European Polymer Journal 49 (2013) 499–509

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European Polymer Journal

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Thermosensitive PNIPAM-peptide conjugate – Synthesis andaggregation

0014-3057/$ - see front matter � 2012 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.eurpolymj.2012.11.005

⇑ Corresponding author. Tel.: +48 32 271 60 77x128; fax: +48 32 271 2969.

E-mail address: adworak@cmpw-pan.edu.pl (A. Dworak).

Barbara Trzebicka a, Barbara Robak a, Roza Trzcinska a, Dawid Szweda a, Piotr Suder b,Jerzy Silberring a,b, Andrzej Dworak a,⇑a Centre of Polymer and Carbon Materials, Polish Academy of Sciences, M. Curie-Sklodowskiej 34, 41-819 Zabrze, Polandb AGH University of Science and Technology, Mickiewicza 30, 30-059 Krakow, Poland

a r t i c l e i n f o

Article history:Received 13 August 2012Received in revised form 12 November 2012Accepted 16 November 2012Available online 29 November 2012

Keywords:PNIPAM-peptide conjugateThermosensitivityAggregationMesoglobulesNanomaterials

a b s t r a c t

This paper describes the synthesis of enzymatically cleavable hybrid biomaterial –poly(N-isopropylacrylamide)-pentapeptide conjugate through atom transfer radicalpolymerization of N-isopropylacrylamide from a resin-loaded peptide macroinitiator. Apentapeptide labeled with a dansyl group (Gly-Arg-Lys-Phe-Gly-dansyl) was synthesizedusing solid-phase peptide synthesis (SPPS) with the use of Fmoc protected amino acids.An ATRP peptide-based macroinitiator was obtained by the coupling of 2-bromopropionicacid to the N-terminus of peptide molecule. The ATRP of NIPAM was performed inheterogenic conditions using macroinitiator anchored on polystyrene resin. Thewell-defined PNIPAM-Gly-Arg-Lys-Phe-Gly-dansyl conjugate with an average molar massof 30,400 g/mol and molar mass dispersity of 1.14 was obtained. The PNIPAM-Gly-Arg-Lys-Phe-Gly-dansyl conjugate underwent a phase transition upon heating in aqueoussolution, with the formation of spherical nanoparticles (mesoglobules) with peptide form-ing the particles’ corona. Tryptic hydrolysis of mesoglobules formed by bioconjugateshowed that the presence of Lys or Arg residues in the structure of the conjugate provideda simple route to cleavage peptide fragments from mesoglobules.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction

Investigations of peptide/polymer materials and theirrole in the material and pharmaceutical sciences have sig-nificantly increased. A large number of papers (for review,see [1–4]) in this field are devoted to the methods of thesynthesis of polymer-peptide conjugates and their self-assembly properties driven by interaction between peptidesegments.

Aside from the well-known coupling reaction betweena peptide and functionalized polymer, methods based onpolymerization with peptide macroinitiators or macromo-nomers are commonly used. Peptide building blocks of awell-defined amino acid sequence are mostly synthesized

through solid-phase peptide synthesis (SPPS) accordingto the Fmoc strategy [5,6]. Conjugation reactions can beperformed either in solution [7–11] or from solid resin[12,13], the latter is more efficient and convenient becausethe isolation of the product is easier.

Few reports deal with the synthesis and characteriza-tion of peptide conjugates [14–20] with poly(N-isopropyl-acrylamide) (PNIPAM), which is the most widely studiedthermosensitive polymer [21].

A combination of ATRP and the ring-opening polymeri-zation of N-carboxy anhydride was used to obtain a dualstimuli-responsive PNIPAM-poly(lysine) conjugate [14]able to form complex morphologies, from micelles andwormlike structures to vesicles, depending on the polymercomposition and type of helicogenic solvent.

Stoica et al. [15] synthesized doubly thermoresponsiveconjugates of PNIPAM with the octapeptide Phe-Glu-Phe-Glu-Phe-Lys-Phe-Lys. The terminal amine groups of the

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peptide were transformed into thiol groups andsubsequently used as a chain transfer agent in the radicalpolymerization of NIPAM. However, the high molar massdispersity of the product indicated low level of control overthe polymerization. The conjugates formed gels at a lowtemperature and underwent a phase-transition inducedby the presence of PNIPAM at a higher temperature.

