Dendrimer-tesaglitazar Conjugate Induces A Phenotype Hloov Ntawm Microglia Thiab Txhim Kho -amyloid Phagocytosis† Part 1

Hloov microglia los ntawm tus kab mob-exacerbating, 'pro-inflammatory' xeev mus rau hauv neuroprotective, 'antiinflammatory' phenotype yog ib lub tswv yim zoo rau kev hais txog ntau yam kab mob neurodegenerative.

Microglia yog ib hom cell hauv lub hauv nruab nrab paj hlwb uas yog lub luag haujlwm tseem ceeb rau kev tswj hwm kev noj qab haus huv thiab kev ua haujlwm ntawm neurons. Lawv tuaj yeem tso tawm ntau yam kev loj hlob thiab cov teeb meem neurotrophic thiab tuaj yeem tswj cov kev ua haujlwm metabolic ntawm cov neurons los ntawm kev tshem cov khoom pov tseg nyob ib ncig ntawm cov neurons. Cov kev tshawb fawb hauv xyoo tas los no tau pom tias microglia muaj feem cuam tshuam nrog kev nco. Cia wb mus saib ua ke.

Ua ntej, microglia tuaj yeem txhawb cov neurons thiab txhawb kev ua haujlwm ntawm cov neurons, yog li txhim kho kev nco. Los ntawm kev tso tawm ntau cov neurotransmitters, xws li glutamate thiab alanine, microglia tuaj yeem txhawb kev sib kis ntawm cov neurons thiab tuaj yeem txhim kho glial-neuron teeb liab resonance ntawm presynaptic daim nyias nyias, ntxiv kev txhawb nqa neuronal excitability thiab nco tsim.

Qhov thib ob, microglia tseem tuaj yeem tshem tawm cov khib nyiab nyob ib puag ncig cov neurons, tswj cov kev ua haujlwm metabolic ntawm cov neurons, thiab txo qis kev tuag neuronal, yog li txhawb kev txhim kho kev nco. Thaum cov khib nyiab ntau dhau nyob ib ncig ntawm cov neurons, nws yuav cuam tshuam rau cov haujlwm metabolic ntawm cov neurons, ua rau kev tuag neuronal thiab ua haujlwm tsis muaj zog, yog li txo qis kev nco. Microglia tuaj yeem tswj hwm qhov ib puag ncig metabolic ntawm cov neurons thiab txo qis kev tuag ntawm cov neuronal los ntawm engulfing thiab decomposing ib puag ncig pov tseg, yog li txhim kho kev nco.

Hauv cov ntsiab lus, microglia muaj feem cuam tshuam nrog kev nco. Los ntawm kev txhawb nqa kev zoo siab ntawm cov neurons thiab tshem tawm cov khib nyiab ib puag ncig, microglia tuaj yeem txhim kho kev nco thiab ua kom peb lub hlwb noj qab haus huv thiab nquag. Peb yuav tsum xyuam xim rau kev tiv thaiv kev noj qab haus huv ntawm microglia kom ua tiav kev nco zoo. Nws tuaj yeem pom tias peb yuav tsum txhim kho kev nco, thiab Cistanche tuaj yeem txhim kho kev nco zoo vim Cistanche yog cov khoom siv tshuaj suav tshuaj suav nrog ntau yam tshwj xeeb, ib qho ntawm kev txhim kho kev nco. Kev ua tau zoo ntawm Cistanche los ntawm ntau yam khoom xyaw uas nws muaj, suav nrog tannic acid, polysaccharides, flavonoid glycosides, thiab lwm yam. Cov khoom xyaw no tuaj yeem txhawb lub hlwb kev noj qab haus huv hauv ntau txoj hauv kev.

Nyem paub 10 txoj hauv kev los txhim kho kev nco

Proinflammatory microglia pab txhawb cov kab mob kev loj hlob los ntawm kev tso tawm cov tshuaj neurotoxic thiab ua kom cov kab mob sib xyaw ua ke ntawm cov protein. PPAR thiab PPAR agonists ob leeg tau pom tias yuav hloov microglia los ntawm kev ua kom zoo ('M1-zoo li') mus rau qhov hloov pauv ('M2-zoo li') phenotype. Cov tswv yim zoo li no tau tshawb nrhiav hauv kev sim tshuaj rau cov kab mob neurological, xws li Alzheimer's thiab Parkinson's kab mob, tab sis tej zaum yuav ua tsis tiav vim lawv qhov tsis zoo ntawm cov hlab ntsha hauv hlwb (BBB) ​​nkag mus.

