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Kev paub

Liposome-Based Co-Immunotherapy Nrog TLR Agonist Thiab CD47-SIRP Checkpoint Blockade Rau Kev Kho Mob Cancer Zoo

Nov 28, 2023

Abstract:

Antitumor tiv thaiv kab mob yog ib qho tseem ceeb ntawm kev kho mob qog noj ntshav thiab feem ntau yog kho los ntawm lub cev tiv thaiv kab mob hauv lub cev, uas ua lub luag haujlwm tseem ceeb hauv kev pib thiab tsim kho lub cev tiv thaiv kab mob. Cov pov thawj tshwm sim tau txheeb xyuas qhov chaw tiv thaiv kab mob hauv lub cev thiab cov qauv lees paub, xws li CD47 thiab Tus Hu Xov Tooj Zoo li 7 (TLR7), raws li kev cog lus kho mob rau kev kho mob qog noj ntshav. Raws li cov fusion protein Fc-CV1, uas suav nrog qhov sib txawv ntawm SIRP qhov sib txawv (CV1), thiab Fc fragment ntawm tib neeg IgG1 cov tshuaj tiv thaiv kab mob, peb tau siv ib qho kev npaj ua ke Fc-CV1 rau imiquimod (TLR7 agonist)-loaded liposomes ( CILPs) kom nquag plias lub hom phiaj CT26. WT syngeneic colon qog qauv. Cov kev tshawb fawb hauv vitro tau qhia tias CILPs tau nthuav tawm cov khoom tso tawm zoo tshaj plaws thiab kev txhawb nqa ntawm tes piv rau imiquimod dawb. Hauv vivo, kev soj ntsuam tau ua pov thawj tias CILPs pom muaj txiaj ntsig zoo dua hauv cov qog nqaij hlav thiab muaj kev cuam tshuam cov qog nqaij hlav tseem ceeb dua li pawg tswj hwm. Qhov kev npaj tshuaj tiv thaiv kab mob no muaj qhov zoo ntawm cov koob tshuaj tsawg thiab tsis muaj tshuaj lom. Cov txiaj ntsig no tau pom tias kev sib xyaw ntawm kev tiv thaiv kab mob tiv thaiv kab mob (ICB) kev kho mob thiab kev tiv thaiv kab mob hauv lub cev, xws li Fc-CV1 thiab imiquimod-loaded liposome npaj siv hauv txoj kev tshawb no, tuaj yeem sawv cev rau lub tswv yim zoo rau kev kho mob qog noj ntshav.

Benefits of cistanche tubulosa-Antitumor

Cov txiaj ntsig ntawm cistanche tubulosa-Antitumor

Ntsiab lus:

imiquimod; liposome; Fc-CV1; immunotherapy; mob qog noj ntshav

1. Taw qhia

Kev mob qog noj ntshav thoob ntiaj teb txuas ntxiv nce txhua xyoo, ua rau muaj kev hem thawj loj rau kev noj qab haus huv ntawm cov pej xeem hauv ntiaj teb thiab ua rau lub nra hnyav rau cov kab ke kho mob. Raws li World Health Organization tsab ntawv ceeb toom, xyoo 2020, 19.29 lab tus neeg mob qog noj ntshav tshiab thiab 9.96 lab tus neeg mob qog noj ntshav tau tshwm sim thoob ntiaj teb [1]. Txawm li cas los xij, nrog kev nce qib txuas ntxiv hauv kev kuaj mob, kev phais, kev kho hluav taws xob, thiab kev siv tshuaj khomob, tag nrho cov ciaj sia taus thiab lub neej zoo ntawm cov neeg mob qog noj ntshav tau zoo tuaj. Qhov tseem ceeb, kev siv tshuaj tiv thaiv kab mob, uas tau pib nrog cov tshuaj tiv thaiv Coley hauv xyoo 1890s [2], ua rau muaj kev kho mob hluav taws xob thiab kws khomob thiab tau ua kom muaj kev vam meej hauv kaum xyoo dhau los, tau txais txiaj ntsig zoo rau cov neeg mob qog noj ntshav.

Tumor immunotherapy tau raug lees paub tias nws zoo heev biocompatibility thiab siab tshwj xeeb thiab muaj peev xwm ua rau lub cev tiv thaiv kab mob kom tshem tawm cov qog hlwb [3–5]. Ob lub tswv yim tseem ceeb rau kev kho mob qog noj ntshav yog kev tiv thaiv kab mob thiab kev tiv thaiv kab mob normalization [6]. Cov qauv ntawm kev tiv thaiv kab mob feem ntau muaj xws li kev kho cytokine, tshuaj tiv thaiv qog nqaij hlav, thiab monoclonal antibodies. Hu xov tooj zoo li receptors (TLRs) yog suav tias yog lub hom phiaj rau kev tiv thaiv kab mob qog noj ntshav vim lawv lub luag haujlwm hauv kev ua kom lub cev tsis muaj zog. TLR agonists tau raug tsim los ua ib qho kev tiv thaiv kab mob, thiab ntau yam ntawm TLR agonists tau ntsib kev sim tshuaj [7]. Monophosphoryl lipid A thiab imiquimod, TLR4, thiab TLR7 agonists, ntsig txog, tau txais kev pom zoo los ntawm Food and Drug Administration rau daim ntawv thov kho mob [7–9]. Imiquimod, TLR7 agonist [9], ua kom lub cev tiv thaiv kab mob thiab txhim kho cov qog nqaij hlav los ntawm kev ua rau TLR7 [10,11]. Lub tswv yim tiv thaiv kab mob normalization yog sawv cev los ntawm cov tshuaj tiv thaiv kab mob tiv thaiv kab mob tiv thaiv kab mob (ICB) uas tswj cov kab mob qog noj ntshav tsis zoo. Txij li thawj zaug tshuaj xyuas qhov inhibitor, ipilimumab tau pom zoo rau kev kho mob hauv Tebchaws Meskas hauv xyoo 2011, cov tshuaj tiv thaiv kab mob tiv thaiv kab mob, xws li PD1 / PDL1 inhibitors, tau ua rau muaj kev cuam tshuam hauv kev kho mob qog noj ntshav [12–14]. Tsuas yog cov chaw kuaj kab mob tau txais txiaj ntsig zoo ib yam li PD1 / PDL1, qee qhov chaw kuaj kab mob hauv lub cev, xws li SIRP -CD47, kuj tau txais kev saib xyuas ntau thiab dhau los ua thaj chaw tseem ceeb hauv kev txhim kho tshuaj tiv thaiv kab mob [15–17].

