Peptide-Directed Self-Assembly of Functionalized Polymeric Nanoparticles. Part II: Effects of Nanoparticle Composition on Assembly Behavior and Multiple Drug Loading Ability

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Peptide-functionalized polymeric nanoparticles were designed and self-assembled into continuous nanoparticle fibers and three-dimensional scaffolds via ionic complementary peptide interaction. Different nanoparticle compositions can be designed to
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  Peptide-Directed Self-Assembly of Functionalized Polymeric Nanoparticles.Part II: Effects of Nanoparticle Compositionon Assembly Behavior and Multiple DrugLoading Ability a Xu Xiang, Xiaochu Ding,* Trevor Moser, Qi Gao, Tolou Shokuhfar,Patricia A. Heiden* Peptide-functionalized polymeric nanoparticles were designed and self-assembled intocontinuous nanoparticle fibers and three-dimensional scaffolds via ionic complementarypeptideinteraction. Differentnanoparticle compositionscanbedesignedtobeappropriate foreach desired drug, so that the release of each drug isindividually controlled and the simultaneous sustainablerelease of multiple drugs is achieved in a single scaffold. Aself-assembled scaffold membrane was incubated withNIH3T3 fibroblast cells in a culture dish that demonstratednon-toxicity and non-inhibition on cell proliferation. Thistype of nanoparticle scaffold combines the advantages of peptide self-assembly and the versatility of polymericnanoparticle controlled release systems for tissue engi-neering. 1. Introduction Efficient tissue regeneration often depends on thedelivery of various drugs (i.e., low or high molecularweight agents including growth factors) at appropriaterates to implanted cells. Therefore, understanding therelationship between tissue engineering, scaffoldingsystems, and drug delivery is crucial for the design of functional scaffolds for the desired applications. [1] Forexample, skin regeneration usually requires the deliveryof a complex mixture of growth factors and cytokines,(e.g., fibroblast growth factor, keratinocyte growth factor,vascular endothelial growth factor, and interleukin 1 a )within the wound to promote cell proliferation andmigration for wound healing. [2,3] Therefore, the fabrica-tion of sophisticated scaffolding systems with the abilitytophysicallysupportcellsandatthesametimedeliveranappropriate ‘‘drug cocktail’’ at an appropriate rate topromote cell growth and health is an important advancefor tissue engineering.Self-assembled peptide nanofiber scaffolds have gainedinpopularitycomparedtoscaffoldingfromotherpolymers,possibly because they are perceived as non-toxic and X. Xiang, X. Ding, P. A. HeidenDepartment of Chemistry, Michigan Technological University,Houghton 49931, MichiganE-mail:, xding@mtu.eduT. Moser, Q. Gao, T. ShokuhfarDepartment of Mechanical Engineering and EngineeringMechanics, Michigan Technological University, Houghton 49931,Michigan a SupportingInformation isavailablefromtheWileyOnlineLibraryorfrom the author. Full Paper   2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim  Macromol. Biosci.  2014,  DOI: 10.1002/mabi.201400438  1   Early View Publication; these are NOT the final page numbers, use DOI for citation !!  