Hydroxyl-terminated polyamidoamine dendrimers (tsis muaj kev sib txuas ntawm ib qho phiaj xwm ligands) tau pom tias hla qhov tsis zoo BBB ntawm qhov chaw ntawm neuroinflammation thiab sib sau ua ke hauv activated microglia.

Yog li, dendrimer conjugation ntawm PPAR / dual agonist tuaj yeem ua rau lub hom phiaj phenotype hloov ntawm activated microglia. Ntawm no peb nthuav qhia cov synthesis thiab characterization ntawm ib tug tshiab dendrimer-PPAR / dual agonist conjugate (D-tesaglitazar).

Hauv vitro, D-tesaglitazar induces ib qho 'M1 mus rau M2' phenotype hloov, txo qis cov pa oxygen reactive hom, nce kev qhia ntawm cov noob rau phagocytosis thiab enzymatic degradation ntawm cov kab mob pathogenic (xws li -amyloid, -synuclein), thiab nce -amyloid phagocytosis.

Cov txiaj ntsig no txhawb kev txhim kho ntxiv ntawm D-tesaglitazar rau kev txhais lus rau ntau yam kab mob neurodegenerative, tshwj xeeb tshaj yog Alzheimer's thiab Parkinson's Disease.

Taw qhia

Hauv Tebchaws Meskas ib leeg, tam sim no muaj ntau dua 5.3 lab tus neeg mob Alzheimer's disease (AD) thiab 1 lab tus tib neeg uas muaj tus kab mob Parkinson (PD). yuav nce zuj zus ntxiv raws li cov pej xeem tseem loj tuaj.

Tsis tas li ntawd, qhov tsis muaj kev vam meej tsis ntev los no hauv kev tsim cov tshuaj tshiab los kho cov kab mob no tau hais txog qhov xav tau rau kev txhim kho cov kev kho tshiab.2,3 Cov kev kho mob tsis zoo no qhia txog cov teeb meem hauv kev tsim tshuaj, suav nrog kev xa cov tshuaj ntau txaus rau lub hlwb kom ua tau zoo yam tsis muaj ua rau muaj kev phiv tsis zoo.

Cov kab mob neurodegenerative xws li AD thiab PD qhia peb yam tseem ceeb ntawm cov kab mob neuropathological: neuroinflammation, pathogenic protein txuam nrog, thiab neuronal tuag.4-7 Cov neeg noj qab nyob zoo, lub cev tsis muaj zog ntawm lub hlwb (themicroglia) tas li phagocytose cov misfolded proteins (xws li -amyloid, -synuclein) ) uas ua rau neuronal tuag raws li lawv tau tsim, uas tiv thaiv cov cim aggregates los ntawm kev tsim.

Txawm li cas los xij, hauv cov neeg uas thaum kawg tsim cov kab mob neurodegenerative, microglia tsis tshem cov proteins no ntxiv lawm thiab hloov mus rau hauv cov kab mob-exacerbating, proinflammatory phenotype (feem ntau hu ua M1).

Txawm hais tias feem ntau pro-inflammatory/anti-inflammatory (M1/M2) kev faib tawm ntawm microglial activation yog ib qho kev nthuav dav ntawm cov spectrum ntawm macrophage polarization, nws tseem siv los ua ib qho dav nomenclature los piav txog qhov tseem ceeb phenotype ntawm microglia hauv neuroinflammation thiab teb rau kev kho mob.

M1-zoo li microglia tso tawm cov pa oxygen reactive thiab lwm yam inflammatory mediators uas ob qho tib si induce neuronaldeath thiab exacerbate kab mob pathology los ntawm kev tsim cov kab mob pathogenic (xws li -amyloid, -synuclein).

Ntxiv mus, qhov tseem ceeb ntawm kev pheej hmoo ntawm caj ces rau kev txhim kho AD (TREM2 thiab APOE) tau qhia nyob rau theem siab hauv microglia, thiab TREM2 / APOE txoj hauv kev tau pom tias ua rau amicroglial phenotype hloov hauv AD, amyotrophic lateral sclerosis (ALS), thiab ntau yam sclerosis. tsiaj qauv.8

Cov kev tshawb pom ntxiv no qhia txog lub luag haujlwm ntawm microglia hauv pathology ntawm ntau tus neeg mob neurodegenerative kab mob. Ib txoj hauv kev los tsim cov phenotype ntawm microglia yuav ua rau cov kws tshawb fawb nkag siab txog lawv lub luag haujlwm hauv cov kab mob neurodegenerative, ntxiv rau qhov muaj peev xwm ua tau zoo.

