pH-Sensitive Peptide Hydrogels as a Combination Drug Delivery System for Cancer Treatment

  Ci a ion: Liu, Y.; Ran, Y.; Ge, Y.; Raza, F.; Li, S.; Za a , H.; Wu, Y.; Pai a-San os, A.C.; Yu, C.; Sun, M.; e al. pH-Sensi i e Pep ide Hyd ogels as a Combina ion D ug Deli e y Sys em o Cance T ea men . Pha maceu ics 2022,14, 652. h ps://doi.o g/10.3390/ pha maceu ics14030652 Academic Edi o s: Ma ia Nowakowska and F anco Dosio Recei ed: 30 Janua y 2022 Accep ed: 11 Ma ch 2022 Published: 16 Ma ch 2022 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 : © 2022 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/). pha maceu ics A icle pH-Sensi i e Pep ide Hyd ogels as a Combina ion D ug Deli e y Sys em o Cance T ea men Yuan en Liu 1,†, Yingchun Ran 2,†, Yu Ge 3, Faisal Raza 4,* , Shasha Li 5, Haj a Za a 4, Yiqun Wu 3, Ana Cláudia Pai a-San os 6, Chenyang Yu 3, Meng Sun 3, Ying Zhu 3,* and Fei Li 3,* 1Depa men o Pha macy, Jiangsu Heal h Voca ional College, Nanjing 211800, China; [email p o ec ed] 2Na ional Clinical Resea ch Cen e o Child Heal h and Diso de s, Minis y o Educa ion Key Labo a o y o Child De elopmen and Diso de s, Depa men o Eme gency, Child en’s Hospi al o Chongqing Medical Uni e si y, Chongqing 400014, China; [email p o ec ed] 3S a e Key Labo a o y o Na u al Medicines, China Pha maceu ical Uni e si y, Nanjing 210009, China; [email p o ec ed] (Y.G.); [email p o ec ed] (Y.W.); [email p o ec ed] (C.Y.); [email p o ec ed] (M.S.) 4School o Pha macy, Shanghai Jiao Tong Uni e si y, 800 Dongchuan Road, Shanghai 200240, China; [email p o ec ed] 5College o Pha macy, Xinjiang Medical Uni e si y, Ü ümqi 830000, China; [email p o ec ed] 6Depa men o Pha maceu ical Technology, Facul y o Pha macy, Uni e si y o Coimb a, 3000-548 Coimb a, Po ugal; [email p o ec ed] *Co espondence: [email p o ec ed] (F.R.); [email p o ec ed] (Y.Z.); [email p o ec ed] (F.L.) † These au ho s con ibu ed equally o his wo k. Abs ac : Con en ional an i umo chemo he apeu ics gene ally ha e sho comings in e ms o dissolubili y, selec i i y and d ug ac ion ime, and i has been di icul o achie e high an i umo e icacy wi h single-d ug he apy. A p esen , combina ion he apy wi h wo o mo e d ugs is widely used in he ea men o cance , bu a sho coming is ha he d ugs do no each he a ge a he same ime, esul ing in a educ ion in e icacy. The e o e, i is necessa y o design a ca ie ha can elease wo d ugs a he same si e. We designed an injec able pH- esponsi e OE pep ide hyd ogel as a ca ie ma e ial o he an i umo d ugs gemci abine (GEM) and pacli axel (PTX) ha can elease d ugs a he umo si e simul aneously o achie e he an i umo e ec . A e de e mining he op imal gela ion concen a ion o he OE polypep ide, we conduc ed an in i o elease s udy o p o e i s pH sensi i i y. The elease o PTX om he OE hyd ogel in he medium a pH 5.8 and pH 7.4 was 96.90% and 38.98% in 7 days. The elease o GEM om he OE hyd ogel in media wi h pH o 5.8 and 7.4 was 99.99% and 99.63% in 3 days. T ansmission elec on mic oscopy (TEM) and ci cula dich oism (CD) expe imen s we e used o obse e he mic os uc u e o he pep ides. The ci cula dich oism o OE showed a single nega i e peak shape when unde neu al condi ions, indica ing a β - olded s uc u e, while unde acidic condi ions, i p esen ed cha ac e is ics o a andom coil. Rheological expe imen s we e used o in es iga e he mechanical s eng h o his pep ide hyd ogel. Fu he mo e, he ea men e ec o he d ug-loaded pep ide hyd ogel was demons a ed h ough in i o and in i o expe imen s. The esul s show ha he pep ide hyd ogels ha e di e en s uc u es a di e en pH alues and a e highly sensi i e o pH. They can each he umo si e by injec ion and a e induced by he umo mic oen i onmen o elease an i umo d ugs slowly and con inuously. This biologically unc ional ma e ial has a p omising u u e in d ug deli e y o combina ion d ugs. Keywo ds: combina ion he apy; d ug deli e y; hyd ogel; cance ; pep ide 1. In oduc ion Cance has become one o he leading causes o mo ali y in he wo ld. Single-d ug he apy has di icul y achie ing high e icacy [ 1 ]. As a esul , combina ion d ug deli e y