Peptide nanotubes formed by self-assembling of cyclicpeptides were used by Biesalski et al. [16,17] as structur-ally defined templates to prepare nanometer-sized poly-mer-peptide hybrid nanotubes. The surface initiatedATRP of NIPAM caused the coating of the peptide core withcovalently bound thermosensitive polymer shell.

Molawi and Studer [19] applied nitroxide-mediatedradical polymerization (NMP) to conjugate PNIPAM withpeptides sequences consisted of hydrophobic aminoacids.They obtained the thermosensitive hybrid materials withtemperature induced switchable peptide activity. Theactivity of these bioconjugates towards hydrolysis cata-lyzed by chymotrypsin was observed only below theirLCST.

Moller et al. [20] used NMP to synthesize PNIPAM-peptide conjugates containing peptide backbone withdifferent Gly-Lys repeating units and multiple PNIPAM sidechains. Depending on composition the thermosensitivebioconjugates aggregated forming different topologies.

Although, the several papers devoted to PNIPAM-peptide conjugates appeared in literature and revealedthe thermosensitivity of such hybrid structures none ofthem described the aggregation behavior of the block bio-conjugates driven by temperature. Heating of dilute aque-ous solution of thermosensitive polymer above certainthemperature (cloud point temperature - TCP) leads to ther-modynamically metastable system consisting of spherical,colloidally stable and equally sized aggregates called meso-globules formed by the collapsed, dehydratated polymerchains [22]. This phenomenon, induce by simple tempera-ture stimulus, leading to well-defined nanostructure callsfor its investigation in relation to polymer-peptide conju-gates. The detailed study of the formation of mesoglobulesmay comprise a valuable information which can be used toobtain nanocarriers for therapeutic peptides.

In this work, we describe the synthesis of the PNIPAM-Gly-Arg-Lys-Phe-Gly-dansyl block conjugate via ATRPfrom peptide loaded resin and discuss its thermosensitivebehavior. The thermally induced aggregation of the bio-conjugate is used to obtain nanoparticles (mesoglobules)with corona formed by peptide block prone to enzymaticdigestion.

2. Experimental section

2.1. Materials

Dansyl NovaTag™ resin was purchased from Novabio-chem (Merck) and used as received. All protected aminoacids (Fmoc-Gly-OH, P98.0%, Fmoc-Phe-OH, P98%, Fmoc-Lys(Boc)-OH P98.0%, Fmoc-Arg(Pbf)-OH, P98.0%), poly(N-isopropylacrylamide) (PNIPAM, Mn = 30,000 g/mol, GPC),N,N0-diisopropylcarbodiimide (DIC, 99%), 2-bromopropion-

ic acid (99%), copper (I) chloride (CuCl, 99.999%),tris[2-(dimethylamino)ethyl]amine (Me6TREN), triisopro-pylsilane (TIS, 99%), trifluoroacetic acid (TFA, 99%) andpiperidine were purchased from Sigma–Aldrich and usedas received. Ethyl (hydroxyimino)cyanoacetate (OXYMA,97%) was purchased from Alfa Aesar and used as received.N-isopropylacrylamide (NIPAM) was purchased from Sig-ma–Aldrich and purified by crystallization in hexane priorto use. Methanol (MeOH, 99.8%) and dichloromethane(DCM 99.8%) were obtained from POCh and used as re-ceived. N,N-dimethylacrylamide (DMF) was from POChand distilled prior to use. Trypsin (sequencing grade) waspurchased from Promega (USA) and used as received. Dou-bly distilled water was used unless otherwise stated. ForHPLC and mass spectrometry, MilliQ quality water wasused.

2.2. Synthesis of peptide macroinitiator

Standard Fmoc solid phase synthesis [6] of thepentapeptide labeled with a dansyl fluorescent group(Gly-Arg-Lys-Phe-Gly-dansyl) was performed on a DansylNovaTag™ resin via coupling reactions facilitated by DICand OXYMA. Dansyl pre-loaded resin (0.13 g of loadingefficiency at 0.38 mmol/g) with Fmoc-protected aminegroups was suspended in 5 mL of DMF to swell. The Fmocprotecting groups were removed from the resin by piperi-dine solution (20% in DMF). Subsequently, a 3-fold excessof N-protected amino acids, DIC and Oxyma were used ineach coupling step. The absence of amine groups after cou-pling was confirmed by the Kaiser test [23]. Each step wasfollowed by the subsequent washing of the resin withDMF, MeOH, and DCM to remove all traces of impurities.