Hloov microglia los ntawm M1 mus rau cov tshuaj tiv thaiv kab mob thiab neuroprotective (M2) phenotype tau raug npaj los ua ib txoj hauv kev kho mob los kho ntau yam kab mob neurodegenerative.5,6Ob tam sim no FDA pom zoo PPAR agonists, hom II mob ntshav qab zib hom rosiglitazone thiab pioglitazone, tau pom tias yuav ua rau muaj kev cuam tshuam loj heev. M1 rau M2 phenotype hloov pauv hauv macrophages thiab microglia hauv vitro thiab hauv vivo.

9,10 PPAR agonists tau raug qhia kom txo qis LPS-vim tso tawm ntawm cov pa oxygen reactive los ntawm kev txo cov kev ua ntawm NF-κB los ntawm inducing NF-κBdegradation thiab xa tawm los ntawm lub nucleus, nrog rau ligand-dependent transrepression.11

Ntxiv mus, cov kev tshawb fawb txog kab mob kis tau pom tias cov neeg mob ntshav qab zib uas noj rosiglitazone lossis pioglitazone yog qhov txo qis rau kev tsim ADand PD.12 Tom qab ntawd, rosiglitazone thiab pioglitazone tau tshawb xyuas los ntawm theem III kev sim tshuaj rau AD, butfailed.13

Ib qho yuav piav qhia txog qhov tsis ua tiav ntawm qhov kev sim tshuaj tau hais los saum no yog qhov kev thauj tsis zoo ntawm cov tshuaj no hla cov ntshav-hlwb barrier (BBB), yog li txwv cov tshuaj uas nkag mus rau lub hlwb ntawm cov neeg mob rau npe hauv cov kev sim tshuaj no.14

Tseeb tiag, nws tau kwv yees tias BBB tiv thaiv kwv yees li 98% ntawm tag nrho cov tshuaj molecule me me los ntawm kev ncav cuag lub hlwb, thiab tsuas yog ib feem ntawm cov tshuaj nkag mus rau hauv lub hlwb mus txog themicroglia.15

Tsis tas li ntawd, PPAR yog lwm cov khoom siv hluav taws xob hauv tsev neeg PPAR.16 Nws nthuav tawm lub luag haujlwm hauv lipid homeostasis thiab tswj kev mob, thiab PPAR agonists kuj tau tshwm sim los ua pov thawj los tiv thaiv cov teebmeem hauv microglia.

Cov kev tshawb fawb soj ntsuam siv cov neuroimaging, post-mortem tissueanalysis, thiab CSF biomarkers tau muab pov thawj tias BBB muaj kev cuam tshuam hauv AD thiab PD, nrog rau lwm yam kab mob neurodegenerative.

17 Generation-4 hydroxyl-terminated polyamidoamine (G4-PAMAM-OH) dendrimers tau pom tias muaj kev cuam tshuam los ntawm qhov tsis zoo ntawm BBB thiab sib sau ua ke hauv activatedmicroglia yam tsis tas yuav tsum tau tsom ligands, tom qab kev tswj hwm hauv ntau hom kab mob neuroinflammationdisease. , suav nrog hauv cov nas, luav, dev, thiab cov tsiaj tsis yog neeg.18–28 Qhov tseem ceeb, G4-PAMAM-OH tuaj yeem ua haujlwm tau zoo thiab hla BBB hauv cov kab mob uas muaj kev cuam tshuam me me BBB xws li Rett Syndrome.29

Tsis tas li ntawd, qhov nce ntawm G4-PAMAM-OH mus rau hauv lub hlwb yog ncaj qha proportional rau tus kab mob hnyav nyob rau hauv ib tug luav qauv ntawm cerebral palsy.30 Hydroxyl-terminated dendrimershave qhov zoo ntawm kev xa tawm noninvasively piv rau qhov cuam tshuam loj heev, xa hauv zos los ntawm lub pob txha taub hau yuav tsum tau nyob rau hauv cov kev tshawb fawb yav dhau los nrog rau lwm yam nanoparticles xws li poly-εcaprolactone thiab PEG, tsis zoo them PAMAM dendrimers, quantum dots, thiab nanoformulations muaj li ntawm polyethyleneimine (PEI) thiab dextran sulfate.31–35