has been widely used in cance ea men . To enhance he he apeu ic e ec o combina ion Pha maceu ics 2022,14, 652. h ps://doi.o g/10.3390/pha maceu ics14030652 h ps://www.mdpi.com/jou nal/pha maceu ics Pha maceu ics 2022,14, 652 2 o 13 d ugs, wo d ugs can be co-loaded and deli e ed o umo issues o achie e syne gis ic d ug deli e y [ 2 , 3 ]. GEM is a wa e -soluble di luo ina ed nucleoside an ime aboli e d ug. I is ema kably e ec i e o a wide ange o solid umo s when used alone, bu i s oxici y is ela i ely high due o i s sho hal -li e and he need o con inuous high doses [ 4 ]. PTX is a s ongly lipid-soluble di e pene alkaloid d ug. I is less oxic han some o he an i umo he apies, bu i has o he p oblems, such as poo bioa ailabili y [ 5 ]. The e o e, o educe side e ec s and imp o e e icacy, PTX in combina ion wi h GEM is o en used o cance ea men , such as b eas cance , panc ea ic cance and bladde cance . Howe e , his d ug combina ion s ill has se e al p oblems. The mos ema kable one is ha he d ugs canno be deli e ed o he a ge loca ion a he same ime, which p e en s he maximum e icacy om being ealized. In ecen yea s, he main wo-d ug co-deli e y sys ems epo ed in he li e a u e ha e been liposomes [ 6 ], nanopa icles [ 7 ], d ug-ca ie copolyme s [ 8 ] and hyd ogels [ 9 ]. A hyd ogel is a h ee-dimensional ne wo k sys em o med by c oss-linked polyme s o ibe s in an ex ended luid. I can swell in wa e [ 10 ]. Because o i s good wa e abso p ion and e e sibili y, hyd ogels ha e been widely used in biomedical applica ions, such as d ug [ 11 ] o cell deli e y [ 12 ] and issue enginee ing [ 13 ]. Responsi e hyd ogels a e new ypes o hyd ogels ha unde go s uc u al changes in esponse o ex e nal physical o chemical s imuli [ 14 ]. They ha e he ad an ages o good biocompa ibili y and esponses o ex e nal en i onmen al condi ions. They can elease d ugs in he designa ed a ea a he same ime, achie ing he goal o a ge ed d ug deli e y. Acco ding o di e en ex e nal s imuli, esponsi e hyd ogels a e mainly classi ied as empe a u e-sensi i e [ 15 ], pH-sensi i e [ 16 ], ligh -sensi i e [ 17 ], mul i-sensi i e [ 18 ] and elec ical s imuli- esponsi e [ 19 ]. In addi ion, some elease mechanisms a e dose-dependen [20]. Cu en ly, mos polyme hyd ogel ma e ials ha e poo biodeg adabili y when used as d ug deli e y ca ie ma e ials, which may be ha m ul o humans [ 21 ]. In con as , pep ide hyd ogels o e signi ican ad an ages o good biocompa ibili y, con olled deg adabili y, lowe immunogenici y and highe bioa ailabili y. They a e easy o p epa e and modi y and ha e g ea ad an ages in d ug deli e y. Howe e , some pep ide hyd ogels ha e low pH sensi i i y in he umo mic oen i onmen . A e being injec ed in o he body, hey leak a la ge amoun o he d ug be o e o ming he hyd ogel. They also ha e he sho comings o poo mechanical s eng h and low bioa ailabili y [22]. To add ess hese issues, we p epa ed an injec able pH- esponsi e pep ide hyd ogel encapsula ing GEM and PTX as a wo-d ug co-deli e y sys em, which we named OE (VKVKVOVK-V D PPT-KVEVKVKV-NH 2 ). The sequence is composed o al e na e pola and non-pola amino acids, including aline and -V D PPT-. A he pH alue unde phys- iological condi ions, he polypep ide can o m nano ibe s h ough he sel -assembly o β -shee seconda y s uc u es. When he polypep ide concen a ion eaches a ce ain le el, he nano ibe s in he polypep ide s uc u e c oss-link h ough sel -assembly o o m a h ee- dimensional ne wo k s uc u e, which can encapsula e ac i e d ugs. In a sligh ly acidic umo mic oen i onmen , he h ee-dimensional ne wo k is dis up ed, and he encapsu- la ed d ugs a e eleased, he eby inhibi ing umo g ow h. This sys em can con inuously elease ac i e d ugs in a sa e and e ec i e dose a he umo si e, which imp o es he he apeu ic e ec and educes side e ec s o he d ugs. In his s udy, he OE pep ide was designed and syn hesized by a solid-phase pep ide syn hesis me hod. The sys em was able o o m pep ide hyd ogels encapsula ing GEM and PTX in i o wi h a sui able mechanical s eng h unde neu al condi ions. In he p ocess o d ug elease, he hyd ophilic d ug