To obtain an ATRP macroinitiator peptide was modifiedon the solid support by standard coupling reaction with 2-bromopropionic acid in the presence of DIC and OXYMA.The reaction was performed with 5-fold excess of 2-bromopropionic acid and activators for 24 h at ambienttemperature. The absence of amino groups after the reac-tion was confirmed by the Kaiser test [23]. The resin wasthen rinsed with DMF.

A small amount of the peptide macroinitiator wascleaved from the resin for analysis. This reaction wasaccomplished by treating the resin for 3 h with a cleavagemixture (95% TFA, 2.5% TIS and 2.5% H2O, v/v/v). The pep-tide was isolated by precipitation in diethyl ether, followedby centrifugation.

2.3. Synthesis of poly(N-isopropylacrylamide)-peptideconjugate

The PNIPAM-Gly-Arg-Lys-Phe-Gly-dansyl conjugatewas obtained via ATRP polymerization performed on thesolid resin pre-loaded with a peptide macroinitiator.Monomer (NIPAM, 8.65 mmol) and ligand (Me6TREN,0.048 mmol) dissolved in 8 mL of DMF/water solution(3:1, v/v) were placed in a round bottom reactor equippedwith a stirrer and an argon/vacuum inlet valve. The solutionwas deoxygenated and the catalyst (CuCl, 0.048 mmol) wasintroduced to the system under an argon atmosphere. Themonomer:initiator:CuCl:ME6TREN molar ratio was

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180:1:1:1. The resin was placed in the reactor when theseoperations were completed. The polymerization lasted for21 h and was accomplished by purging the system withair. Next, the system was rinsed with DMF. Cleavage ofthe PNIPAM-peptide conjugate from the resin with simul-taneous deprotection of Lys and Arg pendant groups (Bocand Pbf, respectively) was carried out in the presence of amixture 95% TFA, 2.5% TIS and 2.5% H2O (v/v/v) for 3 h.The crude product cleaved from the resin was isolated byprecipitation in diethyl ether, followed by centrifugationand purification by dialysis against water for 7 daysthrough a membrane (cut-off of 5000–8000 Da).

2.4. Tryptic cleavage of PNIPAM-peptide conjugate

Tryptic digestion of the PNIPAM-peptide conjugate wasstudied in water (pH = 6.0) at 45 �C, above phase transitionof the bioconjugate, for concentration 0.1 g/L. Bioconjugatesolution (2 mL) was abruptly heated at 70 �C, thermostatedfor one hour, and then thermostated at 45 �C for one hourbefore enzyme solution (100 lL) was added. The enzyme-to-peptide molar ratio was 1:100. The resulting solutionwas incubated overnight.

The same procedure of enzymatic hydrolysis of the bio-conjugate was also performed at 25 �C, below its TCP, inpure water (pH = 6.0).

The samples containing the enzymatic reaction prod-ucts were separated from the polymeric residues by centri-fugation at 50 �C and were analyzed by ESI-MS and HPLC.

2.5. Characterization

2.5.1. ESI Mass spectrometryThe mass spectrum of the Gly-Arg-Lys-Phe-Gly-dansyl

and conjugate digestion products were run on an Esquire3000 quadrupole ion-trap mass spectrometer (Bruker-Dal-tonics, Bremen, Germany). The instrument was operated ina positive-ion mode. The flow-rate was maintained at 3 lL/min, and the mobile phase consisted of 30% methanol inwater, supplemented with 0.1% formic acid. The acquiredspectra were analyzed using Bruker Data Analysis software(ver. 3.0).

2.5.2. Nuclear magnetic resonance (NMR)1H NMR spectrum of the conjugate was recorded in D2O

on a Bruker Ultrashield spectrometer operating at600 MHz.

