Corecell M Foam_CN_v5

ASSEMBLY & PACKING INSTRUCTIONS FOR THE MULTICHAIR 5100txmultichair 5100txmultiCHAIR 5100txASSEMBLY INSTRUCTIONS Figure 1Figure 2Figure 4s lide boltsFigure 3E Turn the chair right side up and install the backrest assem-bly. Slide the two backposts into the back of the seat frame andtighten the two socket head cap screws using the yellow T-han-dle Allen wrench (Refer to Figures 5 & 6). Also tighten the fourbutton head capscrews that attach the back cushion to theback posts using the 5/32” Allen wrench (Refer to Figure 7).Figure 5Figure 6Figure 7ADDITIONAL SAFETY PRECAUTIONSFigure 11Figure 10Figure 9LIMITED WARRANTYA WARRANTY ACTIVATION Please read this warranty before operating or using your multiCHAIR. To activate the warranty on your multiCHAIR register it online or by phone. By operating or using the chair, you agree to the terms of this warranty.B WARRANTY: Nuprodx, Inc. warrants this product against defects in material and workmanship as follows: There is a 10-Day conditional money-back warranty. During this time period, the customer is permitted to try out the multiCHAIR, fully-clothed, keeping the chair in “like new” condition (NOTE: “Like new” condition, in terms of the multiCHAIR, is defined by Nuprodx, Inc. as no visible wear/usage/water marks for the multiCHAIR to be accepted and refunds issued when returned to Nuprodx, Inc.). If the customer decides that the multiCHAIR will not work for them, before the 10-Day period has passed and with the authorization of Nuprodx, the chair can be returned for a full refund minus a 10% restocking charge (NOTE: The customer is responsible for both in-bound and out-bound freight). After the initial 10-Day period has passed, there is a two-year limited warranty for all parts of the chair, with the exception of the seat and back cushions (NOTE: Because of the fragile nature of the foam, there is no warranty for the cushions). The warranty does not cover normal “wear and tear” from everday use of the product and custom parts/custom cushions are also excluded from the warranty and cannot be returned for a refund under any circumstances. Please see section E WARRANTY LIMITATION AND EXCLUSIONS for more info on warranty exclusions.C The warranty period begins on the date you receive the chair. For warranty service, please contact Nuprodx, Inc. no later than one month following the applicable warranty term. The chair will be repaired or replaced at the discretion of Nuprodx, Inc. with no charges to you for parts and labor, provided you have proof of purchase and of purchase date.D DISCLAIMER: Except for the above warranty, and the acknowledgement by Nuprodx, Inc. that the chair, as manu-factured by it, is fit for the general purpose for which most persons acquire a chair of its kind, Nuprodx, Inc. provides that you accept the chair as is, without warranties, either express or implied. Nuprodx, Inc. makes no warranty of fit-ness for your particular purpose and no warranty of merchantability beyond that already stated. No warranties extend beyond the duration of the express warranty stated above.E WARRANTY LIMITATIONS AND EXCLUSIONS: The only obligation of Nuprodx, Inc. is to provide the purchaser with free repair and replacement as described above. This exclusive warranty remedy will not have failed as long as Nuprodx, Inc. is willing and able to repair or replace as described, but if this remedy should be held to have failed, the only remaining warranty obligation of Nuprodx, Inc. shall be to refund the acts beyond the control of Nuprodx, Inc. The warranty does not cover normal “wear and tear” from everday use of the product. Standard seat/back cushions/custom parts/custom cushions are not covered under the warranty.F This warranty gives you specific legal rights, and you may have other rights that may vary from state to state.G This warranty does not apply to problems arising from normal wear, improper operation, improper maintenance, improper storage or similar disclaimer of implied warranties, and some do not allow limitations on how long an implied warranty may last. Some do not allow exclusion or limitation of incidental or consequential damages. So the above limitations or exclusions may not apply to you.H RETURN INSTRUCTIONS In the event that you need to return the Nuprodx multiCHAIR, please follow the instruc-tions below:1) Review the information contained in your warranty to see if this applies to you2) Obtain a Return Authorization # from Nuprodx, Inc.3) Re-package the entire chair and its contents in the original packaging and ship to the following address:Figure A - Drape the black strap across the bottom of thecarrying case (not shown for clarity). Place the invertedshuttle & track frame inside the case. Locate the plasticpanel at the bottom of the frame.Figure B- Place the legs in the inverted cushionand frame.PACKING INSTRUCTIONSFigure C- Place the foam on top of the legs.Figure D- Place the two side arms on top of thefoam.Figure F- Tighten the cloth beltaround the complete frame.Figure E- Place a second piece of foam on the arms,and lay the back posts with rolled up cloth as shown.。

发布日期: 01-五月-2020修订日期 20-一月-2023SDS 编号: 15522产品名称： Ellamera BI-THIN™ 602, 聚合物版本号: 3.0化学品安全技术说明书按照GB/T 163483、GB/T 17519编制。