GEM is i s eleased in la ge quan i ies and can be comple ely eleased in he i s h ee days, while he hyd ophobic d ug PTX con inues o be eleased slowly un il i is comple ely eleased on he se en h day. This no only p olongs he du a ion o ac ion o he d ug bu also enhances he he apeu ic e ec o he d ug and ealizes an an i-b eas cance e ec [ 23 ]. In addi ion, h ough in i o and in i o s udies on i s an i umo ac i i y, we u he success ully con i med ha his sys em has a s ong inhibi o y e ec on umo g ow h and me as asis, which enhances he he apeu ic e ec o he d ugs. In addi ion, we Pha maceu ics 2022,14, 652 3 o 13 also con i med ha he blank pep ide hyd ogel in he sys em is sa e and non- oxic and has good de elopmen p ospec s. 2. Ma e ial and Me hods 2.1. Ma e ials Pep ides we e pu chased om Pep ide Syn hesis Company. GEM and PTX we e e- cei ed om he Na ional Ins i u e o Food and D ugs (Shanghai, China). Unless o he wise speci ied, all o he eagen s we e ob ained om Sigma-Ald ich (S . Louis, MO, USA). 2.2. Me hods 2.2.1. Design, Syn hesis, Sepa a ion and Pu i ica ion o he Pep ide Fi s , based on MAX1 (VKVKVKVK–V D PPT–KVKVKV–NH 2 ) [ 24 ], we designed a pep- ide sequence wi h p ope ies mo e sui able o he deli e y o an icance d ugs. Acco ding o p e ious expe imen s [ 25 ], we conside ed eplacing lysine (K) wi h glu amic acid (E) in he MAX1 sequence, which would educe he cha ge o he pep ide, inc ease he mechanical s eng h o he gel o med by he pep ide and imp o e he s abili y o he d ug-loaded gel. The isoelec ic poin o o ni hine (O) is 10.80 and ha o lysine (K) is 9.74. We eplaced K in he MAX1 sequence wi h O o make he amino acid side chain mo e sensi i e o p o ona ion as well as pH. Th ough he p e ious in es iga ion, i was disco e ed ha he 15 h amino acid in MAX1 had a signi ican impac on he p ope ies o he pep ide [ 26 ]. In addi ion, p e ious esea ch [ 27 ] showed ha he subs i u ion o K a posi ion 6 in he MAX1 sequence by O could imp o e he acid esponse. Finally, a new pep ide sequence based on MAX1 was designed as ollows. O ni hine was used o subs i u e he amino acid a posi ion 6, and glu amic acid was used o subs i u e he amino acid a posi ion 15. The comple e pep ide sequence o his new pep ide was (VKVKVOVK-V D PPT-KVEKVKV-NH 2 ), and we named i OE. The Fmoc o hogonal p o ec ion s a egy was adop ed in he solid-phase pep ide syn hesis me hod, and a mic owa e syn hesize (CEM) was used o syn hesize OE pep ides [ 28 , 29 ]. We employed RHPLC (SHIMADZU, HPLC LC-10ATVP, Tokyo, Japan) o sepa a e and pu i y he OE pep ide. A e pu i ica ion, a liquid-phase mass spec ome e (LC-MS, ACQUITY UPLC H-Class-XEVO TQD, Wa e s, Mil o d, MA, USA) was used o cha ac e ize he p oduc ob ained by p epa ing he liquid phase. 2.2.2. P epa a ion o GEM+PTX-Loaded Pep ide Hyd ogels In o de o p epa e a GEM+PTX suspension, pacli axel and gemci abine wi h a mass a io o 1:4 [ 30 ] we e dissol ed in a 150 mM sodium chlo ide solu ion. The OE pep ide was dissol ed in he abo e suspension, and a e i was en i ely dissol ed, we adjus ed he pH o he solu ion o 7.4. A e s anding o a pe iod o ime, he d ug-loaded hyd ogel was mo e s able. 2.2.3. In es iga ion o he Gela ion Fac o s o OE Pep ide The e ec s o OE concen a ion and solu ion pH on he gela ion o OE polypep ide we e in es iga ed. We p epa ed OE polypep ide hyd ogels wi h concen a ions o 10, 15 and 20 mg/mL and obse ed hei gel o ma ion unde neu al condi ions. A e ha , we p epa ed 15 mg/mL OE polypep ide hyd ogel and obse ed i s gel o ma ion unde di e en pH condi ions. 2.2.4. pH Sensi i i y o he OE Pep ide Hyd ogel We conduc ed an in i o elease s udy on he OE polypep ide hyd ogel o con i m i s pH esponsi eness. A 15 mg/mL OE polypep ide hyd ogel was p epa ed acco ding o he abo e gel p epa a ion me hod, and 0.25 mg/mL GEM and 1 mg/mL PTX we e loaded in o he OE hyd ogel. The s able d ug-loaded hyd ogel was cen i uged a 1000 pm o 4 min o emo e ai bubbles and s o ed in a e ige a o o e nigh . We washed he su ace o he hyd ogel wice wi h pH 7.4 phospha e bu e . Subsequen ly, we added 1 mL o pH 7.4 phospha e bu e o 1 mL o pH 