R  biodegradable, and the porousfibril structuresare thoughtto be similar to extracellular matrix (ECM) and so will aidcellattachment. [4–16] Also,thesematerialscanassembleinsituintoahydrogelatphysiologicalenvironment. [15,16] Thefirst reported self-assembling ‘‘designer peptide’’ wasEAK16-II (AEAEAKAKAEAEAKAK), isolated from a yeastprotein, zuotin. [17] EAK16-II spontaneously self-assemblesinto stable  b -sheets in aqueous conditions across a broadrange of temperature and pH, even in the presence of significantquantitiesofadenaturingagent,suchasureaorguanidium hydrochloride. [18,19] The self-assembling process is driven by the hydro-phobic interactions of alanine (A) domains and ion-pairinteractions between negatively charged glutamic acid(E) side chains with positively charged lysine (K) sidechains. The importance of the strong ion-pair interactionfor successfully forming the  b -sheet was supported byinvestigation of a complementary pair of designerpeptides, that were self-repulsing but possessed strongmutually attracting peptide sequences where one of thepair possessed a sequence with positive charges(Ac-WKVKVKVKVK-amide) and the other sequence withnegative charges (Ac-EWEVEVEVEV-amide). [20] On mixingthis pair of complementary peptides, a rapid assemblyinto a viscoelastic hydrogel occurred at a concentration aslow as 0.25wt.-%. This hydrogel retained mechanicalstrength, even after repeated shear-induced breakdowns,due to the electrostatic interactions. The strong electro-static and selective interaction between the oppositecharges demonstrated one of the key merits of using ion-complementary  b -sheet motifs.Other designer self-assembling peptide hydrogels (e.g.,RADA16-IorII)havealsobeenstudiedandthey,alongwithEAK16-II, have been widely used as both controlled drugdelivery systems and 3D scaffolds. [16–23] The drug deliverymethodistypicallyasimplephysicalmixingofadrugintothe peptide mixture during the gel formation, [21,22] but itcan also be chemically bonded onto the C-terminal orN-terminal. [23] Sometimes a combination of both methodsis used to load multiple active agents. [24,25] However, these methods all have significant limitationswith respect to the quantity of drug(s) that can beincorporated, as well as the real possibility that theincorporated drug(s) may have a detrimental effect onthe subsequent self-assembly process or the mechanicalstabilityoftheformedhydrogel.Thereisalsoaverylimitedability to control the release rate of drugs with differentproperties.Therefore,thepeptideitselfmustbedesignedinconjunction with the specific drug(s) that will be incorpo-rated.Moreover,theeffectivedistributionofmultipledrugsin scaffolds is not easy to achieve.In Part I of this series we demonstrated a new approachto fabricate tissue scaffolding, prepared by peptide-directed self-assembly of polymeric nanoparticles intofibers and 3D scaffolds, to address these limitations. [26] That paper described the synthesis and assembly of thenanoparticle fiber scaffolds, demonstrated the ability tocontain low and high molecular weight model drugs, andproved the incorporation and controlled release of insulinfrom the peptide conjugate nanoparticles and theassembled scaffold. The first paper also described otherpotential variables that affected scaffold morphology andthe advantage of being able to design and makeassembled nanoparticle scaffolds where different desireddrugs can be incorporated into a nanoparticle with thedesired size and composition for that drug. The size of theparticles is controllable and can range from nano- tomicroscale, and can be designed to swell significantly oronly a little in aqueous media depending on thecomposition and size of the shell in core–shell particlescaffolds.The nanoparticle self-assembly approach is expected topossess the ability to: (i) incorporate multiple drugs,regardless of that drug’s hydrophilicity or hydrophobicity,within nanoparticles in the scaffolding; (ii) independentlycontrolthereleaseofeachdrugfromananoparticlewhosecomposition