第1部分 化学品及企业标识Ellamera BI-THIN™ 602, 聚合物化学品中文名Ellamera BI-THIN™ 602化学品英文名制造商或供应商美国总部名称Kraton Corporation地址15710 John F Kennedy Blvd., Suite 300Houston, TX 77032, 美国电话号码+1 281 504 4700中国名称科腾聚合物贸易(上海)有限公司地址南京西路688号22楼2201室中国上海市静安区，邮编200041电话号码+86 21 20823888Technical Support Line -International+1 800 4 Kraton (572866) ; +1 281 504 4950Technical Support Line -EU +31 (0) 36 546 2800网站EMERGENCY RESPONSE NUMBERS:美国化学品运输紧急应变中心(CHEMTREC):+1 703 527 3887SGS Ewacs NV:+32 35 75 5555推荐用途及限制用途化妆品和个人护理产品中的一种成分。

推荐用途01-五月-2020发布日期20-一月-2023修订日期03-五月-2022更新日期15522SDS 编号第2部分 危险性概述紧急情况概述GHS 危险性类别未分类。

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Nuclear Extraction Kit100 ExtractionsCat. No. 2900FOR RESEARCH USE ONLYNot for use in diagnostic procedures.USA & CanadaPhone: +1(800) 437-7500 • Fax: +1 (951) 676-9209 • Europe +44 (0) 23 8026 2233 Australia +61 3 9839 2000 • Germany +49-6192-207300 • ISO Registered worldwide •*********************•*********************This page left intentionally blank .ApplicationCHEMICON®’s Nuclear Extraction Kit (Catalog No. 2900) provides a simple and convenient method for the isolation of cytoplasmic and nuclear samples from mammalian cell culture or tissue samples. The Nuclear Extraction Kit can be used in the preparation of purified proteins for use in Western blotting, Electrophoretic Mobility Shift Assays (EMSA), and in CHEMICON®’s Transcription Factor Assay product line.For Research Use Only; Not for use in diagnostic procedures.Kit Components1. Cytoplasmic Lysis Buffer, 10x: - (Part No. 90497) One vial containing10mL of a concentrated lysis buffer. Dilute to a 1x in deionized water.2. Nuclear Extraction Buffer: - (Part No. 90498) One vial containing 50mL ofa 1x nuclear extraction buffer.3. PBS Packets: - (Part No. 60093) Two pouches containing enough dryreagent to prepare 1 liter of a 1x PBS solution per pouch. Dilute packet in deionized water.4. DTT, 1M: - (Part No. 90499) One vial containing 100µL of 1MDithiothreitol. Prior to use, dilute to a final concentration of 0.5mM(1:2000) in 1x Cytoplasmic Lysis Buffer and 1x Nuclear Extraction Buffer.5. Protease Inhibitor Cocktail: - (Part No. 90492) One vial containing 100µLof protease inhibitors in DMSO for use with mammalian cell and tissueextract buffers. A mixture of protease inhibitors with broad specificity for the inhibition of serine, cysteine and aspartic acid proteases andaminopeptidases. Contains 4-(2-aminoethyl)benzenesulfonyl fluoride(AEBSF), pepstatin A, E-64, bestatin, leupeptin, and aprotinin. Contains no metal chelators (e.g. EDTA, EGTA). Prior to use, dilute 1/1000 in 1xCytoplasmic Lysis Buffer and 1x Nuclear Extraction Buffer.6. Detergent, 10%: - (Part No. 90500) One vial containing 3mL of 10%IGEPAL CA-630 (detergent). Cytoplasmic Lysis Buffer and NuclearExtraction Buffer already contain detergent, however, under certainconditions, more detergent may be required - refer to Extraction Procedure.1Materials Not Supplied1. Tissue Culture Reagents2. Cell Detachment Buffer (Trypsin) or Cell Scrapers3. Syringes, 1mL – 27 Gauge Needle4. 1.5 mL Microcentrifuge Tubes5. 50 mL Conical Tubes6. Deionized Water7. Phosphatase Inhibitor (if needed)8. Microcentrifuge, 4ºC9. Table-top Centrifuge (capable of 16,000 x g), 4ºC10. Rotator/Orbital Shaker, 4o CStorageThe Nuclear Extraction Kit is shipped and stored at -20ºC.• Cytoplasmic Lysis Buffer (10x), Nuclear Extraction Buffer (1x), 10% Detergent, and the PBS Packets can be stored at 2-8ºC.• DTT, 1M and Protease Inhibitor Cocktail must be stored at -20ºC. Avoid repeated freeze-thaw cycles.Preparation of ReagentsNote: Chill all buffers on ice prior to use.• Rehydrate PBS Packet in 1 liter of deionized water.• Dilute 10x Cytoplasmic Lysis Buffer to a 1x solution with deionized water.• Prior to use, add 0.5mM (final) DTT and 1/1000 dilution of Protease Inhibitor Cocktail to 1x Cytoplasmic Lysis Buffer and/or 1x NuclearExtraction Buffer.2Extraction ProcedureA. Cell Culture1. Grow cells to 70-90% confluency for adherent cells or about 1.5 x106/mL for suspension cells.2. If necessary, treat cells with desired method.B. Cell Disruption1. For adherent cells, wash the cells with 1x PBS solution, remove, andthen add warmed trypsin cell detachment buffer to the culture flask(s).Let the trypsin sit for approximately 2-5 minutes (depending on celltype) and shake cells off.Alternately, a cell scraper may be used instead of trypsin. Collect cellsand transfer to a clean centrifuge tube. Rinse the culture flask with twovolumes of ice cold PBS and add to centrifuge tube. Centrifuge thesample at 250 x g for 5 minutes at 4°C. Discard the supernatant andresuspend the cell pellet in 40 mL of ice-cold PBS to wash. Centrifugethe suspension at 250 x g as before. Repeat. Pour off supernatant.Note: All work done after cell trypsinization/detachment needs to beperformed on ice and/or with chilled buffers. It is imperative that thecell pellets and suspension remain as cold as possible without freezingduring the extraction process.2. Estimate the approximate volume of the cell pellet. This value will beneeded for determining the amount of buffer volume necessary fornuclear extraction. (Two T175 tissue culture flasks of confluent HeLacells will generate a cell pellet of approximately 100µL.)3. Add 5 cell pellet volumes of ice cold 1x Cytoplasmic Lysis Buffercontaining 0.5mM DTT and 1/1000 dilution or inhibitor Cocktail.4. Resuspend the cell pellet by gently inverting the tube. Avoid foamproduction. Do not vortex!5. Incubate the cell suspension