5.8 phospha e bu e o he OE-GEM-PTX Pha maceu ics 2022,14, 652 4 o 13 hyd ogel. In addi ion, 0.5% Tween 80 was added o he bu e o inc ease he solubili y o pacli axel. A 0.5, 1, 2, 6, 12, 24, 48, 72, 96, 120 and 168 h, all elease solu ions we e aspi a ed o de e mine he con en s o GEM and PTX. A he same ime, we added 1 mL o esh elease luid. The con en s o GEM and PTX we e measu ed by HPLC. The condi ions o de e mining he con en o GEM we e as ollows: column: K omasil 100-5C18 ( 4.6 ×250 mm , 5 µ m); de ec ion wa eleng h: 268 nm; column empe a u e: 40 ◦ C; phase A (wa e ) low a e: 0.95 mL/min; phase B (ace oni ile) low a e: 0.05 mL/min; olume: 20 µ L. The condi ions o de e mining he con en o PTX we e as ollows: column: K omasil 100-5C18 ( 4.6 ×250 mm , 5 µ m); de ec ion wa eleng h: 227 nm; column empe a u e: 40 ◦ C; phase A (wa e ) low a e: 0.52 mL/min; phase B (ace oni ile) low a e: 0.48 mL/min; olume: 20 µL. 2.2.5. Seconda y S uc u e In o de o in es iga e he seconda y s uc u e o pep ide molecules in di e en condi- ions, we employed ci cula dich oism (CD) de ec ion. A Jasco J-810 spec opola ime e was used o ga he he a -ul a iole CD spec um o OE pep ide hyd ogels. The pH 7.4 and pH 5.8 bu e s we e used o dissol e OE polypep ides o p epa e hyd ogels, and 100 µ L o each solu ion was placed in a 0.1 mm qua z cell. The pa ame e se ings we e as ollows: wa eleng h ange: 190–260 nm; bandwid h: 1 nm; esponse ime: 1 s; scanning speed: 50 nm/min. 2.2.6. T ansmission Elec on Mic oscopy (TEM) T ansmission elec on mic oscope images (TEM) we e acqui ed o obse e he mi- c os uc u e o he pep ide unde di e en condi ions. Pep ide hyd ogels wi h pH 7.4 and pH 5.8 we e p epa ed. A d op o he dilu ed solu ion was aken and placed e enly on he ca bon-coa ed coppe mesh. We used il e pape o abso b he excess liquid a e 3 min. Then, we used 2% phospho ungs ic acid solu ion o nega i e s aining o 3–5 min. A e d ying unde in a ed ligh , a TEM (Hi achi, HT-7700, Tokyo, Japan) was used o obse e he mic os uc u e o he OE pep ide sample. 2.2.7. Rheological S udy The pep ide hyd ogel used o an i umo d ug deli e y equi es high mechanical s eng h o p e en i s collapse a he umo si e and p e en elease and leakage o he d ugs. Since his deli e y ca ie o ms a gel in i o , i is necessa y o deli e i o he umo si e by injec ion. The e o e, he gel also needs o be dilu ed o he injec ion. A he same ime, i mus quickly e o m in o a hyd ogel wi h sui able mechanical s eng h a e being injec ed in o he body. In his s udy, we measu ed he mechanical s eng h and injec able p ope ies o he hyd ogel ca ie by measu ing iscoelas ic pa ame e s such as he modulus o s o age (G 0 ) and loss modulus (G) o polypep ide hyd ogels by o a ional heome e . In o de o s udy he mechanical s eng h as well as he injec abili y o he hyd ogel sys em, he iscoelas ic pa ame e s we e measu ed by he HAAKE600 o a y heome e (Japan Jasco Company, Tokyo, Japan). We p epa ed blank OE pep ide hyd ogels and OE pep ide hyd ogels loaded wi h GEM+PTX. Dynamic equency scanning and dynamic ime scanning we e pe o med on blank OE hyd ogel and d ug-loaded OE polypep ide hyd ogel, espec i ely. The pa ame e s o he dynamic equency sweep we e se as ollows: he equency was 0.1–100 ad/s, and he shea o ce was 1%. The pa ame e s o he dynamic ime sweep we e se as ollows: ixed equency: 1 Hz; shea o ce: 1% a 0–3 min, 100% a 3–5 min, and 1% a 5–37 min. Then, he abo e p ocess was epea ed a 37–70 min. 2.2.8. In Vi o Cy o oxici y S udies Cell Cul u e The mouse b eas cance cell line (4T1) was pu chased om he Cell Resou ce Cen e o Shanghai Ins i u e o Biological Sciences, Shanghai, China. 