2.5.3. Reverse phase high pressure liquid chromatography(RP-HPLC)

RP-HPLC was applied to determine the macroinitiatorpurity and to analyse the enzymatic digestion products.The Agilent system (1260 Infinity) was equipped with anEclipse XDB-C18 column (84.6 � 150 mm, Agilent) and aUV–VIS diode array detector (Agilent, 1260 DAD VL). Tomeasure the macroinitiator’s purity, a linear gradient from10% to 95% B over 45 min was applied (A: 0.1% TFA inwater; B: 0.1% TFA in acetonitrile). The products of enzy-matic hydrolysis were measured using a linear gradientfrom 10% to 95% B over 25 min. The flow rate was main-tained at 0.5 mL/min. The analyzed peptides were moni-

tored at 220 nm. The chromatograms were recordedusing the ChemStation software (Agilent).

2.5.4. Gel permeation chromatographyGPC measurement of the PNIPAM-peptide conjugate

was performed at 45 �C in DMF with a nominal flow rateof 1 mL/min. A multiangle light scattering detector (DAWNEOS, Wyatt Technology, k = 658 nm), refractive indexdetector (Dn-2010 RI, WGE Dr. Bures) and column system(PSS, 100 Å, and two PL gel MIXED-C) were used. The re-sults were evaluated using ASTRA 5.3.4.10 software fromWyatt Technologies and WINGPC 6.0 software from PSS.The refractive index increment (dn/dc) of PNIPAM inDMF equal to 0.075 mL/g determined in separated experi-ment was used for calculations.

2.5.5. Fluorescence spectroscopyFluorescence spectrum of the PNIPAM-peptide conju-

gate was recorded on fluorescence Hitachi F-2500 spectro-photometer. The dansyl group was used as a fluorescentprobe. The measurements were carried out at room tem-perature with excitation at 340 nm. The excitation andemission band widths were both 5 nm.

2.5.6. Cloud point measurementsThe cloud point temperatures (TCP) of the conjugate and

respective PNIPAM homopolymer were determined on aJasco V-530 UV–VIS spectrophotometer with a cuvettethermostated by a Medson MTC-P1 Peltier thermocontrol-ler. The transmittance of bioconjugate and homopolymersolutions of 1 g/L concentration were monitored atk = 500 nm as a function of temperature. Solutions wereheated step-wise. Once the desired temperature wasreached, the samples were equilibrated for 5 min beforemeasurement and then heated to the next temperatureat a heating rate of 0.2 �C/min.

2.5.7. Light scatteringDynamic and static light scattering measurements were

performed on a Brookhaven BI-200 goniometer with verti-cally polarized incident light of wavelength k = 632.8 nmsupplied by a He-Ne laser operating at 35 mW andequipped with a Brookhaven BI-9000 AT digital autocorre-lator. The samples, water solutions of the PNIPAM-peptideconjugate, were stabilized at 70 �C for 1 h before datacollection.

The scattering from aqueous solution of PNIPAM-pep-tide conjugate (c = 0.025–0.1 g/L) was measured at anglesh ranging from 40� to 140� at 70 �C. Because the mesoglo-bules sizes depend on concentration of the polymer in thesolution below the phase transition, the solutions were ob-tained by the dilution of the initial 0.1 g/L solution of 70 �Cwith water at the same temperature what ensured that thesize of particles was preserved. The autocorrelation func-tions were analyzed using the constrained regularizedalgorithm CONTIN to obtain distributions of relaxationtimes (s). The relaxation rates, C (=s�1), give the distribu-tions of the apparent diffusion coefficients (D = C/q2). Here,q is the magnitude of the scattering vector given byq = (4pn sin(h/2))/k, and n is the refractive index of themedium. The apparent diffusion coefficients were plotted

Fig. 2. ESI-MS spectrum of Br-Gly-Arg-Lys-Phe-Gly-dansyl macroini-tiator.

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against concentrations and after extrapolation to infinitedilution the D0 was calculated. The hydrodynamic radiusis obtained by the Stokes–Einstein equation:

Rh ¼ kT=ð6pgD0Þ ð1Þ

where k is the Boltzmann constant, g is the solvent viscos-ity at temperature T in Kelvin and D0 is the diffusion coef-ficient at infinite dilution.

The concentration induced changes of particles appar-ent hydrodynamic radii (R90

h ¼ kT=ð6pgDÞ) in aqueoussolutions at 70 �C were assessed by performing measure-ments at a single angle h = 90�.