on ice for 15 minutes.6. Centrifuge the cell suspension at 250 x g for 5 minutes at 4°C. Discardsupernatant and resuspend the cell pellet in two volumes of ice cold 1xCytoplasmic Lysis Buffer.3C. Cell Lysis1. Using a syringe with a small gauge needle (27 gauge), draw the cellsuspension prepared in Section B from the sample tube into the syringeand then eject the contents back into the sample tube. Repeatapproximately 5 times (drawing and ejecting). If the cells “clump” andyou are not able to draw them into the syringe, more Detergent, 10%may be added.Note: Lysis and extraction buffers already contain detergent. However,if cell clumping occurs during the lysis procedure it may be necessaryto add additional detergent to the Cytoplasmic Lysis Buffer andNuclear Extraction Buffer.2. Centrifuge the disrupted cell suspension at 8,000 x g for 20 minutes at4°C.3. The supernatant contains the cytosolic portion of the cell lysate.Transfer the supernatant to a fresh tube. To keep the cytosolic fraction,aliquot, snap-freeze and store at -80°C. Avoid repeated freeze-thawcycles.4. The remaining pellet contains the nuclear portion of the cell lysate.D. Nuclear Extraction1. Resuspend the nuclear pellet in 2/3 of the original cell pellet volume(determined in step B.2) of ice cold Nuclear Extraction Buffercontaining 0.5mM DTT and 1/1000 Protease Inhibitor Cocktail.2. Using a fresh syringe, with a 27-gauge needle, repeat Step C.1. todisrupt the nuclei, add more Detergent, 10% if necessary.Note: The nuclear extract sample can be stored at –80°C at this point ifneeded.3. Use a rotator or orbital shaker (low speed) to gently agitate the nuclearsuspension at 4°C for 30-60 minutes.4. Centrifuge the nuclear suspension at 16,000 x g for 5 minutes at 4°C.5. Transfer the supernatant to a fresh tube. This fraction is the nuclearextract.6. Determine protein concentration.Note: If additional detergent was added during cell lysis (step C.1 orD.2) it may interfere with certain methods of protein concentrationdetermination. Extract may need to be diluted 10-fold or moredepending upon final detergent concentration.7. Snap-freeze the nuclear extract in aliquots and store at –80°C. Avoidrepeated freezing and thawing of nuclear extract.4WarrantyThese products are warranted to perform as described in their labeling and in CHEMICON literature when used in accordance with their instructions. THERE ARE NO WARRANTIES, WHICH EXTEND BEYOND THIS EXPRESSED WARRANTY AND CHEMICON DISCLAIMS ANY IMPLIED WARRANTY OF MERCHANTABILITY OR WARRANTY OF FITNESS FOR PARTICULAR PURPOSE. CHEMICON ’s sole obligation and purchaser’s exclusive remedy for breach of this warranty shall be, at the option of CHEMICON , to repair or replace the products. In no event shall CHEMICON be liable for any proximate, incidental or consequential damages in connection with the products.2003: CHEMICON International, Inc. - By CHEMICON International, Inc. All rights reserved. No part of these works may be reproduced in any form without permissions in writing.5Cat No. 2900December 2003Revision A: 41599。

Functionalized micelles from block copolymer of polyphosphoester and poly(ɛ-caprolactone)for receptor-mediated drug deliveryYu-Cai Wang a,1,Xi-Qiu Liu b,1,Tian-Meng Sun b ,Meng-Hua Xiong a ,Jun Wang a,b,⁎aDepartment of Polymer Science and Engineering,University of Science and Technology of China,Hefei,Anhui 230026,PR ChinabHefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences,University of Science and Technology of China,Hefei,Anhui 230027,PR ChinaReceived 23October 2007;accepted 29January 2008Available online 16February 2008AbstractCellular specific micellar systems from functional amphiphilic block copolymers are attractive for targeted intracellular drug delivery.In this study,we developed reactive micelles based on diblock copolymer of poly(ethyl ethylene phosphate)and poly(ɛ-caprolactone).The micelles were further surface conjugated with galactosamine to target asialoglycoprotein receptor (ASGP-R)of HepG2cells.The size of micellar nanoparticles was about 70nm in diameter,and nanoparticles were negatively charged in aqueous solution.Through recognition between galactose ligands with ASGP-R of HepG2cells,cell surface binding and internalization of galactosamine-conjugated micelles were significantly promoted,which were demonstrated by flow cytometric analyses using rhodamine 123fluorescent dye.Paclitaxel-loaded micelles with galactose ligands exhibited comparable activity to free paclitaxel in inhibiting HepG2cell proliferation,in contrast to the poor inhibition activity of micelles without galactose ligands particularly at lower paclitaxel doses.In addition,population of HepG2cells arrested in G2/M phase was in positive response to paclitaxel dose when cells were incubated with paclitaxel-loaded micelles with galactosamine conjugation,which was against the performance of micelles without galactose ligand,owing to the ligand –receptor interaction.The surface functionalized micellar system is promising for specific anticancer drug transportation and intracellular drug release.