4T1 cells we e cul u ed in Pha maceu ics 2022,14, 652 5 o 13 DMEM medium con aining 10% e al bo ine se um and double an ibodies in a 5% CO 2 incuba o a 37 ◦C. Biocompa ibili y o Blank Pep ide Hyd ogel In o de o con i m he sa e y and non- oxici y o he gel ca ie , a biocompa ibili y es o he OE hyd ogel was ca ied ou h ough he Cell Coun ing Ki -8 (CCK8). The OE polypep ide solu ions we e p epa ed wi h concen a ions o 0, 1, 10, 50, 100 and 500 µ g/mL, and he pH o he solu ion was adjus ed o o m a s able hyd ogel. 4T1 cells we e cul u ed in he abo e-men ioned medium. When he densi y eached 90%, he cells we e diges ed o ob ain a esuspension. In a 96-well pla e, 4T1 cells we e seeded a a concen a ion o 5×104 cells/mL wi h 100 mic oli e s pe well. A e s abilizing o 24 h, he p epa ed OE polypep ide solu ion was dilu ed wi h cul u e medium. The o iginal medium was emo ed om a se o 96-well pla es, and esh medium con aining polypep ides was added as a con ol g oup. F esh medium was added o ano he se o 96-well pla es. A e 24 h, we added 10 mic oli e s o CCK8 solu ion o each well as he expe imen al g oup. A e incuba ing he cells o 1–2 h, we used a mic opla e eade o ead a 450 nm. Ano he g oup con aining only medium was used as he blank g oup. The cell su i al a io was hen calcula ed. The calcula ion o mula o he cell su i al a e o each g oup was as ollows: cell iabili y(%)=Aexpe imen al g oup −Ablank g oup Acon ol g oup −Ablank g oup ×100% In Vi o An i umo E icacy In his pa , we s udied he abili y o di e en concen a ions o GEM and PTX o inhibi cell p oli e a ion. Solu ions o 15 mg/mL OE pep ide we e p epa ed. G oups we e de e mined as ollows: (1) no mal saline a a ce ain concen a ion (con ol g oup); (2) OE pep ide hyd ogel wi h he GEM solu ions a concen a ions o 0.025, 0.25, 1.25, 2.5 and 12.5 µ g/mL (GEM@H g oup); (3) OE pep ide hyd ogel wi h he PTX solu ions a concen- a ions o 0.1, 1, 5, 10 and 50 µ g/mL (PTX@H g oup); (4) ee GEM+PTX solu ion wi h he same concen a ions as he abo e g ouping (GEM+PTX g oup); (5) OE pep ide hyd o- gel wi h GEM+PTX wi h he same concen a ions as he abo e g ouping (GEM+PTX@H g oup). A e 4T1 cells we e cul u ed o 48 h, in i o cy o oxici y was de e mined by he CCK-8 me hod [31]. 2.2.9. In Vi o An i umo Resea ch S a emen All animal expe imen s complied wi h he ARRIVE guidelines and we e ca ied ou in acco dance wi h he U.K. Animals (Scien i ic P ocedu es) Ac , 1986. Es ablishmen o he Tumo Model We ob ained BALB/c emale mice om Nanjing Jilin Bio echnology De elopmen Co., L d. (Nanjing, China). Animal expe imen s we e ca ied ou acco ding o he guidelines o he Eu opean Communi y. A e disin ec ing he skin o he igh a mpi o emale BALB/c mice, hey we e inocula ed wi h 4T1 mouse b eas cance cell suspension wi h a concen a ion o 5 × 107 cells/mL. Guidelines o ca e and use o labo a o y animals o China Pha maceu ical Uni e si y we e used o pe o m animal s udies, and hese s udies we e duly app o ed by he animal e hics commi ee o China Pha maceu ical Uni e si y (no.: 2021-12-018; da e: 16 Decembe 2021). In Vi o D ug E icacy E alua ion Twen y- i e umo -bea ing mice we e andomly di ided in o i e g oups. The umo s o hese mice we e abou 100 mm 3 and weighed 16~18 g. The 5 g oups o mice we e subcu aneously injec ed as ollows: (1) 100 µ L/20 g saline (con ol g oup); (2) 100 µ L/20 g Pha maceu ics 2022,14, 652 6 o 13 GEM suspension, in which he GEM dose was 10 mg/kg (GEM@H g oup); (3) 100 µ L/20 g PTX suspension, in which he PTX dose was 10 mg/kg (PTX@H g oup); (4) 100 µ L/20 g GEM+PTX suspension, in which he GEM dose was 1.25 mg/kg and he dose o PTX was 5 mg/kg (GEM+PTX g oup); (5) 100 µ L/20 g OE pep ide hyd ogel loaded wi h GEM+PTX (GEM+PTX@H g oup). A e 0, 1, 4 and 7 days, we measu ed he body weigh as well as he umo olume o umo -bea ing mice. The umo olume was calcula ed as ollows: Tumo olumemm3=Longes umo diame e −Sho es umo diame e 2 2 On he se en h day, all mice we e eu hanized. Thei umo s we e emo ed o measu e hei weigh and olume. TUNEL analysis was also pe o med on umo sec ions om he i e g oups o mice. In addi ion, he hea s, li e s, spleens, lungs and kidneys o mice in he con ol g oup, GEM+PTX g oup and GEM+PTX@H g oup we e emo ed and examined by H&E s aining. Biodis ibu ion o he Pep ide Hyd ogel The in i o imaging sys em can obse e he dis ibu ion o Cy5 luo escen p obes in umo -bea ing mice, which can be used o in es iga e he biodis ibu ion o d ug-loaded hyd ogels. We andomly di ided six umo -bea ing mice in o wo g oups, wi h h ee mice in each g oup. These mice had simila