is designed for that specific drug; (iii) controlthe distribution of the different drugs within the scaffold,bytheassemblysequence;and(iv)beabletobeintroducedinto a patient by injection followed by controlled self-assemblyofthescaffoldinsitu.Theseabilitiesprovidethistypeofassembledfibersystemwithsignificantadvantagesover electrospun fiber scaffolding as well as peptide-onlyscaffolding.Here, we illustrate the self-assembly behavior andversatility of peptide-functionalized nanoparticles usingdifferent particle sizes, that can alter scaffold porosity andmechanical stability, and also show the ability to load andindependently control the simultaneous release of threedrugs within a single scaffold. We used two hydrophobicmodel drugs (4 0 ,5 0 -dibromofluorecein as moderately andfluoresceinassomewhatmorehydrophobic),andaslightlyhydrophilic model drug (nitrofurazone). We also showedthebiocompatibilityofthesenanoparticlefiberscaffoldsbyincubating a self-assembled 2D scaffold membrane withfibroblastcells(NIH3T3celllines)inahumidifiedincubatorat 37 8 C for one week. 2. Experimental Section 2.1. Materials All reagents for peptide synthesis were purchased from AAPPTecLLC (Louisville, KY) and used as received. Synthesis of ioniccomplementarypeptides(P1:H 2 N-TTTT-AEAEAEAE-amideandP2:H 2 N-TTTT-AKAKAKAK-amide) was described in previous work. [26] 1-Vinyl-2-pyrrolidinone(VP)(  99%),2-hydroxyethylmethacrylate(HEMA) (97%), methyl methacrylate (MMA) (99%), 1,4-dioxane www.mbs-journal.deX. Xiang, X. Ding, T. Moser, Q. Gao, T. Shokuhfar, P. A. Heiden 2 Macromol. Biosci.  2014,  DOI: 10.1002/mabi.201400438 2   2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 2       R Early View Publication; these are NOT the final page numbers, use DOI for citation !!  (99  þ  %), 2,2 0 -azobisisobutyronitrile (AIBN) (98%), dimethyl for-mamide (DMF, 99.9%), phosphate buffered saline (Biotech) andmodel drugs of nitrofurazone (NF), 4 0 ,5 0 -dibromofluorescein (DBF),fluorescein (FL), and cell adhesion peptide (RGDS) were purchasedfromSigma–Aldrich.Ethylalcohol(200proof,anhydrous)wasfromPHARMCO-AAPER, and diethyl ether (anhydrous) and dimethylsulfoxide (DMSO) were from Mallinckrodt Baker Inc. (Phillipsburg,USA).VPandHEMAwerepurifiedpriortousebypassingthroughaneutral alumina column. MMA was distilled before use. All otherreagents were used as received. Fibroblast cells (NIH3T3 cell line)were purchased from ATCC and cell media (DMEM/high glucose)was from Hyclone Laboratories, Inc. (Utah, USA). Ultrapuredeionized water ( > 17.6 M V  cm) was obtained from MEG-PURESYSTEM (MP-190 LC). 2.2. Synthesis and Self-Assembly of IonicComplementary Peptides A detailed peptide synthesis for P1 (H 2 N-TTTT-AEAEAEAE-amide)andP2(H 2 N-TTTT-AKAKAKAK-amide),characterizationbyMALDI-TOFMS(MicroflexLRF,BrukerDaltonics,Billerica,USA)and 1 HNMR(Varian Unity INOVA 400MHz, McKinley Scientific, Sparta, NJ,USA),andtheself-assemblybehaviorofthedesignerpeptideswerealreadyreportedinpreviousstudyandSupportingInformation. [26] 2.3. Synthesis of Amphiphilic Triblock Copolymers Amphiphilic triblock copolymers of PVP-b-PMMA-b-PVP andPHEMA-b-PMMA-b-PHEMA, with reactive carboxylic acid termi-nals(HOOC-ABA-COOH),weresynthesizedbyRAFTpolymerizationusing S,S 0 -bis( a , a 0 -dimethylacetic acid) trithiocarbonate (BDAT) asachaintransferagent(CTA)and2,2 0 -azobisisobutyronitrile(AIBN)as the initiator, as described in previous work. [26] In the presentwork, two hydrophilic shells (PVP and PHEMA) were tested andusedatdifferentblockratiosofPVPorPHEMAtoPMMA,asshownin Table 1. The detailed synthesis procedure is described inSupportingInformation.Thesevariablesallowedanassessmentof how nanoparticle composition and size affected the subsequentself-assembly behavior as well as drug loading abilities andcontrolled release behavior. 