SIRP -CD47 qhov chaw kuaj xyuas tau pom thawj zaug hauv xyoo 1999 [18,19]. SIRP yog CD47 receptor qhia nyob rau hauv nruab nrab paj hlwb neurons [20] thiab myeloid hlwb, xws li monocytes, macrophages, granulocytes, thiab dendritic hlwb. CD47 yog "tus kheej-labeling molecule" qhia nyob rau ntawm yuav luag txhua lub cell ib txwm thiab overexpressed ntawm feem ntau qog hlwb [21]. Nyob rau saum npoo ntawm cov qog hlwb, CD47 khi rau SIRP, inhibiting macrophage-mediated phagocytosis, uas yog ib qho tseem ceeb ntawm cov qog tiv thaiv kab mob [22]. Nws tau raug pom tias phagocytosis ntawm macrophages nyob rau hauv cov qog microenvironment tuaj yeem txhim kho thaum kev khi ntawm CD47 thiab SIRP raug thaiv [20–25]. Yog li, cov tshuaj tiv thaiv kab mob tiv thaiv kab mob uas thaiv CD47 / SIRP txoj hauv kev tau tsim thiab siv hauv kev kho mob qog noj ntshav [26].

Desert ginseng—Improve immunity (2)

cistanche tubulosa- txhim kho lub cev tiv thaiv kab mob

Nyem qhov no mus saib Cistanche Enhance Immunity khoom

【Nug ntxiv】 Email: cindy.xue@wecistanche.com / Whats App: 0086 18599088692 / Wechat: 18599088692

Hauv txoj kev tshawb no, peb tau siv kev sib xyaw ntawm imiquimod thiab fusion protein (Fc-CV1) los kho mob qog noj ntshav. CV1 yog ib qho kev hloov kho tib neeg recombinant SIRP protein uas muaj 50, 000-fold ntau dua affinity rau CD47 dua li qub protein [27]. Fc-CV1 tau tsim los siv "Knobs-hauv-qhov" thev naus laus zis, raws li CV1 monomer thiab Fc fragment ntawm tib neeg IgG1. Txhawm rau kov yeej cov kev txwv ntawm kev kho mob tsis zoo thiab cov kab mob toxicity cuam tshuam nrog imiquimod [28,29], peb npaj nanoliposomes los ntawm txoj kev txhaj tshuaj ethanol. Cov nanoliposomes muaj cov yam ntxwv ntawm cov tshuaj toxicity tsawg hauv kev xa tshuaj [30,31]. Tom qab ntawd, imiquimod tau encapsulated hauv liposomes uas nto conjugated Fc-CV1. Thaum kawg, qhov kev npaj nano tshiab no tuaj yeem xa cov TLR7 agonists mus rau cov qog nqaij hlav thiab muaj ob txoj haujlwm ua ICB tshuaj thiab TLR agonist. Nws tau siv los kho CD47+ CT26.WT syngeneic colon qog qauv [32].

2. Cov txiaj ntsig thiab kev sib tham

2.1. Tus cwj pwm ntawm LPs (Blank Liposomes), ILPs (Non-CD47-Targeted Imiquimod-Encapsulated Liposomes), CLPs (CD47 Targeted Liposomes), thiab CILPs (Coupled Fc-CV1 rau Imiquimod (TLR7 agonist)-Loaded Liposomes)

CILPs tau ua tiav tiav los ntawm kev txhaj tshuaj ethanol thiab ammonium sulfate gradient txoj kev. Daim duab 1A qhia txog cov txheej txheem synthesis ntawm CILPs. TEM tsom xam pom tau tias CILPs yog kheej kheej thiab zoo ib yam (Daim duab 1B). Tsis tas li ntawd, qhov txiaj ntsig ntawm DLS tau qhia tias qhov loj me me ntawm plaub liposomes yog unimodal faib (Daim duab 1C). Tus nqi khi (BR) tau txheeb xyuas los ntawm SDS-PAGE, uas tau pom nyob rau hauv daim duab 1D, E. Daim duab 1D qhia qhov hnyav molecular ntawm Fc-CV1 thiab cov txheeb ze band brightness ntawm dialyzed thiab pre-dialyzed CILPs. Lub BR tau suav tias yog 67.13 ± 37.10% los ntawm Duab J's scanning densitometry ntawm electrophoretic bands (Daim duab 1E), muab qhov ua tau rau CD47- tsom rau cov qog hlwb.