body weigh s and umo sizes. The 2 g oups o mice we e subcu aneously injec ed as ollows: (1) 100 µ L/20 g Cy5 p obe, in which he dose was 1 mg/kg (Cy5 g oup); (2) 100 µ L/20 g Cy5-loaded OE pep ide hyd ogel, in which he OE dose was 1 mg/kg (OE-Cy5 g oup). Fluo escence imaging o anes he ized umo -bea ing mice was pe o med 1, 6, 12, 24, 48 and 96 h a e adminis a ion. A e ha , all mice we e eu hanized, and hei hea s, lungs, li e s, spleens, kidneys and umo s we e emo ed. We employed an image o obse e he dis ibu ion o he Cy5 p obes in bo h g oups. Biocompa ibili y o OE Pep ide Hyd ogels In Vi o The in i o biocompa ibili y o OE polypep ide hyd ogels was in es iga ed o de- e mine whe he he ma e ials a e sa e in i o . Two g oups o nude mice we e injec ed wi h no mal saline o 15 mg/mL blank OE polypep ide hyd ogel subcu aneously in hei backs. The injec ion dose was 100 µ L/20 g. A e 96 h, all mice we e eu hanized. We e- mo ed he subcu aneous issues a ound he injec ion si e on he back. In o de o obse e whe he i had caused in lamma ion, skin nec osis o o he ad e se eac ions, H&E s aining was employed. 3. Resul s and Discussion 3.1. P epa a ion o GEM+PTX-Loaded Pep ide Hyd ogel The gela ion o OE a di e en concen a ions and pH is shown in Tables 1and 2. Table 1. Gela ion o OE pep ides a di e en concen a ions. Concen a ion o Pep ide (mg/mL) Resul 10 UG 15 SG: 30 s 20 SG: immedia ely SG: The ime equi ed o s able gel o ma ion; UG: unable o o m a s able gel. Table 2. Gela ion o OE pep ides a di e en pH. pH Resul 6.0 UG 7.4 SG: 30 s 8.0 UG SG: The ime equi ed o s able gel o ma ion; UG: unable o o m a s able gel. Pha maceu ics 2022,14, 652 7 o 13 The esul s showed ha a s able hyd ogel could be o med when he concen a ion o he OE pep ide eached a ce ain le el. I was also ound ha he hyd ogel o med mo e quickly when he concen a ion o he OE pep ide was highe . This is shown in Tables 1and 2 . The OE pep ide o med a s able hyd ogel unde neu al condi ions. The e- o e, i is a pH-sensi i e ca ie ma e ial and can be loaded wi h an i umo d ugs. 3.2. pH Sensi i i y o he OE Pep ide Hyd ogel In his pa , we pe o med an in i o elease expe imen using he OE pep ide. Figu e 1A shows he esul s. The o al elease o PTX om he OE hyd ogel in he medium a pH 5.8 was 96.90% in 7 days, and in he medium a pH 7.4, i was 38.98%. The o al elease o GEM om he OE hyd ogel in he medium a pH 5.8 and pH 7.4 was 99.99% and 99.63% in 3 days. The eason o his esul is ha GEM is highly hyd ophilic and can quickly di use in o aqueous solu ions. Pha maceu ics2022,14,x 8o 14    Figu e1.Cumula i e eleaseo GEMandPTX omOEhyd ogelindi e en bu e solu ionswi h pH5.8andpH7.4(A).Ci cula dich oismch oma og amso blankOEpep ideinpH5.8andpH 7.4bu e s(B).T ansmissionelec onmic oscopyo blankOEpep idea pH5.8(C)andpH7.4 (D).Dynamic equencyscanningo blankOEhyd ogelsandGEM+PTX‐loadedhyd ogels(E). Dynamic imescanningo OEpep idehyd ogels(F). 3.3.Seconda yS uc u e Theci cula dich oism esul sa eshowninFigu e1B.Thepic u esshow ha when OEisunde neu alcondi ionso pH7.4,i hasaβ‐ oldeds uc u e,asindica edby he singlenega i epeakshape,whileunde acidiccondi ionso pH5.8,i p esen scha ac‐ e is icso a andomcoil, ha is,nega i epeaksa sho wa eleng hs. These esul sindica e ha  hispep idesequencecan o mas ablehyd ogelbyβ‐ oldingunde neu alcondi ionsandisdes oyedunde acidiccondi ions.Thisshows ha  heOEpep ideispH‐ esponsi e. 3.4.TEM Figu e1C,Dshows he esul so elec onmic oscopyo  he15mg/mLneu aland acidicOEpep idehyd ogel. Figu e 1. Cumula i e elease o GEM and PTX om OE hyd ogel in di e en bu e solu ions wi h pH 5.8 and pH 7.4 ( A ). Ci cula dich oism ch oma og ams o blank OE pep ide in pH 5.8 and pH 7.4 bu e s ( B ). T ansmission elec on mic oscopy o blank OE pep ide a pH 5.8 ( C ) and pH 7.4 ( D ). Dynamic equency scanning o blank OE hyd ogels and GEM+PTX-loaded hyd ogels ( E ). Dynamic ime scanning o OE pep ide hyd ogels (F). Pha maceu ics 2022,14, 652 8 o 13 Acco ding o he esul s, he OE hyd ogel is esponsi e o pH. I can con inuously elease PTX unde acidic condi ions, and acidic condi ions a e simila o he umo mi- c oen i onmen . The e o e, he OE pep ide no only has good gela ion p ope ies bu also esponds in a pH-sensi i e manne . The elease speed can be con olled by egula ing he pep ide concen a ion. The esul s show ha OE has he desi ed pep ide sequence. 