2.4. Characterization of Macro-CTA and TriblockCopolymers The number average molecular weight (  Mn ) of the hydrophilicblock(PVPandPHEMA)wasdeterminedbyMALDI-TOFMSandthenumberaveragedegreeofpolymerization(X N )wascalculatedfromthe measured  Mn . These results are given in Table 1. A detailedanalysisoftheMALDI-TOFMSspectraisdescribedelsewhere. [26]1 HNMR spectra (performed in DMSO-d 6 ) and peak assignments areshown in Figure 1. The integrated peak area from the PVP andPMMA blocks gives the block ratio X PVP /X PMMA  and thus thenumber average molecular weight of the triblock copolymer isdetermined. For example, analysis of PVP-b-3PMMA-b-PVP showsthattheintegratedareaofthepeakat d :3.14(–CH 2 –N,fromthePVPblock) and the peak areas at d : 3.56 (–OCH 3  from the PMMA block,alsocontains–CH–fromthePVPbackbone)areusedtocalculatetheblock ratio of X PVP /X PMMA ¼ 1:1.10 according to Equation (1).2  X  PVP 3  X  PMMA þ  X  PVP ¼  Area ð CH 2  N Þ Area ð OCH 3 ;  CH Þ ¼ 11 : 4124 : 49  ð 1 Þ The actual block ratios of other copolymers (PVP- b -6PMMA- b -PVP,PVP- b -12PMMA- b -PVP, and PHEMA- b -6PMMA- b -PHEMA) are cal-culatedto be 1:2.38,1:6.72,and1:1.60.Accordingto theknown  X  N andtheactualblockratioscalculated,the  X  N and  Mn ofthetriblockcopolymers are thus determined and given in Table 1.The peak assignments for the  1 H NMR spectra of copolymerswere made as shown below. PVP-b-PMMA-b-PVP (in DMSO-d 6 ):  d (ppm) ¼ 0.73–1.04 (–CH 3 ), 1.40–1.82 (–CH 2 –), 3.34 (–CH 2 –N–), 3.53(–OCH 3 ,fromPMMAblock, alsocontainssome–CH–fromthePVPblock). PHEMA-b-PMMA-b-PHEMA (in DMSO-d 6 ):  d (ppm) ¼ 0.72–1.13(–CH 3 ),1.42–1.77(–CH 2 –),3.53(–OCH 3 ,fromPMMAblock,alsocontains –CH 2 –OH from the PHEMA block), 3.87 (–CH 2 –O–C ¼ O). 2.5. Coupling Reaction of Copolymer with Peptide(P1 and P2) Thesynthesizedpeptides(P1andP2)werecoupledwiththedesiredamphiphiliccopolymertoformpeptide-copolymerconjugates.Thecoupling reaction was performed between the carboxylic acidterminals of the polymer and the amine terminals of the desired Table 1.  Reactant ratios and products of the macro-CTA and triblock copolymer. Macro-CTA [M] o  /[CTA] o  /[I] o  X  Na)  Mn a) [Da] Yield [%] PVP 1 000:5:1 78 8 954 84PHEMA 1 000:5:1 64 8 557 93Copolymer Mass ratio b)(Macro-CTA) o / (MMA) o  X  N /  X  Mc)  Mn c) [Da] Yield [%]PVP- b -3PMMA- b -PVP 1:1.5 78/86 17 600 73PVP- b -6PMMA- b -PVP 1:3 78/186 27 600 70PVP- b -12PMMA- b -PVP 1:6 78/524 61 400 54PHEMA- b -6PMMA- b -PHEMA 1:3 64/102 18 800 66 a)  Mn  tested by MALDI-TOF;  X  N  ¼  M  n   M  BDAT =  M  Monomer ;  b) Theoretical mass ratio of reactants used to prepare triblock copolymer;  c)  Mn calculated from  1 H NMR by ratio of integrated peaks. So total hydrophilic  X  N  is 78 and 64, and each block is e.g., PVP 39 -PMMA 86 -PVP 39  orPVP 39 -PMMA 186 -PVP 39 . Peptide-Directed Self-Assembly of Functionalized Polymeric Nanoparticles. Part II: Effects of Nanoparticle Composition on Assembly Behavior and Multiple Drug Loading Abilityw Macromol. Biosci.  2014,  DOI: 10.1002/mabi.201400438  3   2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim   3 Early View Publication; these are NOT the final page numbers, use DOI for citation !!  