Figure 1. (A) Schematic illustration of the synthesis of CILPs. (B) TEM image of CILPs


Daim duab 1. (A) Schematic illustration of the synthesis of CILPs. (B) TEM duab ntawm CILPs

HPLC tau siv los txheeb xyuas qhov ua tau zoo ntawm cov khoom siv encapsulation (EE) ntawm imiquimod (Cov Duab Ntxiv S1). Raws li tau pom nyob rau hauv Daim Duab Ntxiv S1A, qhov dawb imiquimod tau pom ib lub ncov tseem ceeb, thaum CLPs tsis pom qhov ncov nyob rau hauv tib yam kev sim (Cov Duab Ntxiv S1B). Yog li, nws muaj peev xwm txheeb xyuas cov CILPs ncaj qha raws li qhov kev xeem. Raws li qhov xav tau, qhov kev tshawb pom qhov tseem ceeb ntawm CILPs ua ntej thiab tom qab kev lim ntshav tau suav tias yog tag nrho cov ntsiab lus ntawm cov tshuaj thiab cov ntsiab lus ntawm cov tshuaj liposomes, raws li (Cov duab ntxiv S1C, D). EE ntawm imiquimod tau suav tias yog 85.44 ± 4.11%. Piv nrog rau kev tshaj tawm passive-loading drug-xa system [33–35], qhov kev tshawb fawb no tau ua tiav kev ua haujlwm ntawm encapsulation ntau dua thiab cov txheej txheem npaj tau yooj yim dua.

DLS tau pom tias cov diameters ntawm LPs yog 111.567 ± 0.655 nm, thaum CLPs, ILPs, thiab CILPs pom qhov loj me me ntawm 117.467 ± 0.34{{10} } nm, 120.233 ± 0.776 nm, thiab 13{{30}}}033 ± 0.974 nm, ntsig txog (Table 1). Kev hloov pauv ntawm qhov loj ntawm cov liposomes no tuaj yeem raug ntaus nqi los ntawm kev hloov kho Fc-CV1 thiab imiquimod. PDI tau ze rau 0.1 (Table 1), qhia tias cov tsheb xa tshuaj no tau zoo sib xws hauv lawv qhov kev faib tawm. Tsis tas li ntawd, qhov zeta-potential qhov tseem ceeb ntawm LPs, CLPs, ILPs, thiab CILPs raug ntsuas los ntawm −2.231 ± 0.167 mV, −2.492 ± 0.033 mV, −2.383 ± 0.070 mV, thiab 5-7 ± 0.07mV (5-7 ± 0.07mV) , tawm tswv yim hais tias qhov chaw-conjugated Fc-CV1 thiab encapsulated imiquimod muaj me ntsis cuam tshuam rau tus nqi ntawm liposomes.

Table 1. Qhov diameters, zeta-potential, PDI, thiab EE ntawm LPs, CLPs, ILPs, thiab CILPs, feem.

Table 1. The diameters, zeta-potential, PDI, and EE of LPs, CLPs, ILPs, and CILPs, respectively.


2.2. Hauv Vitro Cell Uptake Efficiency

Lub cell uptake efficiency ntawm CILPs tau soj ntsuam los ntawm fluorescence siv ntawm Cy5 coj los ntawm CT26.WT hlwb siv lub High-Content Imaging System. Raws li pom nyob rau hauv daim duab 2, piv nrog dawb Cy5 thiab ILP-Cy5 pawg, CILPs-Cy5 pawg tau nthuav tawm lub peev xwm ntawm cov cell muaj zog dua tom qab 30 min ntawm incubation. Cov txiaj ntsig no tau lees paub tias Fc-CV1 txhim kho lub peev xwm ntawm lub cev ntawm imiquimod-loaded liposomes vim qhov tshwj xeeb affinity rau Fc-CV1 receptor, ua rau muaj zog tiv thaiv qog noj ntshav.


Figure 2. The images of CT26.WT cells after incubation with the free Cy5, ILPs-Cy5, and CILPs-Cy5 (red) for 0.5 h, and staining with DAPI (blue). Scale bar: 50 µm. (n = 3). * p < 0.05; **** p < 0.0001


Daim duab 2. Cov duab ntawm CT26.WT hlwb tom qab incubation nrog dawb Cy5, ILPs-Cy5, thiab CILPs-Cy5 (liab) rau 0.5 h, thiab staining nrog DAPI (xiav). Scale bar: 50 µm. (n {{10}}). p <0.05; **** p <0.0001

2.3. Tso Curves ntawm Liposomes

Cov tshuaj tso tawm nkhaus ntawm cov dawb imiquimod, ILPs, thiab CILPs tau ntsuas nyob rau hauv ib qho kev simulated hauv vivo ib puag ncig. Daim duab 3 qhia tau hais tias qhov dawb imiquimod tau tso tawm tag nrho tom qab 1 h, thaum qhov tso tawm tus nqi (RR) ntawm imiquimod uas encapsulated nrog ILPs thiab CILPs tsuas yog 54.75 ± 4.08% thiab 39.70 ± 0.05% ntawm 12 h , raws. Tsis tas li ntawd, kev tso tawm qeeb ntawm imiquimod hauv ILPs thiab CILPs tuaj yeem raug kaw nrog cov tshuaj tswj cov khoom tso tawm ntawm liposomes. RR ntawm imiquimod hauv ILPs thiab CILPs pom tias tsis muaj qhov sib txawv ntawm 96 h, qhia tias "post insertion" pom qhov cuam tshuam tsis zoo rau daim nyias nyias ntawm liposomes.