3.3. Seconda y S uc u e The ci cula dich oism esul s a e shown in Figu e 1B. The pic u es show ha when OE is unde neu al condi ions o pH 7.4, i has a β - olded s uc u e, as indica ed by he single nega i e peak shape, while unde acidic condi ions o pH 5.8, i p esen s cha ac e is ics o a andom coil, ha is, nega i e peaks a sho wa eleng hs. These esul s indica e ha his pep ide sequence can o m a s able hyd ogel by β - olding unde neu al condi ions and is des oyed unde acidic condi ions. This shows ha he OE pep ide is pH- esponsi e. 3.4. TEM Figu e 1C,D shows he esul s o elec on mic oscopy o he 15 mg/mL neu al and acidic OE pep ide hyd ogel. Compa ing Figu e 1C,D shows ha unde neu al condi ions, OE o ms a slende nanome e ibe ne wo k. The illus a ed ne wo k is sui able o packaging an i umo d ugs. Unde acid condi ions, elec on ansmission mic oscopy did no show a nano ib ous pep ide. The pep ide is sho e and sca e ed, which is di e en om i s o m unde neu al condi ions. I can be concluded ha his pep ide has good pH sensi i i y. 3.5. Rheological S udy The scanning esul s o OE sequences o blank hyd ogels and d ug-loading gels a e shown in Figu e 1E. Figu e 1F shows he esul s a e he hyd ogel was injec ed in o he speci ied pa by exe ing h us in he sy inge. A e 3 min o shea ing, he shea o ce suddenly ose o 100% and hen e u ned o 1%. The esul s o dynamic ime scanning showed ha when he shea o ce inc eased o 100% o 3 min, G 0 changed om 135 Pa o 143 Pa, while when he shea o ce dec eased o 1% o 5 min, he G 0 o he pep ide hyd ogel quickly eco e ed o mo e han 4000 Pa. In Figu e 1E, he e is no signi ican change in heological p ope ies, ega dless o whe he o no he d ug is loaded. The alue o he s o age modulus (G 0 ) is abou 10 imes la ge han ha o he loss modulus (G 00 ), which p o es ha i is a s able ca ie ma e ial wi h s ong igidi y and solid-like p ope ies. The esul s in Figu e 1F indica e ha his hyd ogel can p oduce a ce ain deg ee o luidi y when subjec ed o shea o ce and can quickly e o m in o a s able pep ide hyd ogel a e he shea o ce is emo ed. This indica es ha he pep ide gel can be injec ed, and hus, he pain caused by su gical implan a ion can be educed. 3.6. In Vi o Cy o oxici y S udies Based on he cha ac e iza ion, he cy o oxici y o d ug-loaded OE was u he s udied. The CCK-8 assay was used o in es iga e he cy o oxici y o blank hyd ogels and o explo e hei biocompa ibili y. In Figu e 2A, he su i al a es o 4T1 cells a e 48 h o co-incuba ion wi h OE polypep ide hyd ogels wi h di e en concen a ions a e all abo e 97%. The esul s show ha concen a ions wi hin his ange a e sa e. The ma e ial may be sui able o an i umo d ug deli e y. Figu e 2B shows he supp ession o 4T1 cell p oli e a ion by GEM and PTX a di e en concen a ions. As shown in he igu e, he cy o oxici y o OE hyd ogels wi h d ugs was simila o ha o ee GEM and PTX. This indica es ha GEM and PTX can s ill exe an i umo ac i i y a e being inco po a ed in o pep ide hyd ogels. Compa ing he esul s o bo h GEM and PTX encapsula ed by hyd ogels and he esul s o he indi idual d ugs encapsula ed by hyd ogels, i is con i med ha he combined ea men has a be e umo inhibi ion e ec . Pha maceu ics 2022,14, 652 9 o 13 Pha maceu ics2022,14,x 9o 14   Compa ingFigu e1C,Dshows ha unde neu alcondi ions,OE o msaslende  nanome e  ibe ne wo k.Theillus a edne wo kissui able o packagingan i umo  d ugs.Unde acidcondi ions,elec on ansmissionmic oscopydidno showanano‐ ib ouspep ide.Thepep ideissho e andsca e ed,whichisdi e en  omi s o m unde neu alcondi ions.I canbeconcluded ha  hispep idehasgoodpHsensi i i y. 3.5.RheologicalS udy Thescanning esul so OEsequenceso blankhyd ogelsandd ug‐loadinggelsa e showninFigu e1E.Figu e1Fshows he esul sa e  hehyd ogelwasinjec edin o he speci iedpa byexe ing h us in hesy inge.A e 3mino shea ing, heshea  o ce suddenly ose o100%and hen e u ned