R  peptide using 2-(7-aza-1-H-benzotriazol-1-yl)-1,1,3,3-tetramethy-laminium(HATU)asthecouplingagent,asreportedpreviously. [26] However,herethepolymermolecularweightsaredifferent,sothereactant quantities also differ. A detailed description of the coupling reaction is provided in the Supporting Information.The peptide-copolymer conjugates were characterized by FT-IR(Spectrum One, Perkin Elmer, Massachusetts, USA) to verify thecoupling reaction. The sample designations for the peptide-copolymer conjugates are shown in Table 2. 2.6. Self-Assembly Study of Peptide-CopolymerConjugates in Aqueous Solution All peptide-copolymer conjugates were separately self-assembledin aqueous solution to form the peptide-functionalized nano-particles (P1-NP-P1 and P2-NP-P2). Then, the individual nano-particles bearing complementary peptides (P1-NP-P1 andP2-NP-P2) were combined together where they then assembledinto 1D nanoparticle fibers and eventually formed 3D scaffolds asdirectedbytheioniccomplementaryassemblybetweenP1andP2.The assembly of the nanoparticles into nanoparticle fibers wasaccomplished in three steps as follows. First the P1-copolymerconjugates (20mg) were dissolved in DMSO (2mL) to give a clearprecursorsolution,andthisprecursorsolutionwasslowlyinjected(0.4mL  min  1 ) into weakly basic deionized water (10mL, pH 9.0,adjusted by 1m NaOH solution),using a 31G syringe, while beingstirred at 700rpm by a magnetic stirrer. This resulted in the firstlevelofassemblytogiveP1-NP-P1suspension.Aftercompletingtheinjection of the precursor solution, the magnetic stirring wascontinued for 30min and then sonicated 3min to form a stablepeptide-nanoparticle suspension. Separately, in the second step,the P2-copolymer conjugates were dissolved in DMSO to formanother precursor solution and then injected into weak acidic D.I.H 2 O(10mL,pH5.4,adjustedby1mHCl)alsosonicatedfor3mintogive a P2-NP-P2 suspension.The nanoparticle suspensions are formed in basic (pH 9.0) andacidic (pH 5.4) D.I. H 2 O to better ionize the carboxylic acid sidegroups of P1 and amine side groups of P2 to bear negative andpositivecharges, respectively. Thisensures that the newly formednanoparticlesbearcomplementarypeptidesthatareself-repulsive.However, we can also accomplish assembly at neutral pH or PBS(pH7.4). [26] Thetypicalyieldforthesescaffoldsystemsrangesfrom90 to 95%.Table2showsthenamesoftheindividualpeptide-nanoparticleconjugates(i.e.,beforebeingcombinedtoassembleintoascaffold),andthetimeallottedforself-assemblyofthescaffoldafterthetwonanoparticle suspensions are combined. Qualitative observationdemonstrated that longer time was needed for the larger nano-particles to assemble (the nanoparticles used for S-1 were bothtypically ca. 165–175nm by DLS, while those in S-3 were ca.565–580nm,withthesizeincreasingasthesizeofthehydrophobiccoreincreased).Forexample,thestableP1-NP1-P1suspensionwasinjectedintothestableP2-NP1-P2suspensionwithgentlemagneticstirring at 400rpm for 30min to give a uniformly mixedsuspension. Within 10min after the stirring was reduced from400 to 100rpm, there was visual evidence of assembly with theappearance of a ‘‘sponge-like’’ phase thateventually settled at thebottomofthevial.Theallowedassemblytimewas3h.Theprocesswas same for P1-NP3-P1 assembling with P2-NP3-P2 except 10hwas allowed for the assembly.The nanoparticle scaffolds from smaller peptide-nanoparticles(from P1-NP1-P1 and P2-NP1-P2) and larger peptide-nanoparticles(fromP1-NP3-P1andP2-NP3-P2)wereappliedtosiliconwafersandallowed to dry before observation by FESEM. This was done byapplying an aliquot (100 m L) of the assembled nanoparticles in athinlayerontoacleanedsiliconwafer.Thenanoparticlelayerwasair dried to leave an assembled nanoparticle membrane (des-ignatedasa2Dscaffold)andthenvacuumdriedat50 8 Cfor6h.Thenanoparticle membrane was coated with platinum/palladium(Pt/Pd,  5nm) prior to FESEM characterization. 