figure 3. Imiquimod release curves of the free imiquimod, ILPs, and CILPs in PBS at 37 ◦C. Data are presented as means ± standard deviation (n = 3). * p < 0.05, ** p < 0.01

daim duab 3. Imiquimod tso cov nkhaus ntawm cov dawb imiquimod, ILPs, thiab CILPs hauv PBS ntawm 37 ◦C. Cov ntaub ntawv raug nthuav tawm raws li txhais tau tias ± tus qauv sib txawv (n=3). * p < 0 05, ** p < 0.01

2.4. Cytotoxicity Study

Txhawm rau kawm txog cytotoxicity ntawm CILPs hauv vitro, CCK{{0}} kev soj ntsuam tau ua rau ntawm CT26.WT hlwb. Raws li pom nyob rau hauv daim duab 4, CILPs ntawm tsawg concentrations (0.1–1 µg/mL) tsis pom cytotoxicity, uas yog raws li kev ruaj ntseg ntawm kev txhaj tshuaj. Cell viability tau qis me ntsis tom qab 5 µg / mL imiquimod kev kho mob, tej zaum vim yog ROS thiab endoplasmic reticulum-mediated immunogenic cell tuag vim yog imiquimod [36]. Ntxiv mus, CILPs ua rau txo qis hauv cell viability piv rau cov pab pawg tswj hwm, uas yog vim muaj kev sib koom ua ke ntawm imiquimod thiab Fc-CV1.


Figure 4. The viability of the cells treated with Fc-CV1, imiquimod, CLPs, ILPs, and CILPs for 24 h. The concentration of the Fc-CV1 and imiquimod were both 0–10 µg/mL. * p < 0.05, ** p < 0.01, ns = no significant differences

Daim duab 4. Kev muaj peev xwm ntawm cov hlwb kho nrog Fc-CV1, imiquimod, CLPs, ILPs, thiab CILPs rau 24 teev. Qhov concentration ntawm Fc-CV1 thiab imiquimod yog ob qho tib si 0–10 µg/mL. * p < 0.05, ** p < 0.01, ns=tsis muaj qhov sib txawv loj

2.5. Biodistribution Study

Kev faib tawm thiab sib sau ua ke ntawm cov qauv sib txawv hauv cov qog thiab cov kabmob loj ncaj qha cuam tshuam rau kev kho mob. Tom qab kev tswj hwm cov hlab ntsha, cov teeb liab fluorescence tau saib xyuas siv qhov ze-infrared vivisection system los ntsuas qhov kev faib tawm thiab sib sau ua ke ntawm cov qauv sib txawv hauv cov qog thiab cov kabmob loj tom qab 24 teev. Raws li pom nyob rau hauv daim duab 5 tag nrho cov fluorescence siv ntawm ILPs-Cy5 thiab CILPs-Cy5 yog siab tshaj dawb Cy5, qhia tau hais tias cov liposomes muaj ib tug ntev-kwv yees nyob rau hauv vivo. Tsis tas li ntawd, kev siv tshuaj fluorescence ntawm CILPs-Cy5 pab pawg kho mob yog qhov siab tshaj plaws hauv cov qog nqaij hlav, uas tuaj yeem raug sau tseg rau cov qog-targeting nyhuv ntawm Fc-CV1 ligands, yog li txhim kho cov nyhuv tiv thaiv qog. Strikingly, fluorescence siv ntawm daim siab thiab spleens muaj zog dua lwm yam kabmob, uas yog tej zaum vim phagocytosis ntawm liposomes los ntawm lub reticuloendothelial system (RES) nyob rau hauv daim siab thiab lub raum [37].

Figure 5. The distribution of Cy5-fluorescence in tumors and major organs at 24 h after intravenous administration. Data are shown as means ± standard deviation (n = 3). **** p < 0.0001


Daim duab 5. Kev faib tawm ntawm Cy5-fluorescence nyob rau hauv cov qog thiab cov kabmob loj ntawm 24 teev tom qab kev txhaj tshuaj. Cov ntaub ntawv qhia raws li txhais tau tias ± tus qauv sib txawv (n=3). **** p < 0.0001

2.6. Hauv Vivo Tumor Inhibition Study

Lub sijhawm kho mob (Daim duab 6A) thiab cov teebmeem ntawm kev npaj sib txawv tau pom nyob rau hauv daim duab 6. Piv nrog rau pawg PBS thiab LP, lwm pab pawg kho mob tau nthuav tawm qee yam qog-suppressor. Qhov tseem ceeb, cov qog nqaij hlav me me tom qab kev kho mob nrog CILPs, qhia tias CILPs muaj cov qog nqaij hlav zoo heev (Daim duab 6B).

Figure 6. (A) Schematic illustration of anti-colon cancer treatment on the CT26.WT tumor model. (B) The growth curves of tumor volume during the treatment period. (C) Relative tumor photographs and (D) weight of the tumor collected from different treatment groups on day 17. (E) The relative body weight ratio of the body weight compared with 0 day. Data are presented as mean ± standard deviation (n = 5). * p < 0.05, ** p < 0.01, *** p < 0.001.


Daim duab 6. (A) Schematic illustration of anti-colon cancer treatment on the CT26.WT qog model. (B) Kev loj hlob nkhaus ntawm cov qog ntim thaum lub sijhawm kho. (C) Cov duab qog cov txheeb ze thiab (D) qhov hnyav ntawm cov qog uas tau sau los ntawm cov pab pawg kho mob sib txawv rau hnub 17. (E) Tus txheeb ze lub cev qhov hnyav ntawm lub cev hnyav piv nrog 0 hnub. Cov ntaub ntawv raug nthuav tawm raws li txhais tau tias ± tus qauv sib txawv (n=5). * p < 0 05, ** p < 0.01, *** p < 0.001.