Tsis tas li ntawd, cov hydroxyyl PAMAM dendrimers no yog qhov zoo tshaj plaws rau kev txhais lus vim lawv qhov scalability thiab ua tau zoo nyob rau hauv vivo safetyprofile.36–38 Vim cov ntaub ntawv ua ntej kev kho mob zoo, a (G4-PAM-OH)-N- acetyl-cysteine ​​conjugate tam sim no tau raug tshuaj xyuas hauv kev sim tshuaj thaum ntxov rau menyuam yaus lub paj hlwb adrenoleukodystrophy (NCT03500627) thiab kab mob coronavirus hnyav 2019 (COVID{11}}) cuam tshuam o (NCT04458298).

Peb kawm ib tug dendrimer-drug conjugate ntawm tesaglitazar (Tesa) txuas rau tiam -4 hydroxyl-terminated PAMAMdendrimer. Tesaglitazar yog ib qho muaj zog PPAR / dual agonist uas sib txuas cov txiaj ntsig zoo ntawm PPAR thiab PPAR agonists. Nws muaj cov pab pawg carboxylic acid ua haujlwm rau covalent conjugation rau dendrimer thiab rau kev tso tawm tom ntej.

Ntxiv covguitars tau tsim thiab kuaj hauv tsev kho mob, tab sis Tesa raug xaiv vim nws qhov loj dua PPAR rau PPAR kev ua piv txwv, cov qauv tshuaj lom neeg yooj yim, thiab kev nyab xeeb profile.39–43

Tesa yav dhau los tau mus txog theem III kev kuaj mob rau hom 2 mob ntshav qab zib hauv Tebchaws Meskas tab sis ua tsis tiav vim kev siv tshuaj toxicity, uas tuaj yeem tiv thaiv los ntawm kev txo qis qhov tsim nyog ntawm kev tswj hwm los ntawm kev tswj cov dendrimer xa.39,40,44Vim Tesa yog ib qho PPAR / dual agonist, nws lub hom phiaj xa mus rau activated microglia ntawm qhov chaw ntawm neuroinflammation tuaj yeem muaj txiaj ntsig zoo.

Ntawm no, peb ua kom pom qhov sib xyaw thiab cov yam ntxwv ntawm ib qho dendrimer-tesaglitazar conjugate (D-Tesa) thiab ua kom pom qhov sib xyaw no muaj peev xwm ua kom muaj 'M1 toM2' phenotype hloov hauv microglia thiab txhim kho phagocytosis ntawm fluorescently sau npe -amyloid.

Cov ntaub ntawv thiab cov txheej txheem

Khoom siv

1-[3-(Dimethylamino)propyl]-3-ethyl carbodiimide methiodide(EDC), 4(dimethylamino)pyridine (DMAP), CuSO4·5H2O, sodium ascorbate, hexynoic acid thiab bovine serum albumin (BSA) tau yuav los ntawm Sigma Aldrich US thiab siv tau txais (St Louis, MO).

Tesaglitazar tau txais los ntawm AstaTech Inc. (Bristol, PA). Ethylenediamine-core PAMAM dendrimer (tiam 4 nrog 64 hydroxyl kawg-pab pawg) tau txais los ntawm Dendritech Inc. (Midland, MI) ua cov tshuaj inmethanol.

Lub dendrimer tau khaws cia hauv methanol ntawm 4 degree thiab methanol tau evaporated ua ntej siv. Lub tshuab lim dej nrog lub cev hnyav molecular txiav tawm (MWCO) ntawm 1 kDa tau yuav los ntawm Spectrum Laboratories Inc. (New Brunswick, NJ).

Tag nrho lwm cov kuab tshuaj tau siv raws li tau txais hauv lawv cov ntaub ntawv tsis muaj dej.Txhua qhov kev tshwm sim, tshwj tsis yog tooj liab (I), catalyzed alkyne-azide cycloaddition (CuAAC) nyem cov tshuaj tiv thaiv, tau ua nyob rau hauv cov dej tsis muaj dej hauv cov organic nruab nrab nrog qhov cub-qhuav iav nyob rau hauv inert nitrogen. huab cua.

