLL- -PLL)) we e p epa ed by Hu e al. lum- b okinase as an enzyme which has a hal li e ha is oo sho o be used in combina ion wi h o he d ugs. Due o i s nega i e cha ge, i combined wi h PLL b ush block ia elec os a ic in e ac ion. PTX wi h hyd ophobici y p ope y is able o combine PZLL h ough hyd opho- bic in e ac ion, he eby enhancemen o he hal -li e and bioa ailabili y o he d ugs could be de eloped ia he sus ained elease and enhancemen o umo si e en ichmen h ough passi e a ge ing. They ound ha (PEG-b-(PELG-g-(PZLL- -PLL)) polyme s p olonged he hal -li e and bioac i i y o LK and PTX in Sp ague-Dawley a s [ 155 ]. Tao e al. de eloped inc easing deli e y e iciency wi h u ilizing di e en deg ee o choles e ol subs i u ed pullulan polyme (CHPS). The bes esul s we e p o ided when he deg ee o choles e ol was high wi h he smalles size and e icien loading. The esul demons a ed ha CHp-3 wi h la ges size had s onges abili y o p e en o cells mig a ion. Addi ionally, all h ee ype o NP which con aining d ugs could inhibi MB49 cells [156]. 3.2.4. Applica ions o Liposome o T ea men o BC Liposomes a e syn he ic phospholipid nano esicles wi h a bilaye memb ane shape, i s de eloped by Alec Bngham in 1961 [ 138 ]. Liposomes ha e been used in ca ying a ious biological and biochemical molecula such as d ug, nucleo ides, p o ein, e c. The binding o liposomes o human BC cell lines a ec s he usage o liposomes in BC he apy. The u iliza ion o liposomes show g ea ad an ages as nano ehicles, such as being capable o solubilizing bo h hyd ophilic and hyd ophobic molecules in an aqueous co e and phospholipid bilaye , espec i ely; biocompa ibili y because o hei o igin om choles e ol and phospholipids; good s abili y and capabili y o anspo d ugs by sui able selec ion o p epa a ion p ocedu e [ 42 ]. Nogawa e al. epo ed ha polo-like kinase 1 (PLK-1) is ela ed o g ades and su i al a e o BC pa ien s. They ca ied ou , in a esically, ins illing a complex o PLK-1 siRNA/liposomes in o he o ho opic umo bladde o he mouse. They success ully exhibi ed ans ec ing PLK-1 siRNA in o umo ic cells and diminished PLK-1 exp ession and supp ession o cance p oli e a ion in his mouse model [ 138 ]. In a esical adminis a ion o an i-p oli e a i e ma e ial seems one o he common he apeu ic me hods o ea men o BC. Liposomes encapsula ed wi h he apeu ic agen s ha e been con i med o enhance he e icacy o in a esical he apy. Hsu e al. epo ed ha ecombinan human in e e on alpha (IFN- α ) loaded in liposomes inhibi s g ow h o BC cell line 253J mo e han ee IFN- α alone o liposomes wi h saline. Fu he mo e, he same in es iga ion also es ablished ha a subline o 253J, which is esis an o ee IFN- α , became esponsi e o IFN- α loaded in liposomes [ 138 ]. In a esical ins illa ion o li e bacillus Calme e Gué in (BCG) bac e ia u ilized as an ideal ea men me hod o high g ade NMIBC. Howe e , se ious ad e se e ec s we e epo ed, including con aining BCG in ec ions, sepsis, and e en dea h. Repackaging pa s o he Cance s 2021,13, 2214 19 o 29 BCG bac e ia has been ca ied ou in o de o subs i u e o li e bac e ia wi h he p omise o achie e sa e and sui able e icacy. Fo ins ance, he cell wall skele on (CWS) o BCG was in es iga ed o clinical applica ion. Howe e , i was seems o be mo e ha d o dissol e in wa e and deli e o he si e o he a ge . By encapsula ing BCGCWS wi hin liposomes, whose size was 166 nm, Nakamu a e al. we e able o epo sui able e icacy in a models agains he de elopmen o BC [123]. The mycobac e ium bacillus calme e Gué in cell wall skele on (BCG-CWS) was con- side ed he impo an immune ac i e pa o BCG. So, i is a g ea sugges ion as a non- in ec ious immuno he apeu ic d ug, and also as a subs i u e o li e BCG o u ilize agains u o helial cance . Howe e , i s usage as an i-cance he apy is es ic ed due o he ac ha BCG-CWS can agg ega e in all aqueous and non-aqueous sol en s. To inc ease he in e naliza ion o BCG-CWS in o BC cells wi hou agg ega ion, Whang and cowo ke s in 2020 designed an in e es