Thaum kawg ntawm qhov kev sim, cov nas tau muab txi thiab lawv cov qog tau raug tshem tawm, thaij duab, thiab ntsuas qhov ntsuas qhov ua tau zoo ntawm kev npaj sib txawv. Raws li pom nyob rau hauv daim duab 6C, D, pab pawg kho mob ntawm CILPs muaj cov qog nqaij hlav me tshaj plaws thiab lub teeb tshaj plaws piv rau lwm pab pawg, qhia tias CILPs muaj cov qog nqaij hlav loj tshaj plaws. Table 2 qhia tau hais tias cov qog loj hlob inhibition tus nqi ntawm CILPs tau suav tias yog 84.35%. Tsis tas li ntawd, 84.35% qog nqaij hlav qog tau nce siab dua li ntawm pawg CLPs thiab ILPs, thiab qhov no txhais tau hais tias muaj kev sib koom ua ke ntawm kev thaiv cov "tsis txhob noj kuv" axis thiab ua kom lub cev tiv thaiv kab mob. . Piv nrog rau qhov sib npaug ntawm kev npaj hauv vitro (2.5-5 µg / mL), qhov siab dua inhibition tus nqi yog ze ze rau ntawm cov tshuaj tiv thaiv kab mob tiv thaiv kab mob thiab thaiv lub cev tiv thaiv kab mob hauv vivo, uas tsis muaj nyob hauv vitro.

Table 2. Cov ntaub ntawv ntawm qog hnyav thiab qog loj hlob inhibition (TGI) ntawm ntau pawg kho mob.

Table 2. The data of tumor weight and tumor growth inhibition (TGI) of various treatment groups.


2.7. CILPs nce T-Cell Infiltration thiab Secretion ntawm IFN

Txhawm rau tshawb xyuas lub cev tiv thaiv kab mob ntawm cov qog nqaij hlav, lub cev tiv thaiv kab mob tshwm sim los ntawm CILPs tau txheeb xyuas cov tshuaj immunohistochemically tom kawg ntawm kev tshuaj xyuas qog nqaij hlav. Cov CD4+ thiab CD8+ T hlwb tau nce hauv cov qog nqaij hlav ntawm pawg CILPs (Daim duab 7). Kev sib koom ua ke ntawm Fc-CV1 thiab imiquimod txoj kev kho tau ua rau muaj CD4+ thiab CD8+ T cell infiltration. Tsis tas li ntawd, muaj ib qho tseem ceeb IFN secretion nyob rau hauv cov qog uas tau kho CILPs, uas yog vim CILPs ua rau tus xov tooj hu-zoo li receptor 7 thiab ua kom lub cev tiv thaiv kab mob.

Figure 7. Immunohistochemistry of mouse tumor tissues; positive cells are stained brown. Scale bar, 50 µm (n = 5).

Daim duab 7. Immunohistochemistry ntawm nas qog nqaij hlav; cov hlwb zoo yog stained xim av. Scale bar, 50 µm (n=5).

2.8. Biosecurity Assessment of CILPs

Lub biosafety ntawm CILPs-mediated therapy kuj tseem yog qhov tseem ceeb ntawm kev nkag mus rau qhov kev tshuaj ntsuam xyuas. Thaum lub sij hawm txoj kev tshawb fawb qog nqaij hlav hauv vivo, CILPs tsis ua kom poob ceeb thawj hauv lub sijhawm kho (Daim duab 6D). Cov kab mob biochemical ntawm lub siab thiab lub raum yog tag nrho nyob rau hauv ib txwm muaj raws li tus qauv siv ntau yam ntawm cov nas (Table 3), thiab cov kab mob hauv nruab nrog cev tsis pom muaj kev hloov pauv tseem ceeb, qhia tias CILPs muaj biosafety zoo heev (Daim duab 8). Cov kev tshawb pom no yog qhov tseem ceeb hauv kev ntsuam xyuas lub peev xwm ntawm CILPs uas muaj kev nyab xeeb thiab muaj txiaj ntsig zoo tiv thaiv kab mob rau kev kho mob qog noj ntshav.

Table 3. Cov kab mob biochemical ntawm daim siab thiab lub raum

Table 3. The biochemical parameters of the liver and kidney

Figure 8. H&E (hematoxylin and eosin) staining of the main organs. Scale bar, 50 µm (n = 3)


Daim duab 8. H&E (hematoxylin thiab eosin) staining ntawm lub cev tseem ceeb. Scale bar, 50 µm (n=3)