Rau kab lis kev cai ntawm tes: Dulbecco's Modified Eagle Medium (DMEM), fetal bovineserum (FBS), penicillin-streptomycin (P/S), 0.05% trypsin-EDTA, thiab MTT reagent tau txais los ntawm Invitrogen (Carlsbad, CA , USA).

Griess reagent tau txais los ntawm Promega (Madison, WI) thiab TNF- ELISA tau los ntawm R&D Systems (Minneapolis, MN). Analytical qib methanol tau yuav los ntawm Sigma-Aldrich. Trypan xiav tau txais los ntawm Corning (Manassas, VA, USA).

Synthesis cov txheej txheem rau D-Tesa conjugates
Tetraethylene glycol mono azide (2) tau tsim los siv ua ntej tshaj tawm raws tu qauv.45

Synthesis thiab purification ntawm Tesa-TEG-azide (3).
Tesa (950 mg, 2.32 mmol) tau yaj hauv 10 ml dimethylformamide (DMF). Rau qhov kev sib tov sib xyaw no, tetramethylene glycol mono-azide (2, 662.1 mg, 3.02 mmol) hauv DMF (1 ml) tau ntxiv rau hauv cov dej. Tom qab ntawd DMAP (255.4 mg, 2.09 mmol) thiab EDC (577.5 mg, 3.02 mmol) tau ntxiv rau cov tshuaj tiv thaiv sib xyaw, thiab qhov tshwm sim tau nplawm hauv qab nitrogen ntxuav ntawm chav tsev kub rau 24 teev.

Cov tshuaj tiv thaiv tau saib xyuas siv nyias-layerchromatography thiab kev ua haujlwm siab ua kua chromatography (HPLC). Cov tshuaj tiv thaiv sib xyaw yog diluted nrog 100 ml dichloromethane (DCM) thiab cov tshuaj tiv thaiv crude sib tov tau hloov mus rau hauv ib qho kev sib cais funnel, thiab cov organic txheej tau tom qab ntxuav peb zaug nrog saturated sodium bicarbonate tov, tom qab ntawd saturated ammonium chloride tov thiab thaum kawg nrog brine.

Tom qab ntawd cov organic txheej tau qhuav nrog anhydroussodium sulfate. Cov kuab tshuaj nyob rau hauv cov organic txheej yog ces tshem tawm siv ib tug teb evaporator, thiab cov tshuaj yog redissolved nyob rau hauv 3 ml DCM thiab absorbed rau silica gel kom huv si nrog ib tug CombiFlash® chromatography system siv ib tug gradient txoj kev nrog ethyl acetate / hexane ua cov kuab tshuaj los tsim 3as ntshiab. - daj yog '.

Cov khoom uas xav tau, ntshiab yog eluted ntawm kwv yees li 30-40% ethyl acetate. (Yield: 70%.) 1H NMR (500 MHz, CDCl3) δ 7.27 (d, J=8.6 Hz, 2H), 7.15(d, J=1.9 Hz, 2H), 7.08 (d, J=8.6 Hz, 2H), 6.73 (d, J=8.6 Hz, 2H), 4.25–4.14 (m, 2H), 4.07 (t, J {{ 33}}.8 Hz, 2H), 3.94 (dd, J =7.8, 5.2 Hz, 1H), 3.67–3.47 (m, 14H), 3.30 (dd, J=8.7 3.8 Hz, 2H), 3.06 (s, 3H), 3.02 (t, J=6.7 Hz, 2H), 2.91–2.84 (m, 2H), 1.08 (t, J=7} .0 Hz, 3H).ESI-MS: theoretical C28H39N3O10S: 609.24, tau (M + 1): 610.13.

Synthesis thiab purification ntawm D-YNE (5).

(480 mg, 4.22 mmol) tau ntxiv rau hauv ib qho tshuaj tov ntawm G4-PAMAM-OH (2.5 g, 0.176 mmol) hauv 20 ml anhydrous DMF. Ntxiv rau qhov sib xyaw no, DMAP (430 mg, 3.52 mmol) thiab EDC (1 g, 5.28 mmol) tau ntxiv.

Cov tshuaj tiv thaiv sib tov yog stirred nyob rau hauv nitrogen ntxuav rau 24 teev nyob rau hauv chav tsev kub. Thaum ua tiav cov tshuaj tiv thaiv, cov tshuaj tiv thaiv sib xyaw tau raug xa mus rau 1000 MWCO dialysis tube DMF lim dej tau ua tiav rau 24 teev, thiab DMF tau hloov txhua rau 6 teev.