ing nanosys em consis o liposome which loaded nanosized BCG-CWS (180 nm) o in a esical ins illa ion in BC. This nanosys em displayed an an i- umo al e ec in an o ho opic BC mouse model, and he BCG-CWS NPs can be u ilized as a non- oxic subs i u e o li e BCG wi h imp o ed s abili y, and size compa ibili y. They used an in a esical way by applying a ca he e in he o ho opic BC mouse model in o de o in a esical deli e y o BCG-CWS mouse o selec i ely a ge umo cells. In i o esul s indica ed ha BCG-CWS nanosize, encapsula ed wi h con en ional liposomes (CWS-Nano-CL), was mo e e ec i e a inhibi ing BC cell g ow h compa ed o nonencapsu- la ed BCG-CWS. T ea men wi h CWS-Nano-CL p omp ed he inhibi ion o he mammalian a ge o apamycin (mTOR) pa hway and also he ac i a ion o AMP-ac i a ed p o ein kinase (AMPK) phospho yla ion, cause o apop osis, bo h in i o (5637 cell lines) and in i o . So, hey sugges ed ha he in a esical ins illa ion o CWS-Nano-CL can help BCG-CW cellula endocy osis and supply a sma d ug-deli e y sys em as a he apeu ic me hod o BCG-media ed BC ea men [133]. Recen ly scien is s designed a d ug deli e y sys em consis o maleimide-modi ied PEGyla ed (Mal-PEG) liposomes we e known as mucoadhesi e nanoca ie o in a esical he apy o BC. Mal-PEG liposomes loaded luo escein sodium (FS) displayed mo e pene- a ion and e en ion ime on bladde mucosal compa ed o wi hou liposomes. In ano he wo k a g oup o scien is s ha e enginee ed a pla o m by u ilizing luidizing liposomes inco po a ed in o gellan hyd ogel which p o ide an in si u-gelling liposome-in-gel (LP-Gel) sys em. LP-Gel employs u ine o unde go ion- igge ed gela ion o cons uc a c oss-linked gellan ma ix. The pla o m mimics he bladde mucosa, so esul ing in sui able in e ac ion and adhesion o he bladde wall. A e LP-Gel pla o m is ins illed in o he bladde o a , he ion- igge ed gela ion binds o he u o helium, a e ha , he luidizing liposomes pen- e a e ia he u o helial ba ie and he d ug localiza ion in umo lesions we e p olonged. Fu he mo e, ins illa ion o pacli axel-loaded LP-Gel showed p o ac ed d ug localiza ion in he bladde almos se en days, o e ing po en ial usage in clinical p ac ice [157]. In a ecen s udy, Valenbe g e al used 1.2-dipalmi oylsn-glyce o-3-phosphodiglyce o (DPPG2) based he mosensi i e liposomes (TSL) wi h loaded DOX combined wi h hy- pe he mia (HT) caused g ea e amoun s o DOX in he bladde . In his wo k, hey used 21 pigs in which, a e anes hesia, hey we e placed in a u ina y ca he e equipped wi h a adio equency an enna o HT (1 hou s), and hen adminis a ed di e en doses o DPPG2-TSL-DOX and ee DOX wi h o wi hou HT. A e ha , he pigs we e immedia ely sac i iced. HPLC was u ilized o measu e DOX concen a ions in he bladde , i al o gans and se um, and luo escence mic oscopy was used o es ima es he dis ibu ion o DOX in he bladde wall. They concluded ha his me hod compa ed o con en ional DOX, inc eases he le el o DOX in he bladde wall while diminishing DOX in he majo o gans. DPPG2-TSL-DOX accompanied wi h HT p o ided mino DOX accumula ion in he hea and kidney o pigs, compa ed o IV ee DOX [139]. In ano he simila s udy, Mikhail e al used lyso- he mosensi i e liposomal doxo u- bicin (LTLD) in combina ion wi h local mild HT, in o de o a ge ed d ug deli e y o he bladde and po en ial BC he apy. This s udy also pe o med on po cine wi h he ollowing Cance s 2021,13, 2214 20 o 29 h ee ca ego ies: (I) IV adminis a ion o LTLD wi h HT, (II) IV ee DOX wi h HT, and (III) IV LTLD wi hou HT. Fo mula ions o he d ug we e deli e ed h ough IV in usion which p olonged 30 min, simul aneous wi h 1-h bladde i iga ion, 45 ◦ C wa e o HT ca ego y, 37 ◦ C o non-HT ca ego y, hen immedia ely bladde esec ion. DOX le els we e in es iga ed in consecu i e pa s pa allel o he bladde lumen by liquid ch oma og aphy ollowing d ug ex ac ion. As an ou come, hey es ablished ha DOX accumula ion and dis ibu ion wi hin he bladde was ob ained a highe amoun s han wi h ee IV DOX by mild bladde hype he mia accompanied by sys emic deli e y o LTLD [158]. 