o1%.The esul so dynamic imescanning showed ha when heshea  o ceinc eased o100% o 3min,G′changed om135Pa o143Pa,whilewhen heshea  o cedec eased o1% o 5min, heG′o  hepep ide hyd ogelquickly eco e ed omo e han4000Pa. InFigu e1E, he eisnosigni ican changein heologicalp ope ies, ega dlesso  whe he o no  hed ugisloaded.The alueo  hes o agemodulus(G′)isabou 10 imesla ge  han ha o  helossmodulus(G″),whichp o es ha i isas ableca ie  ma e ialwi hs ong igidi yandsolid‐likep ope ies.The esul sinFigu e1Findica e ha  hishyd ogelcanp oduceace aindeg eeo  luidi ywhensubjec ed oshea  o ce andcanquickly e o min oas ablepep idehyd ogela e  heshea  o ceis emo ed. Thisindica es ha  hepep idegelcanbeinjec ed,and hus, hepaincausedbysu gical implan a ioncanbe educed. 3.6.InVi oCy o oxici yS udies Basedon hecha ac e iza ion, hecy o oxici yo d ug‐loadedOEwas u he s ud‐ ied.TheCCK‐8assaywasused oin es iga e hecy o oxici yo blankhyd ogelsand o explo e hei biocompa ibili y.InFigu e2A, hesu i al a eso 4T1cellsa e 48ho  co‐incuba ionwi hOEpolypep idehyd ogelswi hdi e en concen a ionsa eallabo e 97%. The esul sshow ha concen a ionswi hin his angea esa e.Thema e ialmaybe sui able o an i umo d ugdeli e y.Figu e2Bshows hesupp essiono 4T1cellp oli ‐ e a ionbyGEMandPTXa di e en concen a ions.Asshownin he igu e, hecy o‐ oxici yo OEhyd ogelswi hd ugswassimila  o ha o  eeGEMandPTX.Thisindi‐ ca es ha GEMandPTXcans illexe an i umo ac i i ya e beinginco po a edin o pep idehyd ogels.Compa ing he esul so bo hGEMandPTXencapsula edbyhy‐ d ogelsand he esul so  heindi iduald ugsencapsula edbyhyd ogels,i iscon‐ i med ha  hecombined ea men hasabe e  umo inhibi ione ec .  Figu e 2. Cell iabili y o blank OE hyd ogel ( A ). The cell inhibi ion a e o GEM-loaded OE pep- ide hyd ogel, PTX-loaded OE pep ide hyd ogel, GEM+PTX-loaded OE pep ide hyd ogel and ee GEM+PTX (B). ** p< 0.01. 3.7. In Vi o An i umo Resea ch 3.7.1. In Vi o E alua ion o he E icacy o Pep ide Hyd ogels The umo -bea ing mice used o he in i o an i umo s udy we e di ided andomly in o i e g oups, and g oups we e adminis e ed physiological saline (con ol g oup), OE pep ide hyd ogel loaded wi h GEM (GEM@H g oup), OE pep ide hyd ogel loaded wi h PTX (PTX@H g oup), GEM and PTX suspension (GEM+PTX g oup) o OE pep ide hyd ogel loaded wi h PTX and GEM (GEM+PTX@H g oup). The weigh o umo -bea ing mice was moni o ed, and he umo olume was measu ed and calcula ed on days 0, 1, 4 and 7 a e adminis a ion. As obse ed in Figu e 3A, he body weigh o umo -bea ing mice sligh ly dec eased 1 day a e subcu aneous injec ion wi h PTX and GEM suspension, and he a e age weigh o umo -bea ing mice d opped om 16.5 g o 13.7 g in 6 days. The body weigh o mice in he GEM@H and PTX@H g oups was mo e s able; wi hin 7 days a e adminis a ion, he change end o he body weigh o he umo -bea ing mice in he GEM+PTX@H g oup showed no signi ican di e ence om he blank g oup. The umo olume changes in mice wi hin 7 days a e adminis a ion a e shown in Figu e 3B, om which i can be seen ha he umo s in he con ol g oup g ew apidly, wi h he umo olume inc easing apidly om 97.4 mm 3 o 743.9 mm 3 wi hin 7 days. In con as , he umo s in he GEM+PTX@H g oup showed a mo e p onounced inhibi o y e ec a e adminis a ion han he GEM+PTX g oup and e en ended o become g adually smalle . The umo was inhibi ed a e 7 days, and he inhibi ion a e was 90.84%. Figu e 3C,D show he umo weigh and olume o umo -bea ing mice. The minimum a e age weigh o he umo s, which was 0.19 g, was obse ed in he GEM+PTX@H g oup. Thus, he inhibi ion o umo s was highes in he GEM+PTX@H g oup. These esul s show ha when ac ing as a d ug deli e y ehicle, he OE pep ide hyd ogel can elease a la ge amoun o GEM and slowly elease PTX. Figu e 3E shows he esul s o hema oxylin and eosin (H&E) s aining o he hea , li e , spleen, lung and kidney o he umo -bea ing mice. I shows ha in he GEM+PTX g oup, no mal issues we e damaged o some ex en . Figu e 3F shows he TdT-media ed dUTP nick-end labeling (TUNEL) assay esul s o he umo sec ions om each g oup o mice. I is e iden ha he la ges luo escen a ea is ound in he GEM+PTX@H g oup.