2.7. Controlled Release Testing Before focusing on the study of the multiple drugs delivery, wecheckedthedrugreleaseprofilesusingsinglemodeldrugloadedinassembled scaffolds with different particle composition and sizeover a 23d period, as shown in previous work and SupportingInformation(TableS1andFigureS3). [26] Here,wefocusedonusingscaffold S-2 to demonstrate multiple drug loading and thecontrolled release behavior.Controlled release studies using scaffolds loaded with multiplemodeldrugswereperformedat37 8 CinPBSbuffer(pH7.4).Bothtwoand three drug-loaded scaffolds were prepared to test the multipledrug loading and simultaneous sustained release from the scaffold.The model drugs used were nitrofurazone (NF) as a slightlyhydrophilic model drug, 4 0 ,5 0 -dibromofluorescein (DBF) as a Table 2.  Peptide-copolymer conjugate nanoparticles with scaffold names and assembly times. Peptide-copolymer conjugates Scaffold Assembled NP name Scaffold assembly time [h] P1-PVP- b -3PMMA- b -PVP-P1 S-1 P1-NP1-P1 3P2-PVP- b -3PMMA- b -PVP-P2 P2-NP1-P2P1-PVP- b -6PMMA- b -PVP-P1 S-2 P1-NP2-P1 6P2-PVP- b -6PMMA- b -PVP-P2 P2-NP2-P2P1-PVP- b -12PMMA- b -PVP-P1 S-3 P1-NP3-P1 10P2-PVP- b -12PMMA- b -PVP-P2 P2-NP3-P2P1-PHEMA- b -6PMMA- b -PHEMA-P1 S-4 P1-NP4-P1 6P2-PHEMA- b -6PMMA- b -PHEMA-P2 P2-NP4-P2 www.mbs-journal.deX. Xiang, X. Ding, T. Moser, Q. Gao, T. Shokuhfar, P. A. Heiden 4 Macromol. Biosci.  2014,  DOI: 10.1002/mabi.201400438 4   2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 4       R Early View Publication; these are NOT the final page numbers, use DOI for citation !!  moderately hydrophobic model drug, and fluorescein (FL) as asomewhat more hydrophobic model drug. General procedures forpreparation of two and three drug nanoparticle scaffolds are givenbelow.The selected peptide-copolymer conjugate is dissolved inDMSO together with the desired drug to give a precursorsolution. For example, for a two-drug scaffold, we prepared NF/P1-PVP-b-6PMMA-b-PVP-P1 (38mg þ 5.0wt.-% NF) in 1.5mLDMSO and DBF/P2-PVP-b-6PMMA-b-PVP-P2 (38mg þ 5.0wt.-%DBF) in 1.5mL DMSO. The NF/P1-copolymer precursor solutionwas then slowly injected into deionized water (10mL) to give anNF-loaded core–shell nanoparticle suspension (NF-loadedP1-NP2-P1). A DBF/P2-copolymer precursor solution was sim-ilarlypreparedandinjectedintodeionizedwater(10mL)togiveaDBF-loaded core–shell nanoparticle suspension (DBF-loadedP2-NP2-P2). The DBF-loaded P2-NP2-P2 suspension was thenslowly injected (23 G syringe) into the NF-loaded P1-NP2-P1suspension with gentle magnetic stirring at 400rpm for 1h togiveauniformmixture,whichwasthenallowedtoself-assembleinto a 3D nanoparticle scaffoldwithmagneticstirringat100rpmand settled to the bottom of the vial.Thenanoparticlescaffoldswerecollectedbycentrifugationat4000rpm for 20min to isolate any NF and DBF that were notcaptured by the nanoparticles or just loosely adsorbed onto thenanoparticle surfaces. They were then washed with fresh D.I. H 2 O(2.0mL each time). The centrifugation and washing process wererepeated two additional times. All supernatant fractions werecombined to collect all uncaptured NF and DBF, so that the actualloading (wt.-%) of NF and DBF in nanoparticles was calculatedbasedonEquation(2).Thewashedscaffoldsolidswerethenusedtoset up the controlled release study.Actual wt :  % ¼ W  intial drug   W  uncaptured drug W  drug  loaded nanoparticle  100 % ð 2 Þ The three drug loading process was similarly accomplished,but in this case, the ratio of P2-NP2-P2 (DBF loaded) to P1-NP2-P1(NFloaded)toP2-NP2-P2(FLloaded)wassetupat1:2:1byweighttoachievetheloadingofthreedrugsintoasinglescaffold.Briefly,DBF/P2-PVP- b -6PMMA- b -PVP-P2 (19mg þ 5.0wt.