3.2.5. Applica ions o Exosome as Na u al NP o T ea men o BC Exosomes a e iny endosome-de i ed memb ane nano esicles (app oxima ely 30– 100 nm) sec e ed om a ious ypes o hos cells showing di e en biological unc ion. Exosomes ha e a no ewo hy ole in cellula communica ions as a ehicle o nucleic acids and p o eins. They ha e been eme ged as no el media o s o umo p og ession o e he ecen decade [ 159 ]. Exosomes ha e mul iple and impo an biological oles, such as im- mune esponse egula ion, p esen a ion o an igens o immune cells, in e cellula ela ion ia ans e o p o eins, nucleic acid such as miRNA, mRNA. Exosomes a e a majo sou ce o umo bioma ke s in liquid biopsy samples. Exosomes can sepa a e om he u ine o BC pa ien s wi h MIBC induced epi helial o mesenchymal ansi ion in u o helial cells. A no el obse a ion in o he unc ion o exosomes in he ansi ion o BC in o muscle-in asi e cance was ca ied ou . The e o e, exosome in es iga ion in ad anced BC could be consid- e ed a mode n sys em o p edic ing p og ession and inno a ing a ge ed he apy [ 160 ]. Exosomes c ea e by he inne budding o in a-cellula endosomes, a e ha al e ed in o mul i- esicula bodies (MVB) ha belong o he endosomal pa hway and can use wi h he cellula memb ane and sec e e hei payload in o he ex acellula en i onmen [ 161 ]. Gene ally, exosomes exis en in mos biological luids and show impo an unc ions in cance . So, in es iga ions amoun o exosomes in a ious s eps o BC could be use ul o diagnosis and imely he apy o BC [159]. Newly, scien is s con i m ha exosomes a e a po en ial sou ce o umo bioma ke s in liquid biopsies, such as blood and u ine samples due o con aining RNAs, DNAs, and p o eins. In pa icula , exosomal miRNAs (exomiRs) play an impo an ole as bioma ke s o umo g ow h and p og ession (Figu e 6) [162]. Body luid biopsy is one o he mos p omising me hods in cance esea ch because i is a simple, low-cos , and non-in asi e p ocedu e. So ha , liquid biopsy specimens can show us use ul and comple e in o ma ion abou he gene ics o cance pa ien s. Elsha kawi e al. epo ed ha umo de i ed exosomes (TDE) could be a use ul and sui able biological de ice o ea ly iden i ica ion o BC. They we e measu ed TDEs concen a ion in u ine and se um samples o se en y BC pa ien s om Ta o T3 s ages, and 12 heal hy con ol people using ELISA echnique. As esul s show, exosomes concen a ions in BC pa ien s we e enhanced compa ed o heal hy people in se um and u ine samples a di e en phases o he disease. Mo eo e , hey ound ha se um was mo e pa icula sample o disco e y o exosomes in BC [159]. Hil b unne e al. epo ed ha exosomes wi h p o-ca cinogenic p ope ies could be iden i ied in u ine om his ologically down-s aged BC pa ien s. They demons a ed ha u ina y exosomes om he bladde , e en when no mac oscopic umo obse e. Nex , a combina ion o p ima y ansu e h al esec ion (TUR-B) and neoadju an cispla in-based combina ion chemo he apy (NAC), a y om exosomes ound in u ine om he uppe ac . These exosomes exhibi ed a malignan me abolic manne , which p obably could enhance me as asis and ecu ence. Hil b unne e al. epo ed ha he bladde ’s pe o mance as a si e o exosomes capable o dissemina e o a si es in lymph nodes and o he dis an o gans, whe e hey help dispe sion ia me abolic communica ing [ 163 ]. In ac , hey ound ha why a la ge numbe o MIBC pa ien s ecu ence e en a e NAC and adical cys ec omy (RC). They showed ha exosomes hold a malignan memo y pheno ype in he bladde e en a e (TUR-B) and NAC, con i ming he signi icance o adical cys ec omy Cance s 2021,13, 2214 21 o 29 o e mino su ge y o emo e he o iginal umo -p omo ing exosomes. Howe e , u he s udies on he me abolic index o u ine and bladde muscle a e needed o p o e his ac [163]. Figu e 6. S uc u e o exosomes as po en ial sou ce o umo bioma ke s in liquid biopsies. Goule e al. demons a ed ha ex acellula esicles (EV) including mic o esicles, apop o ic bodies and exosomes de i ed om BC (BCEV) pa ien s