Tom qab ntawd lim dej nrog deionized (DI) dej tau ua tiav rau 24 teev, nrog cov dej hloov txhua rau rau teev. Thaum kawg, qhov tshwm sim ntawm lub raj lim dej muaj cov ntsiab lus lyophilized rau 48 teev, yielding ib tug dawb, fluffy hmoov.(Yield: 61%.) 1H NMR (500 MHz, DMSO) δ 8.10–7.67 (m, dendrimerinternal amide H), 4.72 (s, dendrimer nto OH), 4.01 (t, ester –CH2), 3.32 (m, dendrimer –CH2), 3.06 (m, dendrimerand linker –CH2), 2.85–2.58 (m, dendrimer –CH2), 2.56–1.89 (m, dendrimer thiab linker –CH2), 1.78–1.61 (m, linker –CH2).Synthesis thiab purification ntawm D-Tesa (6).

Tesa-TEG-azide (3,177.8 mg, 0.3{{10}}}3 mmol) tau ntxiv rau qhov sib xyaw ua ke ntawm D-YNE (5, 35{{20) }}} mg, 0.023 mmol) hauv 5 ml ntawm 1: 1 sib tov ntawm tetrahydrofuran (THF) thiab dej nrog 0.5 ml ntawm DMF nyob rau hauv lub microwavereactor muaj kev nyab xeeb 20 ml vial. Rau cov tshuaj tiv thaiv CuAAC nyem, coppersulfate pentahydrate (11.6 mg, 0.0467 mmol) thiab (+)-sodium-Lascorbate (9.3 mg, 0.0467 mmol) tau ntxiv rau cov tshuaj sib xyaw.

Lub vial raug kaw, thiab cov hlab ntsha cov tshuaj tiv thaiv tau muab tso rau hauv Biotage® Initiator microwave reactor thiab reacted nyob rau hauv 20 W microwave hluav taws xob nrog nplawm rau 8 teev ntawm 50 degree. Cov tshuaj tiv thaiv sib xyaw tau raug xa mus rau 1000 MWCOdialysis raj, thiab DMF lim ntshav tau ua tiav rau 24 teev, nrog DMF raug hloov los ntawm cov kuab tshuaj tshiab txhua 4 teev.

Tom qab ntawd, cov ntsiab lus ntawm lub raj lim dej tau hloov mus rau afalcon raj, thiab qhov sib npaug ntawm DI dej tau ntxiv. Tsis tas li ntawd, 200 µl ntawm ethylenediaminetetraacetic acid disodium ntsev tov tau ntxiv rau cov ntsiab lus ntawm lub raj falcon.

Qhov kev sib xyaw no tau muab tso rau hauv 1000 MWCO dialysistube tshiab, thiab kev lim ntshav tau ua rau 12 teev hauv 1000 ml DIwater nrog EDTA cov tshuaj ntxiv tom qab dej lim dej rau 12 teev.

Cov sib tov yog ces lyophilized rau 48 teev thiab ua rau ib tug dawb, fluffy hmoov. (Yield: 64%.) 1H NMR (500 MHz, DMSO) δ 8.2–7.6 (m, dendrimer internalamide H), 7.36 (d, Tesa ArH), 7.21 (d, Tesa ArH), 7.03 (d, TesaArH), 6.76 (d, Tesa ArH), 4.37 (s, Tesa H), 4.18–4.04 (m, linkerH), 3.96 (dd, Tesa thiab linker H), 3.70 (m, Tesa thiab txuas H) , 3.58–3.14 (m, dendrimer –CH2), 3.14–2. , 1.82–1.67 (m, linker –CH2), 0.97 (t, Tesa –CH3).

Cov txheej txheem ua cim

Nuclear magnetic resonance (NMR). NMR spectra raug kaw rau ntawm Bruker 500 MHz spectrometer ntawm chav tsev kub. Proton tshuaj hloov pauv (δ) tau tshaj tawm hauv ppm.

1H NMR tau siv los txiav txim seb tus naj npawb ntawm Tesa molecules txuas rau txhua lub molecule ntawm D-Tesa los ntawm kev sib xyaw ua ke ntawm cov proton, los ntawm kev sib piv cov peaks ntawm cov amide protons ntawm dendrimer atδ 7.6-8.2 ppm nrog cov aromatic protons ntawm Tesa ntawm δ 7.36-6.76 methyl ppman ppm. Tesa hauv thaj av aliphatic.High-performance kua chromatography (HPLC).