©2008Elsevier B.V .All rights reserved.Keywords:Polyphosphoester;Biodegradable block copolymer;Receptor-mediated drug delivery;Reactive micelle;HepG2cells1.IntroductionDrug delivery systems using soluble polymers,liposomes,and self-assembled nanoparticles etc.have been developed to reduce the side effects and increase the therapeutic efficacy of anticancer drugs [1–4].Among them,loading drug molecules into the hydrophobic core of self-assembled polymer micelles in a mesoscopic size range (several tens of nanometers)is one of the resolutions to increase solubility of hydrophobic drug.Such micellar nanoparticles passively accumulate in solid tumortissues via enhanced permeability and retention (EPR)effect and enhance therapeutic efficacy [4,5].Polymeric micellar na-noparticles can be thermodynamically stable,and the hydrophil-ic shell ensures its stable dispersion in aqueous solution through a steric stabilization effect,which facilitates its long term blood circulation following intravenous injection [6].These advan-tages make polymer micellar nanoparticles promising for hy-drophobic drug delivery.Challenge is remained in development of cellular specific polymeric micellar system for targeted drug delivery.Biofunc-tional molecules or ligands are expected to be conjugated to micelle surface for specific cell binding and internalization.In this regard,functionable groups at the end of hydrophilic polymer segments are generally required for bioconjugation.Most reported functionalized polymeric micellar systems are based on block copolymers of hydrophobic aliphatic polyester andAvailable online at Journal of Controlled Release 128(2008)32–40/locate/jconrelCorresponding author.Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences,University of Science and Technology of China,Hefei,Anhui 230027,PR China.Tel.:+865513600335;fax:+865513600402.E-mail address:jwang699@ (J.Wang).1Contribute equally to this work.0168-3659/$-see front matter ©2008Elsevier B.V .All rights reserved.doi:10.1016/j.jconrel.2008.01.021functionalized hydrophilic poly(ethylene glycol).For example, Kataoka et al.reported sugar and small peptidyl ligands modified polymer micelles and reactive polymer micelles based on an aldehyde-ended poly(ethylene glycol)/poly(lactide)block copo-lymer[7,8].Langer et ed carboxy-terminated poly(D,L-lactide-co-glycolide)-block–poly(ethylene glycol)to fabricate micellar nanoparticles for surface conjugation of prostate specific membrane antigen binding aptamer[9–11].Liang et al.reported polymer micelles of poly(γ-benzyl L-glutamate)/poly(ethylene glycol)diblock copolymer end capped with galactose moiety for liver targeted drug delivery[12].Instead of conjugation to the end of hydrophilic polymer segments,biofunctional molecules or ligands have also been conjugated to the side groups of hydrophilic block,such as galactosamine-conjugated micelles based on poly(γ-glutamic acid)(γ-PGA)and poly(lactide)(PLA) block copolymer[13–15].In the past few years,we have developed biocompatible po-lyphosphoesters for drug/gene delivery and tissue engineering, taking their advantages of biodegradability and structural flex-ibility[16–18].We have reported two convenient methods to synthesize structurally and compositionally defined block copolymer of poly(ɛ-caprolactone)(PCL)and polyphosphoester. One method was sequential polymerization ofɛ-caprolactone and cyclic phosphoester monomer with trimer of aluminum isoprop-oxide as the initiator[19].The other method was to use PCL macroinitiator to initiate cyclic phosphoester monomer polymer-ization with stannous octoate as the catalyst[20].Through these two methods,polyphosphoesters with hydroxyl end groups can be conveniently synthesized.On the other hand,we have recently demonstrated in aqueous solution triblock copolymers of PCL and poly(ethyl ethylene phosphate)(PEEP)formed micellar nanoparticles with hydrophobic PCL core and hydrophilic PEEP shell[17].As compared with the well-known poly(ethylene glycol),hydrophilic polyphosphoesters may hold interesting properties for drug delivery system design since polypho-sphoesters are degradable and more structurally flexible for physicochemical property adjustment.The aim of this study is to develop reactive micelles for surface ligands conjugation using block copolymer of PCL and polyphosphoesters and study its potential for cellular specific drug delivery.We synthesized diblock copolymer of PCL and PEEP and further activated the hydroxyl end groups of PEEP by reaction with N,N′-carbonyldii-midazole.In aqueous solution,the activated diblock copolymer assembled into micellar nanoparticles with surface ready to react with amine(s).D-Galactosamine was then conjugated to the surface of these micellar nanoparticles.The potential of such system for targeted anticancer drug delivery to HepG2cells was studied by examining asialoglycoprotein receptor(ASGP-R) mediated cell binding and internalization ability and bioactivity of paclitaxel-loaded micelles.2.Materials and methods2.1.Materials2-Ethoxy-2-oxo-1,3,2-dioxaphospholane(EEP)was synthe-sized and purified as previously reported[20].Tetrahydrofuran (THF)was refluxed over potassium–sodium alloy under N2at-mosphere and distilled out just before use.PCL macroinitiatorbearing one hydroxyl end group per polymer chain(PCL67–OH) was synthesized by ring-opening polymerization ofɛ-caprolac-tone in THF using aluminum isopropoxide as the initiator[19].The polymerization degree of the PCL macroinitiator was67,which was calculated based on the integration ratio of the tripletresonance at4.03ppm(2H)and the singlet resonance at3.66ppm(2H)from its1H NMR.The molecular-weight distribution ofPCL67–OH was1.16which