The values of the radii of gyration, Rg, were obtainedfrom the partial Zimm plot, which can be described bythe following equation:

1=Iex ¼ Cð1þ ðR2g q2Þ=3Þ ð2Þ

where Iex is the excess of the scattered light.

3. Results and discussion

3.1. Synthesis and characterization of peptide macroinitiator

The Gly-Arg(Pfb)-Lys(Boc)-Phe-Gly-dansyl was synthe-sized according to standard solid phase protocol and Fmocchemistry [6] in the presence of OXYMA and DIC as cou-pling agents [24]. The removal of the Fmoc protectinggroup was accomplished by piperidine treatment. To ob-tain the macroinitiator for ATRP of NIPAM, the terminalamine group of glycine located at the N-terminus of thepeptide sequence was reacted with 2-bromopropionicacid. The schematic presentation of macroinitiator synthe-sis is shown in Fig. 1.

The unblocked Br-Gly-Arg-Lys-Phe-Gly-dansyl peptidemacroinitiator separated from solid support was analyzedby ESI-MS, 1H NMR and RP-HPLC. The macroinitiator puritycalculated based on chromatogram peaks integration was97.5%.

In the ESI-MS spectrum of the macroinitiator (Fig. 2) thesignal at m/z = 973.4 corresponds to pseudomolecular ion[M + H]+, which is in agreement with the calculated mono-isotopic mass value of the molecule (972.4 g/mol). Thepeak at m/z of 487.2 corresponds to a doubly protonatedpseudomolecular ion [M + 2H]2+. The presence of brominegroups in the macroinitiator was confirmed by the isotopicprofile of observed in the ESI-MS spectrum both the singly

Fig. 1. Schematic representation of the solid phase synthesis of

(a) and doubly (b) charged signals. The ESI-MS analysisconfirmed proper synthesis of the macroinitiator.

3.2. Solid phase synthesis and characterization of PNIPAM-peptide conjugate

Solid-phase supported atom transfer radical polymeri-zation of different monomers from peptide-loaded resinleading to well-defined bioconjugates was described byWooley [12]. The advantage of using an insoluble supportfor metal-mediated polymerization is that it aids the sepa-ration of product from catalyst residues by a simplesolvent washing procedure. To succeed in polymerizationthe reaction conditions apart from selection of proper cat-alytic system should induce resin swelling, thus makingthe initiating groups accessible to the monomer [25]. Atomtransfer radical polymerization of NIPAM under differentconditions has been reported in the literature [26–28].Masci and co-workers [28] showed that controlled ATRPof NIPAM could be performed through the use of CuCl/Me6-

TREN as a catalyst in water/DMF mixture.In our work, the polymerization of NIPAM was per-

formed similarly as described by Masci [28] using CuCl/Me6TREN as a catalyst in 3:1 (v/v) mixture of DMF/water.The heterogenic ATRP was initiated by, linked to the solidresin, pentapeptide macroinitiator containing 2-bromopro-pionate group. The synthesis of the PNIPAM-peptide conju-gate is schematically shown in Fig. 3.

Br-Gly-Arg(Pbf)-Lys(Boc)-Phe-Gly-dansyl macroinitiator.

Fig. 3. Schematic representation of the solid phase synthesis of PNIPAM-Gly-Arg-Lys-Phe-Gly-dansyl conjugate.

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The GPC-MALLS chromatogram of the bioconjugate sep-arated from the solid resin by an action of cleavage mixtureshowed a monomodal molar mass distribution (Fig. 4). Thenumber average molar mass of the PNIPAM-peptide conju-gate was 30,400 g/mol, meaning 260 of NIPAM units in the

conjugate. The molar mass dispersity Mw/Mn was 1.14. Theobtained molar mass was higher than the theoretical valuecalculated with the assumption of a 100% monomerconversion (Mth = 21,000 g/mol). The higher degree ofpolymerization of the conjugate could be due to the partial

Fig. 4. Chromatogram of PNIPAM-peptide conjugate (RI trace).

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initiator deactivation, which is observed for amide-basedreagents [29] and/or due to the hindered access to the ini-tiating sites immobilized on resin surface [25].