HPLC (Waters Corporation, Milford, Massachusetts) nruab nrog a1525 binary twj tso kua mis, In-Line degasser AF, 717 plus autosampler, 2998 photodiode array detector, thiab 2475 multi λ fluorescence detector interfaced nrog Waters Empower software tau siv.

Symmetry C18 thim rov qab theem kab (Tosoh, Nyiv) muaj 5 μm particle loj, 25 cm ntev, thiab 4.6 mmternal txoj kab uas hla tau siv. Cov tshuaj tau saib xyuas ntawm 210 nm thiab 254 nm siv PDA detectors.

Cov kuab tshuaj A wasHPLC-qib dej nrog 0.1% trifluoroacetic acid (TFA), thiab cov kuab tshuaj B yog acetonitrile (ACN) nrog 5% dej thiab 0.1% TFA.Cov txheej txheem siv pib ntawm 1{ {7}}0 : 0 (ACN: dej), txo qis rau 10: 90 (dej: ACN) hauv 5 feeb, nyob ntawm qhov polarity li 15 feeb, thiab rov qab mus rau 100 : 0 (ACN: dej) hauv 5 feeb.Cov dej ntws tau khaws cia ntawm 1 ml min-1.Mass spectroscopy.

ESI-MS tau ua tiav ntawm BrukermicroTOF-II huab hwm coj spectrometer siv acetonitrile / dej (9: 1) ua kuab tshuaj. Cov ions molecular li protonated peaks [M + nH]n+ lossis adducts [M + nX]n+ (X=Na, K, lossis NH4) tau siv los lees paub cov qauv empirical.

Dynamic light scattering thiab ζ-muaj peev xwm. A Zetasizer NanoZS (Malvern Instrument Ltd, Worchester, UK) nruab nrog a50 mW He-Ne laser (633 nm) tau siv los txiav txim qhov loj me thiab ζ-muaj peev xwm faib. D-Tesa tau yaj hauv dej hauvDI mus rau qhov concentration ntawm 0.2 mg ml−1 rau DLS thiab in10 mM sodium chloride mus rau qhov concentration ntawm 0.1 mg ml−1 rauζ-muaj peev xwm.

Kev ntsuas tau ua ntawm 25 degree, siv lub kaum sab xis ntawm 173 degree raws li tau piav qhia yav dhau los.27,46Drug tso tawm txoj kev tshawb fawb. D-Tesa tau yaj ntawm qhov concentration ntawm 1 mg ml-1 nyob rau hauv ob qho tib si phosphate buffer tov (pH 7.4) tomimic plasma tej yam kev mob los yog sodium citrate tov (pH 5.5) rau mimic lysosomal tej yam kev mob.

Esterases los ntawm daim siab porcine (los ntawm Sigma Aldrich) tau ntxiv rau cov tshuaj sodium citrate thaum pib ntawm kev tso tawm txoj kev tshawb fawb thiab tau rov ua dua txhua txhua 3 hnub thaum kawm.

Txhua lub vial muaj 15 ml qauv thiab lawv tau txuas ntxiv shaken ntawm 37 degree rau lub sijhawm ntawm kev sim. Nyob rau ntawm lub sijhawm sib txawv, sib npaug ntawm 200 µl cov qauv los ntawm txhua qhov pH tau sau thiab cov haujlwm esterase tau tom qab quenched los ntawm kev ntxiv 200 µl ntawm methanol. Zero-teev lub sij hawm taw tes piv txwv tau ua tus tswj.

Cov qauv raug khaws cia ntawm -80 degree kom tsis txhob muaj hydrolysis ntxiv. Cov qauv no tau txheeb xyuas ntxiv los ntawm HPLC thiab thaj tsam hauv qab ntawm qhov nkhaus (ntawm 210 nm) rau qhov siab tshaj tshuaj dawb tau suav. Cov cheeb tsam nyob rau hauv qhov nkhaus tau sib raug zoo nrog cov tshuaj tso tawm los ntawm kev siv lub calibration nkhaus qhov twg paub tias cov ntsiab lus ntawm Tesa dawb tau khiav ntawm HPLC ntawm 210 nm.

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