was determined by gel permeation chromatography(GPC)as described below.Stannous octoate(Sn (Oct)2)was purified according to a method described in literature [20].D-Galactosamine hydrochloride(98%),D-glucosamine hydrochloride(99%),N,N′-carbonyldiimidazole(CDI,98%), and paclitaxel were obtained from Sigma-Aldrich Co.All other solvents were of reagent grade and used as received.Dialysis membrane tubing Spectra/Por®Float-A-Lyzer(MWCO25,000) was obtained from Spectrum Laboratories,Inc.2.2.Syntheses and characterization of polymers2.2.1.Synthesis of block copolymer(PCL–PEEP)Block copolymer PCL–PEEP was obtained by ring-opening polymerization of EEP using PCL67–OH as the initiator and Sn (Oct)2as the catalyst.Briefly,to a solution of EEP(7.60g, 50.0mmol)and PCL67–OH(7.64g,1.0mmol)in THF at35°C was added Sn(Oct)2(0.41g,1.0mmol).After3h reaction,the mixture was concentrated and the polymer was precipitated in cold ethyl ether twice.The obtained block copolymer PCL–PEEP was dried under vacuum to a constant weight at room temperature.The yield was approximately75%.The degree of polymerization(DP)of EEP was calculated based on the integration ratio of resonance at 4.18and4.26ppm(6H),assigned to methylene protons of PEEP block,to resonance at2.35ppm(2H),assigned to the methylene protons of PCL block(Fig.1A).Based on this calculation,the DP of EEP was36,with respect to72%EEP conversion.The mol-ecular-weight distribution was1.40,determined by gel perme-ation chromatography.This copolymer was further denoted as PCL67–PEEP36.Fig.1.1H NMR spectra of PCL67–PEEP36(A),PCL67–PEEP36–CDI(B)and glucose-conjugated block copolymer(C).33Y.-C.Wang et al./Journal of Controlled Release128(2008)32–402.2.2.Synthesis of CDI activated block copolymer(PCL67––PEEP36––CDI)Block copolymer PCL67–PEEP36(1.0g)and CDI(65mg,5.0 equiv mol of hydroxyl groups)were dissolved in10mL of anhydrous THF.The solution was stirred at room temperature for 12h,and then concentrated.The polymer was precipitated into anhydrous ethyl ether.The activated block copolymer PCL67–PEEP36–CDI was obtained by filtration and then dried under vacuum.The yield was approximately90%.2.2.3.Characterization and measurementsBruker A V300NMR spectrometer(300MHz)was used for1H NMR spectrum analyses to determine the structure and composi-tion of block copolymers.Deuterated chloroform containing 0.03v/v%tetramethylsilane was used as the solvent for NMR measurements.Molecular weights and molecular-weight distri-butions were determined by gel permeation chromatography measurements on a Waters system,equipped with a Waters1515 HPLC solvent pump,a Waters2414refractive index detector,and four Waters Styragel columns(HR4,HR2,HR1,HR0.5,effective molecular-weight range5000–500,000,500–20,000,100–5000, 0–1000respectively).HPLC grade chloroform was purchased from J.T.Baker and used as the eluent at40°C,delivered at a flow rate of1.0mL min−1.Monodispersed polystyrene standards obtained from Waters Co.with a molecular-weight range1310–5.51×104were used to generate the calibration curve.2.3.Preparation and characterization of polymer micelles2.3.1.Preparation of micellesMicelles were prepared by a dialysis method.PCL67–PEEP36–CDI(50mg)was dissolved in5mL of THF.To this solution was added dropwise100mL of Milli-Q water(Millipore Milli-Q Synthesis,18.2MΩ)under gentle stirring.After standing at room temperature for3h,THF was removed by dialysis against Milli-Q water for24h.2.3.2.Conjugation of D-galactosamine and D-glucosamine to micelle surfaceD-Galactosamine or D-glucosamine was conjugated to micelle surface via reaction with micelles as described above.D-Galac-tosamine or D-glucosamine was dissolved into micelles at pH9.0. After24h reaction at room temperature,micelles were dialyzed against Milli-Q water for24h to remove free D-galactosamine or D-glucosamine.The contents of D-galactosamine and D-glucosa-mine conjugated to micelles were determined by the colorimetric Morgan Elson assay[21].2.3.3.Characterization of polymer micellesTo confirm the surface functionality of micelles,micelles in aqueous solution was lyophilized and dissolved in CDCl3.1H NMR spectrum was recorded using Bruker A V300NMR spectrometer(300MHz).The methods for critical micelle con-centration(CMC)determination,transmission electron micro-scopy(TEM)observation,and particle size and zeta potential measurements were described in detail in the supplementary information.2.4.Cell binding and internalization studies2.4.1.Preparation of rhodamine123-loaded micellesRhodamine123-loaded micelles were prepared in a similar method as described in Section 2.3.1.PCL67–PEEP36–CDI (10mg)and rhodamine123(0.1mg)were dissolved in THF (5mL),and10mL of Milli-Q water was added dropwise to this solution under gentle stirring.THF was removed by dialysis against water for24h.Conjugation of D-galactosamine or D-glu-cosamine to micelle surface was done as described in Section 2.3.2.Micelle with D-glucosamine or D-galactosamine conjuga-tion was denoted as NP-Glu or NP-Gal,respectively.The control micelles(NP)without any saccharide moiety was made using PCL67–PEEP36in a similar method.2.4.2.Binding of micelles to HepG2cellsHepG2cells(150,000cells per well)in24-well plates were precultured with100μL of rhodamine123-loaded NP,NP-Glu or NP-Gal in phosphate-buffered saline(PBS,1mg mL−1)at4°C for 1h.Cells were washed with ice-cold PBS and further cultured