Fig. 5. 1H NMR spectrum of PNIPAM-pe

The 1H NMR analysis of the bioconjugate confirmed itsexpected structure (Fig. 5). Strong signals at d = 1.18 ppm,d = 1.62 ppm, d = 2.05 ppm and d = 3.94 ppm are assignedto protons of PNIPAM. Characteristic signals in the aro-matic region of the spectrum d = 6.7–8.7 ppm come fromthe aromatic protons of phenylalanine and dansyl frompeptide moiety. The signal at d = 3.3 can be attributed todansyl –CH3 groups (Fig. 5, signal p). Small signals(d = 4.42, 4.24, 4.04, 3.70, 3.56, 3.48, 3.22, 2.94, 2.28, 1.85,1.25 ppm) come from protons in peptide backbone andits side groups. A much smaller quantity of protons origi-nating from amino acids present in the bioconjugate struc-ture in comparison to those coming from PNIPAM causesthat they are barely visible in the 1H NMR spectrum.

The presence of a dansyl group in the conjugate struc-ture was also confirmed by its emission spectrum at anexcitation wavelength of 340 nm (Fig. 6). When analogousmeasurements were performed for the PNIPAM homopol-ymer, no emission signals were observed.

ptide conjugate (600 MHz, D2O).

Fig. 6. Emission spectrum of PNIPAM-peptide conjugate (H2O, wave-length of excitation 340 nm).

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The GPC, NMR and fluorescence analysis confirmed theability of the peptide modified with bromopropionic acidto initiate the polymerization of NIPAM.

3.3. Thermosensitivity and aggregation of PNIPAM-peptideconjugate

The phase transition temperature of obtained PNIPAM-peptide conjugate was determined by UV–VIS spectros-copy. Transmittance changes of water solutions of the PNI-PAM-peptide conjugate and PNIPAM homopolymer with asimilar molar mass of 30,000 g/mol are compared in Fig. 7.The temperature behavior of the conjugate differed fromthat of pure PNIPAM. The bioconjugate exhibited sharpphase transition and higher cloud point temperature thanthat observed for the homopolymer. No hysteresis was ob-served when the bioconjugate solution was cooled from 80to 25 �C.

It is known that the phase temperature of thermosensi-tive polymers is changed by the introduction of hydropho-

Fig. 7. Transmittance versus temperature for PNIPAM and PNIPAM-peptide conjugate with similar molar mass (1 g/L water solutions).

bic or hydrophilic moieties into their structures [30]. Aswas mentioned in the introduction the conjugation ofPNIPAM with peptide sequences consisting of hydrophobicaminoacids (Phe-Phe-Gly, Ala-Ala-Phe) decreased thephase transition temperature of bioconjugates in compari-son with PNIPAM homopolymer [19]. The decrease of tran-sition temperature was observed also for comb-likeconjugates of Gly-Lys backbone connected with PNIPAMside chains through benzoic acid derivative [20]. Here,the presence of the hydrophilic pentapeptide at the endof the PNIPAM chain shifted the cloud point temperatureof the conjugate to higher values by about 3 �C.

In dilute aqueous solution above the phase transitiontemperature thermoresponsive polymers can aggregateto nanoparticles of controlled sizes called mesoglobules[22]. The sizes of mesoglobules strongly depend on differ-ent factors such as solution concentration, heating rate andmolar mass [31]. To decrease the size of mesoglobulesabrupt heating of an aqueous solution of thermoresponsivepolymers was applied [32].

Dynamic light scattering measurements of 0.1 g/L PNI-PAM-peptide solution (Fig. 8A) showed that only one pop-ulation of mesoglobules was formed during the abruptheating of the bioconjugate solution. The size of bioconju-gates‘ mesoglobules was similar to the average size of mes-oglobules formed under the same conditions by PNIPAMhomopolymer. The phenomenon was completely revers-ible; the mesoglobules disaggregated upon cooling thesolution to room temperature.

The initial solution concentration strongly affected thedimensions of mesoglobules formed under abrupt heating.Fig. 8B shows the variation in the particles’ hydrodynamicradii R90

h in abruptly heated solutions as a function of thebioconjugate solution concentration (0.1 to 1.0 g/L). The in-crease of solution concentration led to formation of largermesoglobules.