3. Cov ntaub ntawv thiab cov txheej txheem

3.1. Khoom siv

Imiquimod tau yuav los ntawm MedChem Express (Shanghai, Suav). Fc-CV1 tau muab los ntawm Lub Xeev Lub Chaw Haujlwm Tseem Ceeb ntawm Cov Tshuaj Tiv Thaiv Cov Tshuaj thiab Kev Kho Mob. Cov roj (cholesterol), hydrogenated lecithin (HSPC), DSPE-PEG2000, DSPE-PEG2000-NHS, thiab DSPE-PEGCy5 tau yuav los ntawm Xi'an Ruixi Biological Technology Co., Ltd. (Xi'an, Suav). Lub 40, 6-diamidino-2-phenylindole (DAPI) tau txais los ntawm Keygen Biotech (Nanjing, Suav). Ammonium sulfate tau txais los ntawm Sinopharm (Shanghai, Suav). Methanol (chromatographic qib) thiab acetonitrile (chromatographic qib) tau yuav los ntawm Aladdin Reagent Company (Shanghai, Suav). Lwm cov reagents yog tag nrho cov kev ntsuam xyuas hauv tsev. Mouse Colon Cancer Cell Kab CT26. WT tau muab los ntawm Cell Bank ntawm Suav Academy ntawm Kev Tshawb Fawb. Cov hlwb tau loj hlob hauv lub cell incubator hauv RPMI 1640 nruab nrab uas muaj 10% FBS (Thermo Fisher Scientific, New York, NY, USA) ntawm 5% CO2 thiab 95% huab cua. Poj niam BALB/c nas ntawm 4-6 lub lis piam tau muab los ntawm Pengyue Laboratory Tsiaj Breeding Co., Ltd. (Jinan, Suav).

3.2. Kev npaj ntawm Blank Liposomes

Ua ntej, 13.455 mg ntawm HSPC, 4.3595 mg ntawm DSPE-PEG2000, thiab 4.7095 mg ntawm cov roj cholesterol tau raug hnyav thiab yaj hauv 0.1 mL ethanol (HSPC: DSPE-PEG2000: roj cholesterol=55.5: 5.05:39.3 molar ratio). Qhov thib ob, cov tshuaj ethanol tau txhaj tshuaj sai sai rau hauv cov tshuaj preheated ammonium sulfate (250 mM, 1 mL, 65 ◦C) ntawm lub koob txhaj tshuaj thiab tom qab ntawd incubated rau 30 feeb ntawm 65 ◦C nrog sib nqus stirring. Tom qab ntawd, cov tshuaj npaj tau raug extruded los ntawm polycarbonate daim nyias nyias (Avanti, Alabaster, AL, USA); Qhov luaj li cas yog 200 nm, 100 nm, thiab 50 nm, feem. Thaum kawg, cov kev daws teeb meem yog cov liposomes dawb paug (LPs) nrog lipid concentration ntawm 22.5 mg / mL.

3.3. Kev npaj ntawm CD47 Targeted Imiquimod-Encapsulated Liposomes

Ua ntej, DSPE-PEG2000-NHS tau yaj hauv ddH2O (60 ◦C) thiab tom qab ntawd sib tov nrog Fc-CV1 thiab incubated nyob rau hauv lub rooj co ntawm chav tsev kub rau 5 h (Fc-CV1: DSPPEG2000- NHS=12: 1 molar ratio). Cov tshuaj tiv thaiv kab mob ntawm kev sib txuas ntawm Fc-CV1 thiab DSPE-PEG2000-NHS koom nrog NHS ester reacting nrog cov thawj amine ntawm Fc-CV1 thiab tsim ib lub rooj amine bond conjugate. Cov tshuaj npaj tau muab faib ua ke nrog LPs thiab incubated ntawm 60 ◦C rau 1 h kom ntxig Fc-CV1 rau hauv liposomes, uas yog hu ua "post insertion" [38]. Qhov sib tov sib xyaw yog tias 2.25 mg Fc-CV1 tau hloov kho rau 22.5 mg liposomes. Tom qab ntawd cov CD47 tsom liposomes (CLPs) tau txais.

Tom qab ntawd, CLPs tau muab tso rau hauv ib lub hnab lim ntshav (300 kDa) thiab dialyzed tiv thaiv HEPES (10 mM, pH=7.4) rau 1 h kom tshem tawm ammonium sulfate thiab seem Fc-CV1 nyob rau hauv lub txheej aqueous theem. Cov dialyzed CLPs tau incubated nrog imiquimod tov (5 mg / mL) ntawm qhov ratio ntawm 1 mg rau 7 µmol tag nrho phospholipids nyob rau hauv chav tsev kub rau 24 h thiab ces dialyzed tawm tsam PBS (300 kDa) peb zaug kom tshem tawm unencapsulated imiquimod, uas yog xa mus rau. ammonium sulfate gradient txheej txheem [39] Tom qab ntawd cov CILPs tau txais. Tsis tas li ntawd, cov non-CD47-targeted imiquimod-encapsulated liposomes (ILPs) tau npaj. CLPs thiab ILPs tau suav tias yog kev tswj hwm rau CILPs.

3.4. Cim xeeb

Lub morphology ntawm cov liposomes no tau yees duab nrog ib tug kis tau tus mob electron microscope (TEM, Joel, Tokyo, Nyiv). Qhov loj, zeta-muaj peev xwm, thiab polymer dispersity Performance index (PDI) ntawm cov liposomes tau txiav txim siab los ntawm dynamic light scattering (DLS) thiab electrophoretic light scattering (ELS) siv Malvern's Zeta sizer ZSE (Malvern, UK). Kev sib khi tus nqi (BR) ntawm Fc-CV1 tso rau hauv liposomes tau ntsuas los ntawm 15% sodium lauryl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) thiab Image J software (Bethesda, MD, USA). Lub encapsulation efficiency (EE) ntawm imiquimod tau kuaj pom los ntawm high-performance kua chromatography (HPLC, Thermo Fisher Scientific, New York, NY, USA).