with1mL of complete DMEM(Dulbecco's Modified Eagle's Medium,containing10%Hyclone fetal bovine serum,50units mL−1penicillin and50units mL−1streptomycin)at37°C and5% CO2atmosphere.At different culture intervals,cells were se-parately harvested by trypsinization,washed with PBS and resus-pended in200μL of PBS for flow cytometric analysis using a Becton Dickinson FACSCalibur flow cytometer.2.4.3.Cellular uptake of micelles by HepG2cellsRhodamine123-loaded micelles(100μL,1mg mL−1in PBS)were incubated with150,000HepG2cells in1mL of complete DMEM culture medium.For the competing inhibition study,D-galactosamine was added to reach the final concentra-tion of20mM.After incubation at37°C for4h,cells were trypsinized,washed with PBS twice,resuspended in200μL of PBS and subjected to flow cytometric analysis.For microscopic observation,HepG2cells(5×104)were seeded on coverslip in a24-well tissue culture plate until they were totally adherent.100μL of Rhodamine123-loaded NP or NP-Gal(1mg mL−1in PBS)were added to distinct wells and incubated at37°C for2h in1mL of complete DMEM culture medium.The cells were washed and fixed with4%formalde-hyde and the slides were mounted and observed with a Zeiss LSM510Laser Confocal Scanning Microscope imaging system with an upright confocal microscope and a40×objective.2.5.Drug loading and activity analyses2.5.1.Preparation of paclitaxel-loaded micellesPaclitaxel was loaded into micelles by the dialysis method.In a typical procedure,the block copolymer(10mg)was dissolved in 1.0mL of THF,and to this solution was subsequently added paclitaxel dissolved in DMSO at various weight ratios to block copolymer(paclitaxel/polymer=0.05–0.1).Milli-Q water was then added dropwise to this solution.The mixture was stirred at room temperature for3h and filtered through0.45μm Millipore membrane filter.The solution was dialyzed for24h and freeze-34Y.-C.Wang et al./Journal of Controlled Release128(2008)32–40dried.Paclitaxel-loaded NP-Gal is further denoted as NP-Gal-PTX,while the control NP-PTX was made using PCL67–PEEP36 without D-galactosamine conjugation in a similar method.After dissolving paclitaxel-loaded micelles with acetonitrile–water(50:50,v/v),the loading amount of paclitaxel was deter-mined by HPLC analysis.HPLC analysis was performed on a Waters HPLC system consisting of Waters1525binary pump, Waters24872-channel UV–vis detector,1500column heater and a Symmetry C18column.HPLC grade acetonitrile–water (50:50,v/v)was used as the mobile phase at30°C with a flow rate of1.0mL min−1.UV–vis Detector was set at227nm and linked to Breeze software for data analysis.Linear calibration curves for concentrations in the range of0.098–100μg/mL were constructed using the peak areas by linear regression analysis.The regres-sion equation was calculated as y=42204x+8287.8(R2= 0.9996).The concentrations of paclitaxel were determined by comparing the peak area with the stand curve.The drug loading content(DLC)and drug loading efficiency(DLE)were calculated by the following equations:DLC¼weight of PTX in micellesweight of PTX loaded micellesÂ100kDLE¼weight of PTX in micellesweight of PTX used for encapsulationÂ100k2.5.2.In vitro paclitaxel release from micellesIn vitro release profiles of paclitaxel from micelles were investigated in phosphate-buffered saline(PBS,0.02mol L−1,pH 7.4)using a dialysis-bag diffusion technique.Micelles(1.5mL) were introduced into a dialysis membrane tubing and incubated in 25mL of buffer at37°C with stirring.At predetermined intervals, buffer were drawn and replaced with an equal volume of fresh medium.The concentration of paclitaxel in the solution was measured by HPLC.2.5.3.Viability of HepG2cells treated with paclitaxel-loaded micellesThe cytotoxicity of NP-Gal-PTX or NP-PTX against HepG2 cells was evaluated in vitro by MTT assay,using paclitaxel dissolved in DMSO as the control(the final concentration of DMSO in medium was1%v/v).HepG2cells were seeded in96-well plates at10,000cells per well in100μL of complete DMEM medium and incubated at37°C in5%CO2atmosphere for24h. The culture medium was replaced with100μL of fresh medium containing paclitaxel-loaded micelles.Various PTX concentra-tions were achieved by adding dilution of the micelle formulation with4.0%of drug loading content.Cells were further incubated for72h,followed by addition of25μL of MTT stock solution(5mg mL−1in PBS)to achieve a final con-centration of1mg mL−1.After incubation for an additional2h, 100μL of the extraction buffer(20%SDS in50%DMF,pH4.7, prepared at37°C)was added to the wells and incubated over-night at37°C.The absorbance of the solution was measured at 570nm using a Bio-Rad680microplate reader and cell viability was normalized to that of HepG2cells cultured in the culture medium without paclitaxel.2.5.4.Cell cycle analyses of HepG2cells treated with paclitaxel-loaded micellesHepG2cells cultured in24-well plates were treated for24hwith paclitaxel in DMSO,NP-PTX or NP-Gal-PTX at threedifferent paclitaxel doses(0.075,0.3,and1.2μM).For cellstreated with paclitaxel in DMSO,the final concentration ofDMSO in medium was kept at1%(v/v).The cells were tryp-sinized,washed with PBS,fixed with70%ethanol and cen-trifuged.The cell pellet was suspended with PBS and treatedwith200μL of propidium iodide(PI)staining solution(0.1%Triton X-100,0.2mg mL−1DNase-free RNase A and20μgmL−1PI)for15min at37°C.The fluorescence was