can enhance “ ans- o ma ion” o no mal ib oblas s in o cance -associa ed ib oblas s (CAF). They isola ed ex acellula esicles om se e al cell lines such as T24, RT4, and SW1710 BC and u ilized hem o ea no mal ib oblas s sepa a ed om human bladde biopsies. The ou come showed ha ecipien ib oblas s ob ained CAF composi ion wi h enhanced p oli e a ion and mig a ion abili y, as well as aised exp ession o CAF ma ke s smoo h muscle ac in (SMA), ib oblas mo i a ion p o ein (FAP), and Galec in [164]. In ecen yea s, esea che s ha e epo ed exosomes can be applied as nanoca ie s, which can be u ilized in he ea men o many umo s by eleasing hei ca goes in o he a ge ed si e. exosomes as nanoca ie s o load an i- umo he apeu ic agen s o siRNAs in o exosomes. The ad an ages o his me hod including imp o ed he e icacy o d ugs, enhancing he bioa ailabili y o d ugs; being non- oxic o low- oxic, e c. Fo example, Wang e al es ablished a dual- unc ional exosome based supe pa amagne ic NP sys em employing exosomes as a a ge ed d ug ca ie o he ea men o cance cells [165]. In ano he s udy, Cai e al epo ed ha he e ec o exosomal miR-133b on he p oli e a ion o BC and i s molecula pa hway. Fi s , hey ha e in es iga ed he exp ession o miR-133b in BC and neighbo ing heal hy issues, as well as in body luid exosomes o BC pa ien s and heal hy con ols. Then, he en y o exosomes in cells was con i med ia luo escence localiza ion. Fu he mo e, cy o oxici y and apop osis we e ca ied ou in BC cells ans ec ed wi h mimics and incuba ed wi h exosomes. A e ha , he unc ion o exosomal miR-133b was in es iga ed in nude mice ans-plan umo s. Mo eo e , he a ge gene o miR-133b was es ima ed ia bio-in o ma ics echnique. The amoun o miR- 133b was conside ably educed in BC issues and in exosomes om he luids o pa ien s, co ela ing wi h weak gene al su i al in The Cance Genome A las (TCGA) da abase. Exosomal miR-133b can be ob ained using pos - ans ec ion BC cells by mimicking miR- 133b. MiR-133b exp ession was enhanced a e incuba ion wi h ex e nal miR-133b, leading o inhibi ion o iabili y and inc eased apop osis in BC cells. Exosomal miR-133b can supp ess umo g ow h in i o . In addi ion, we ound ha exosomal miR-133b migh be Cance s 2021,13, 2214 22 o 29 in ol ed in inhibi ing BC p oli e a ion by ea anging he dual-speci ic p o ein phospha ase 1 (DUSP1). These indings could hold new hope o BC ea men di ec ions [ 166 ]. In conclusion, hei esul s p o ide s ong e idence ha exosomal miR-133b ac s as a umo inhibi o y agen a ge ing DUSP1 in BC p oli e a ion. In addi ion, exogenous miR-133b can be inges ed by BC cells, educing he malignan pheno ype o BC cells. Exosomal miR -133b o o he miRNAs be e dis inguished he disease and make speci ic ad ances in he ea men o BC [166]. 4. Conclusion, Challenges, and Fu u e P ospec i e Many a emp s ha e been made in ecen decades o igh cance by combining no el echnology and adi ional app oaches. In his a icle, he applica ions o nano echnology we e discussed. Nano echnology has demons a ed ex ensi e applica ions bo h in he diagnosis and in ea men o a ie y o cance s, including imp o ing he selec i i y and sensi i i y o he ea men o BC. NPs ha e been able o o e coming he limi a ions o cu en medical app oaches in many esea ch and clinical ials. Fo MRI and CT imaging, a ious me allic NPs a e applied as con as agen s, inc easing he likelihood o de ec ing ea ly BC. GNPs’ excellen pho o he mal p ope ies ha e con ibu ed o hei widesp ead use in imaging. Howe e , se e al conce ns emain, such as he p o ec ion o hese NPs, when adminis a ed in he human body. Addi ional esea ch is equi ed o ully unde s and he speci ics o nano echnology-based me hods, and o de elop hese NPs as cance diagnos ic ools. On he o he hand, while NPs ha e he po en ial o ea BC, he e a e s ill a numbe o hu dles o o e come be o e, hey can be success ully ansla ed in o clinics, including esea ch in o he ela ionship be ween NPs and BC cells. 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