-% DBF)/DMSO(0.75mL) solution, NF/P1-PVP- b -6PMMA- b -PVP-P1 (38mg þ 5.0wt.-% NF)/DMSO (1.5mL) solution and FL/P2-PVP- b -6PMMA- b -PVP-P2 (19mg þ 5.0wt.-% FL)/DMSO (0.75mL) solution wereprepared and each was separately injected into D.I. H 2 O (5, 10,and 5mL correspondingly) to give the three model drug-loadednanoparticle suspensions. The DBF-loaded P2-NP2-P2 suspensionwas slowly injected into the NF-loaded P1-NP2-P1 suspensionwithgentlemagneticstirringat400rpmfor1h togivea uniformmixture. Due to the ratio of P1-NP2-P1 to P2-NP2-P2 was 2:1, atthis stage the self-assembled nanoparticle fibers will favor‘‘nanoparticle trimers’’ terminated with P1 on both ends. Then,thethirddrug-loadednanoparticlesuspension(FL-loadedP2-NP2-P2 suspension) was slowly injected into the above P1-terminatednanoparticle fiber suspension, restoring‘‘peptide stoichiometry’’,and gentle magnetic stirring was continued to advance theassembly and give the triple drug loaded scaffolding.The triple drug-loaded scaffold solids were isolated andwashed with fresh D.I. H 2 O (2.0mL each time, washing twoadditional times) to collect all free model drugs in supernatant.Againtheactualdrugloading(wt.-%)wascalculatedaccordingtoEquation (2) after the uncaptured drugs were quantified by UVspectrometry.Both the two and three-drug loaded scaffold solids were set upfor controlled release tests over 24h at 37 8 C in PBS media. Thescaffold solids were re-dispersed in PBS (5.0mL) and then trans-ferredinto dialysis tubing, which was then immersedinto 100mLof PBS buffer in a beaker at 37 8 C. The beaker with dialysis tubingwas sealed to keep total mass constant during controlled releasetests.Ifsolventlosswasdetected,D.I.H 2 Owasaddedtokeeptotalmass consistent. At each time interval, a 1.5mL aliquot of PBSsolutionwithreleaseddrugwasremovedfromthebeakerandthesamevolumeoffreshmediawasaddedtokeepthetotalvolumeat100mL. This procedure was repeated until the controlled releasetest was completed.The quantity of each drug that was not captured duringnanoparticlepreparation,andthequantityofreleaseddrugateachtime interval was determined by UV–Vis spectrometry usingcalibrationcurvespreparedfromstandardsolutionsofNF,DBF,andFL. The drug concentration ranged from 0.001 to 0.005mg  mL  1 ,and was measured against the standard curves using Beer’s lawequations (Figure S3) and Equation (3) and (4) (see SupportingInformation). Table 3 gives the loading efficiency and actualloading (wt.-%) of the model drugs in both the two and three drugloaded scaffolds. 2.8. Cytotoxicity Tests Using NIH3T3 Cell Lines Nanoparticle scaffolds were self-assembled from P1-NP(  )-P1 andP2-NP(  )-P2(Table2,NP(  )representsNP1,NP2,NP3,orNP4).Thescaffolds, containing different particle sizes and compositions,weretestedfor biocompatibilitywithNIH3T3fibroblast cells.This Table 3.  Loading efficiency of model drugs in two-drug and three-drug nanoparticle scaffolds. Scaffold Model drug NP pair Theo. loading wt. % Act. loading wt.% Loading eff. % 2 Drug NF P1-NP2-P1 5.0 1.2 23DBF P2-NP2-P2 5.0 4.7 943 Drug DBF P2-NP2-P2 5.0 4.7 94NF P1-NP2-P1 5.0 0.53 11FL P2-NP2-P2 5.0 4.6 93 Peptide-Directed Self-Assembly of Functionalized Polymeric Nanoparticles. Part II: Effects of Nanoparticle Composition on Assembly Behavior and Multiple Drug Loading Abilityw Macromol. Biosci.  2014,  DOI: 10.1002/mabi.201400438  5   2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim  5 5 Early View Publication; these are NOT the final page numbers, use DOI for citation !!  R
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