measuredusing flow cytometer and cell cycle was analyzed usingWinMDI2.9software.3.Results and discussion3.1.Synthesis and characterization of block copolymersWe have previously reported polyphosphoesters with linearmolecular structure can be synthesized through ring-openingpolymerization of EEP in THF under co-initiation of dodecanoland Sn(Oct)2[20].Instead of dodecanol,we have also used PCLdiol as the initiator to synthesize triblock copolymer of PCL andPEEP[17].In this study,mono hydroxyl-terminated poly(ɛ-caprolactone)PCL67–OH was used as macroinitiator for EEP polymerization to obtain a diblock copolymer(Scheme1).Thefeeding molar ratio of PCL67–OH to EEP was1:50,while the reaction time was limited to3h since extension of reaction time will likely lead to chain exchange side reaction though EEP conversion can be increased[20].Such copolymer chains contain functional hydroxyl groupsat the end of PEEP segments,which was demonstrated by thepresence of resonance appeared at3.82ppm,assigned to me-thylene protons conjoint to hydroxyl end groups of polypho-sphoester block(Fig.1A)as reported by us previously[17,19].These hydroxyl end groups can be conveniently modified forbiofunctional molecules conjugation.As depicted in Scheme1,in this study,coupling reagent CDI was used to activatetheScheme1.Schematic illustration of syntheses of block copolymer and surface functionalized micelles.35Y.-C.Wang et al./Journal of Controlled Release128(2008)32–40hydroxyl groups and generate the carbonylimidazole derivative PCL67–PEEP36–CDI,while imidazole groups are known to be easily substituted under the attack of nucleophiles such as amines.1H NMR analysis of PCL67–PEEP36–CDI demon-strated the successful conversion of hydroxyl groups to car-bonylimidazole moieties.As shown in Fig.1B,no signal at 3.82ppm was further found in1H NMR spectrum of PCL67–PEEP36–CDI.Instead,newly appeared resonance at4.62ppm should be assigned to protons of methylene groups conjoined to the end carbonyl group of PCL67–PEEP36–CDI.In addition,the presence of resonances at7.21,7.45and8.20ppm,should be assigned to protons of imidazole residues,demonstrating the successful activation of hydroxyl groups of PEEP blocks.3.2.Micelle preparation and characterizationPCL67–PEEP36–CDI is amphiphilic and in aqueous medium it self-assembled to form micellar structure.The spherical morphology of micelles was demonstrated by TEM examina-tion,shown in the supplementary information(Fig.S1).The CMC value of PCL67–PEEP36–CDI,which describes the physical properties of the micelles relating to its thermodynamic stability,was determined by the method based on partition of pyrene probe in hydrophobic core against aqueous environment [22].The intensity ratio of the bands at339.0and335.5nm (I339.0/I335.5)as a function of the logarithm of the copolymer concentration was given in supplementary information(Fig.S2). The CMC value of PCL67–PEEP36–CDI,which was8.9×10−4mg mL−1,was taken at the intersection of the tangents to the horizontal line of intensity ratio with relatively constant values and the diagonal line with rapid increased intensity ratio.3.3.Conjugation of D-galactosamine and D-glucosamine to micelle surfaceD-Galactosamine and D-glucosamine were conjugated to mi-celle surface via substitution of imidazole by amino groups.To demonstrate the successful conjugation,micelle in aqueous solution was lyophilized after conjugation and the polymer was extracted into CDCl3for1H NMR analyses.As shown in Fig.1C, resonances corresponding to imidazole residue protons disap-peared.Instead,newly appeared peaks at3.3–3.8,5.01and 6.02ppm were due to the presence of glucosyl protons and its anomeric protons(a and a'),indicating the complete substitution of imidazole by glucosyl residues.The contents of D-galactosa-mine and D-glucosamine conjugated to micelles determined by the colorimetric Morgan Elson assay[21]were59.6±8.2nmol mg−1and65.1±10.2nmol mg−1,corresponding to78.2±10.7mol%and85.4±13.3mol%of total end groups,respectively. The CMC of sugar-conjugated block copolymer was comparable to PCL67–PEEP36and PCL67–PEEP36–CDI as determined by the same method described above.In addition,average size(about 70nm in diameter)and size distribution of micelles were not significantly affected after sugar conjugation that was measured by dynamic light scattering and given in supplemental informa-tion(Fig.S3A).However,with the conjugation of sugar mo-lecules,the micelles were more negatively charged compared with PCL67–PEEP36–CDI micelles.The average zeta potential value was around−20mV(Fig.S3B),which may promote micelle stabilization due to static repellency between particles.3.4.Cell binding and internalizationThe asialoglycoprotein receptor(ASGP-R)on the surface of hepatoma cells is a recycling endocytotic receptor and re-cognizes galactose-and N-acetylgalactosamine-terminaled gly-coproteins[23].To demonstrate the enhanced binding ability of galactosamine-conjugated micelles(NP-Gal)to HepG2cells, the micelles were loaded with rhodamine123fluorescent dye and incubated with HepG2cells at4°C.Micelles not bound to cell surfaces were washed off after1h incubation,and the cells were further cultured under normal cell culture conditions at 37°C.Because cell internalization of nanoparticles via endocy-tosis is an ATP-dependent process,micelles attached to cell surface should not be internalized by cells