effects of cistance-antitumor

Cov txiaj ntsig ntawm cistanche tubulosa-Antitumor

3.5. Hauv Vitro Cell Uptake Study

Lub hauv vitro cell uptake ntawm cov liposomes no tau txiav txim siab los ntawm kev sib xyaw fluorescence siv ntawm Cy5 hauv CT26. WT hlwb. DSPE-PEG-Cy5 tau tsim nrog CILPs thiab ILPs (DSPE-PEG-Cy5: CILPs lossis ILPs=1: 5 huab hwm coj piv) ntawm 60 ◦C rau 1 h, raws li, thiab tom qab ntawd fluorescent liposomes tau txais lub npe CILPs-Cy5 thiab ILPs-Cy5. CT26.WT hlwb tau muab noob rau hauv 96-cov phiaj zoo thiab muab faib ua peb pawg (1 × 104 hlwb/zoo, n=3). Tom qab lub cell confluency mus txog 50-70%, txhua pab pawg tau incubated nrog dawb Cy5, ILPs-Cy5, thiab CILPs-Cy5, raws li, rau 30 min ntawm 37 ◦C, nyob rau hauv uas qhov kawg concentrations ntawm Cy5 yog 1 µg / mL nyob rau hauv 0.1 mL RPMI 1640 nruab nrab. Tom qab ntawd, cov hlwb raug immobilized los ntawm 4% paraformaldehyde rau 15 feeb tom qab ntxuav ob zaug nrog PBS thiab tom qab ntawd stained nrog 10 µL DAPI (1 µg / µL) rau 10 min tom qab ntxuav ob zaug nrog PBS. Thaum kawg, kev txhawb nqa ntawm txhua pab pawg hlwb tau sau tseg los ntawm High-Content Imaging System (Molecular Devices, Sunnyvale, CA, USA) tom qab ntxuav ob zaug nrog PBS.

3.6. Txoj Kev Kawm Tshaj Tawm Imiquimod

Kev tso tawm nkhaus ntawm ntau yam tshuaj-loaded liposomes tau ua los ntawm kev lim ntshav (300} kDa), thiab cov txiaj ntsig tau muab piv nrog cov tshuaj dawb imiquimod. Luv luv, 0.6 mL ILPs, CILPs, thiab cov tshuaj imiquimod tau cais ntim rau hauv lub hnab lim ntshav thiab muab tso rau hauv 500 mL ntawm PBS tov (pH=7.4, 37 ◦C). Qhov sib npaug ntawm cov qauv raug muab rho tawm los ntawm lub hnab ntawm lub sijhawm teem tseg (0, 0.5, 1, 2, 4, 6, 8, 10, 12, 24, 48, 72, thiab 96 h), thiab tus nqi tso tawm (RR) ntawm imiquimod tau suav nrog kev sib npaug hauv qab no:

image


qhov twg Sund thiab Sd sawv cev rau thaj tsam ntawm HPLC ncov ntawm imiquimod hauv kev npaj ua ntej dialyzed thiab dialyzed ntawm ntau lub sijhawm, raws li (n=3).

3.7. Cytotoxicity ntawm CILPs

CT{{0}}.WT hlwb tau cog rau hauv 96- lub phaj zoo (1 × 104 / qhov zoo, n=3) thiab cog rau hauv 100 µL ntawm RPMI 1640 nruab nrab kom txog thaum qhov sib xyaw ze li ntawm 70%. Txhawm rau txiav txim siab cytotoxicity ntawm CILPs, RPMI 1640 nruab nrab tau raug tshem tawm los ntawm txhua qhov dej thiab hloov nrog cov kev daws teeb meem ntawm CILPs thiab RPMI 1640 nruab nrab, nrog rau qhov ntau ntawm Fc-CV1 thiab imiquimod li ntawm 0 txog 10 µg / mL. Cov hlwb tau incubated nrog cov tshuaj complex rau 24 teev. Tsis tas li ntawd, cov hlwb uas tau kho nrog Fc-CV1, CLPs, imiquimod tov, thiab ILPs hauv qhov zoo sib xws tau suav tias yog pawg tswj hwm. Cov hlwb tseem muaj sia nyob tau kuaj pom nrog CCK-8 kev soj ntsuam tom qab 24 teev, thiab kev muaj peev xwm ntawm cov hlwb tsis kho tau suav tias yog 100%.

3.8. Biodistribution Study

Cuaj Poj Niam BALB/c nas tau txhaj tshuaj subcutaneously nrog CT26. WT hlwb (5 × 105 ) nyob rau hauv txoj cai flank thiab cov qog nqaij hlav nas no tau muab faib ua peb pawg (n=3). Cov qog nqaij hlav tau ntsuas los ntawm kev siv calipers thiab suav nrog cov qauv hauv qab no:

image


Thaum cov qog nqaij hlav mus txog 100 mm3, dawb Cy5, ILP-Cy5, thiab CILPs-Cy5 tau txhaj tshuaj intravenously ntawm ib koob ntawm Cy5 ntawm 0.4 mg / kg. Nees nkaum plaub teev tom qab txhaj tshuaj, cov qog, lub plawv, lub siab, tus po, lub ntsws, thiab lub raum raug tshem tawm. Cov teeb liab fluorescence ntawm cov qog thiab cov kabmob tseem ceeb tau kuaj pom los ntawm IVIS qhov muag pom qhov system (IVIS Lumina LT III, PerkinElmer, Waltham, MA, USA).