at4°C[24].Further incubation of cells with attached nanoparticles on cell membrane at37°C allowed micelle internalization into cells and rhodamine 123release.As shown in Fig.2A,the fluorescent intensity of HepG2cells right after1h treatment with NP-Gal micelles at4°C was slightly higher than those treated with non-modified micelles(NP)or glucosamine-conjugated micelles(NP-Glu), which was also indicated as the relative geometrical mean fluorescence intensity(GMFI)shown as the inset.The fluo-rescent positive cell population was around18%after incubation at4°C with NP-Gal.It is worth noting that high concentration of rhodamine123in micellar core might result influorescenceFig.2.Flow cytometric analyses of binding ability of non-modified micelles (NP,blue),glucosamine-conjugated micelles(NP-Glu,green),and galactosa-mine-conjugated micelles(NP-Gla,red)to HepG2cells.Cells were mixed with micelles at4°C for1h and further incubated at37°C for0(A),75(B),150(C) and180min(D).Blank cells incubated with micelle free medium were used as the control(purple).The relative geometrical mean fluorescence intensity (GMFI)of cells incubated with NP,NP-Glu and NP-Gal are shown as insets. (For interpretation of the references to colour in this figure legend,the reader is referred to the web version of this article.)36Y.-C.Wang et al./Journal of Controlled Release128(2008)32–40quenching therefore fluorescence detected by flow cytometry could be relatively weak.Fig.2B –D shows the results analyzed by flow cytometry when HepG2cells were further incubated at 37°C following the micelle attachments to cell membrane.It is obvious that with elongation of incubation time from 75to 180min,positive cell population (with log mean fluorescence intensity more than 101)increased from 28.9%to 76.6%,while the relative GMFI increased from 1.65to 6.39in the group treated with NP-Gal,indicating more rhodamine 123-loaded micelles attached to HepG2cells at 4°C were internalized and the cargo was released.In contrast,fluorescence of cells treated with NP or NP-Glu was much less pronounced due to their poor attachment to HepG2cells at 4°C.This phenomenon demon-strated that the ligand –receptor recognition between galactosyl residue and ASGP-R mediated the surface binding of micelles to HepG2cells.It is possible that negatively charged surface of micelles resulted in electrostatic repulsion to cells,which in fact is unfavorable to micelle binding to cell surface.Therefore,without receptor mediation,NP-Glu and NP had less chance to reach HepG2cell surface and be internalized into cells.In another experiment,HepG2cells were directly incubated at 37°C with rhodamine 123-loaded micelles.Cellular accumula-tion of rhodamine 123was analyzed and results were shown in Fig.3A –C.Incubation of NP-Gal with HepG2cells at 37°C for 4h resulted in increased cell population with high fluorescence (66.2%with log mean fluorescence intensity more than 102),indicating significant micelle internalization and rhodamine 123release.On the contrast,the percentage of cell population with log mean fluorescence intensity more than 102were less than 7%when cells were incubated with NP or NP-Glu,demonstrating only minimal micelles were taken up by HepG2cells without receptor mediation.In the presence of 20mM galactosamine,the uptake of NP-Gal was significantly inhibited,which was char-acterized by the relative GMFI of HepG2cells incubated with orwithout galactosamine (Fig.3D),suggesting that cellular uptake of NP-Gal was mediated by the asialoglycoprotein receptors.Fig.4showed the differential interference contrast (DIC),fluorescence and merged images of HepG2cells after 2h incubation at 37°C with rhodamine-123loaded micelles with or without galactosamine conjugation.The intensity of fluores-cence observed in HepG2cells incubated with NP-Gal markedly increased when compared with that of HepG2cells incubated with non-modified NP.It further confirmed the preponderance of NP-Gal on cellular uptake due to the interaction between galac-tosyl moieties with ASGP-R of HepG2cells.3.5.Delivery of paclitaxel to HepG2cellsPaclitaxel is a highly hydrophobic anticancer drug that has a poor solubility (approximately 1μg/mL in aqueous solution at pH 7.4)[25,26].Amphiphilic block polymers,which self-assemble to nanoparticles in aqueous solution,have been often employed as the vehicle to load paclitaxel into the hydrophobic core for enhanced delivery efficiency [27].In paclitaxel loadingprocedureFig. 3.Flow cytometric analyses of cellular rhodamine 123fluorescence intensity of HepG2cells incubated with galactosamine-conjugated micelles (NP-Gal),glucosamine-conjugated micelles (NP-Glu)or non-modified micelles (NP)at 37°C for 4h (A –C),and the relative geometrical mean fluorescence intensity (GMFI)of HepG2cells cultured with NP-Gal in the absence (−Gal)or presence (+Gal)of 20mM of galactosamine(D).Fig.4.Differential interference contrast (A),fluorescence (B)and merged (C)images of HepG2cells after 2h incubation with non-modified micelles (NP)or galactosamine-conjugated micelles (NP-Gal)at 37°C.Table 1Drug loading efficiency (DLE)and drug loading content (DLC)of micelles prepared at various feeding weight ratios of paclitaxel to block copolymerFeed weight ratio of polymer to PTX DLE (%)DLC (%)10:144.2 4.4220:157.5 2.8750:177.4 1.55200:192.90.4737Y.-C.Wang et al./Journal of Controlled Release 128(2008)32–40。