3.9. Hauv Vivo Tumor Inhibition Study

Peb caug-tsib poj niam BALB/c nas tau muab faib ua xya pawg (n=5), thiab 5 × 105 CT26. WT hlwb tau txhaj tshuaj subcutaneously rau sab xis ntawm txhua tus nas. Thaum tus nas qog loj txog 100 mm3, kev kho mob tau pib. PBS, LPs, imiquimod, Fc-CV1, ILPs, CLPs, thiab CILPs tau txhaj tshuaj intravenously hnub 0, 4, 8, thiab 12. Cov koob tshuaj imiquimod yog 2.5 mg / kg hauv thawj zaug thiab thib ob txhaj tshuaj thiab 5 mg / kg. nyob rau hauv peb thiab plaub txhaj tshuaj. Hauv txhua qhov kev txhaj tshuaj, koob tshuaj Fc-CV1 yog 5 mg / kg. Ntawm qhov koob tshuaj no, cov tshuaj concentration hauv lub cev yog 2.5-5 µg / mL, uas ua rau cytotoxicity ntawm cov hlwb hauv kev sim hauv vitro. Cov kab mob qog thiab lub cev hnyav tau ntsuas txhua hnub thaum kho. Hnub tim 17, cov nas cov qog raug tshem tawm thiab ntsuas lawv qhov hnyav. Tumor growth inhibition (TGI) raug xam raws li cov qauv hauv qab no:


image


3.10. Immunohistochemistry

Immunohistochemistry staining ntawm CD4, CD8, thiab IFN tau ua hauv cov ntaub so ntswg raws li tus qauv txheej txheem. Tom qab deparaffinizing thiab rehydrating paraffin seem ntawm cov qog nqaij hlav, 3% BSA tau ntxiv rau lub voj voog kom npog cov ntaub so ntswg, thiab tom qab ntawd nws raug kaw rau 30 min ntawm chav tsev kub. Cov seem tau stained nrog CD4, CD8, thiab IFN cov tshuaj tiv thaiv (Servicebio, Wuhan, Tuam Tshoj) thaum hmo ntuj ntawm 4 ◦C, thiab tom qab ntawd incubated nrog cov tshuaj tiv thaiv kab mob thib ob (HRP-labeled, Servicebio, Wuhan, Tuam Tshoj) ntawm chav sov li 50 min. Tom qab ob zaug ntxuav nrog PBS. Thaum kawg, cov seem tau pom nyob rau hauv lub tshuab tsom iav (Nikon, E100, Tokyo, Nyiv) tom qab ua haujlwm nrog DAB tus neeg sawv cev chromogenic.

Desert ginseng—Improve immunity (9)

cistanche tubulosa- txhim kho lub cev tiv thaiv kab mob

3.11. Biosecurity Assessment of CILPs

Thaum kawg ntawm txoj kev tshawb nrhiav qog-inhibition, tag nrho cov qog tau sau, thiab qhov hnyav ntawm cov qog no tau ntsuas. Cov kab mob biochemical, suav nrog alanine transaminase (ALT), aspartate transaminase (AST), creatinine (CREA), thiab urea (UREA), tau kuaj pom hauv cov ntshav ntawm cov nas. Cov qog nqaij hlav tshiab thiab cov kabmob loj tau kho nrog 4% paraformaldehyde thiab muab tso rau hauv paraffin siv lub tshuab embedding (Wuhan Junjie Electronics, JB-P5, Wuhan, Suav). Cov ntaub so ntswg nrog lub thickness ntawm 5 µm raug txiav los ntawm microtome (Leica Instrument, RM2016, Shanghai, Tuam Tshoj), thiab ces stained nrog hematoxylin thiab eosin (H&E) raws li txoj cai. Tom qab ntawd, cov staining slices raug soj ntsuam nyob rau hauv ib lub microscope (Nikon, E100, Tokyo, Nyiv).

3.12. Kev txheeb cais

Tag nrho cov ntaub ntawv yog qhia raws li txhais tau tias ± SD. Cov theem tseem ceeb ntawm pawg tau sim los ntawm ib-txoj kev ANOVA siv Graphpad Prism 8.0.2. p < 0.05 tau txhais tias yog qhov sib txawv tseem ceeb.

3.13. Tsiaj Ethics

Tag nrho cov txheej txheem thiab cov lus pom zoo txog cov tsiaj tau pom zoo los ntawm Pawg Neeg Saib Xyuas Kev Tshawb Fawb Txog Kev Tshawb Fawb ntawm Liaocheng University ua raws li cov lus qhia hauv lub tebchaws rau kev saib xyuas thiab siv cov tsiaj txhu (pom zoo Code: 2022111010; Hnub Pom Zoo: 1 Kaum Ib Hlis 2022).

Cistanche deserticola—improve immunity (6)

cistanche cog-nce kev tiv thaiv kab mob

4. Cov lus xaus

Hauv cov ntsiab lus, imiquimod-encapsulated CD47- lub hom phiaj liposomes tau tsim tiav, thiab cov txiaj ntsig hauv vitro tau ua pov thawj tias CILPs muaj kev tso tawm zoo dua thiab cov txiaj ntsig tshwj xeeb. Cov txiaj ntsig hauv vivo tau pom tias qhov kev npaj tuaj yeem ua tau zoo los ntawm cov qog hlwb thiab nws tau nthuav tawm cov qog kho mob zoo heev thiab biosafety vim muaj ob txoj haujlwm ntawm kev tiv thaiv kab mob thiab kev tiv thaiv kab mob hauv lub cev. Yog li, imiquimod-encapsulated liposomes ua ke rau Fc-CV1 zoo li tau cog lus tshiab nanomedicine los txhim kho CD47 qhia qog noj ntshav. Immunotherapy raws li kev tiv thaiv kab mob hauv lub cev tuaj yeem ua lub tswv yim dav dav tiv thaiv qog nqaij hlav rau kev sib koom ua ke nrog ICB yav tom ntej.

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Koj Tseem Yuav Zoo Li