To obtain real hydrodynamic radius Rh of bioconjugates‘mesoglobules values of the C function were measured at70 �C for scattering angles between 40� and 140� for concen-trations ranging from 0.025 to 0.1 g/L (Fig. 9A). To eliminatethe influence of the solution concentration on the mesoglo-bules sizes (Fig. 8B), for C measurements the solutions wereobtained by the dilution of the initial 0.1 g/L at 70 �C withwater at the same temperature. For each solution the diffu-sion coefficient (D) was calculated and used to plot the con-centration dependence (Fig. 9B). After extrapolation toinfinite concentration the D0 = 1.09 � 10�11 m2/s wasestimated and Rh = 57 nm was calculated by the Stokes–Einstein equation.

The radius of gyration (Rg) of the obtained mesoglobulesas determined by static light scattering using partial Zimmplot (Fig. 10) was 54 nm. The ratio of shape factor Rg/Rh

was 0.95, which indicated that the particles assumed thestructure of a loose sphere [33].

3.4. Enzymatic hydrolysis of the bioconjugate

It is commonly known that the C-terminal side ofarginine or lysine within the peptide chain provides ahydrolysable peptide bond in presence of trypsin [34].The sequence of amino acids of pentapeptide conjugated

Fig. 8. (A) Size distribution of mesoglobules (DLS) (abrupt heating, c = 0.1 g/L, 70 �C, sample volume 2 mL), (B) mesoglobules hydrodynamic radius as afunction of temperature for aqueous PNIPAM-peptide solutions at various concentrations (abrupt heating, 70 �C, sample volume 2 mL).

Fig. 9. PNIPAM-peptide mesoglobules upon abrupt heating: (A) the C curve at 70 �C (0.1 g/L), (B) the diffusion coefficient D as a function of the bioconjugateconcentration at 70 �C (c = 0.1–0.025 g/L, sample volume = 2 mL).

Fig. 10. Partial Zimmplot of the bioconjugate aqueous solution. The linethrough the data points represents the linear fit (abrupt heating, c = 0.1 g/L, 70 �C, sample volume = 2 mL).

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with PNIPAM contains both arginine and lysine, which en-sures a simple route for the cleavage and release of thepeptide fragments from the polymeric anchor under tryp-sin treatment.

The tryptic digestion of peptide-PNIPAM conjugate wasperformed below its TCP and above when bioconjugate wasaggregated to mesoglobules.

For enzymatic digestion the bioconjugate mesoglobuleswere obtained by abrupt heating to 70 �C of 0.1 g/L watersolution similarly as it was performed for the LS measure-ments and stabilized for one hour at 45 �C. Enzyme wasintroduced to prepared dispersion of mesoglobules. Thescheme of the enzymatic hydrolysis of bioconjugate isshown in Fig. 11.

The presence of digestion products (1 and 2, Fig. 11)was confirmed by ESI-MS analysis of the supernatant solu-tions from the incubation medium as shown in Fig. 12.

The single charged signals [M + H]+ in the ESI-MSspectrum originate from products of the conjugate

Fig. 11. Enzymatic digestion of PNIPAM-peptide conjugate.

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hydrolysis at the C-side arginine (Fig. 11, group 1, m/z = 626.7) and lysine, (Fig. 11, group 2, m/z = 498.6). Twoadditional signals in the ESI-MS spectrum with m/z = 351.5 and 294.5 derive from spontaneous fragmenta-tion of ions m/z = 626.7 and 498.6 in ion source. Eventhought that ESI-MS is a soft ionization technique theapplied ionization energy was high enough to cleavageC–N bonds in peptide chain, what was confirmed byMS/MS and HPLC analysis (data not shown).

To be accessible for enzyme, peptide parts of aggregatedbioconjugate have to be placed on the outer layer of the

mesoglobules as described previously for P(DEGMA-ME)bioconiugate [35]. DLS measurements showed that themesoglobules were stable in presence of enzyme andhydrolysis occurred in heterogenic conditions withoutchanging the mesoglobules sizes.

Tryptic digestion of the peptide-PNIPAM conjugate wasalso performed below its TCP, when dissolved bioconjugatechains were present in solution. The obtained results weresimilar to those of mesoglobules digestion.

For PNIPAM conjugate bearing a hydrophobic peptidesequence Gly-Phe-Phe Studer and Molawi observed a

Fig. 12. ESI-MS spectrum of products of the bioconjugate trypsin hydrolysis.

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different behavior: the peptide block was cleaved only be-low the transition temperature and left unchanged, whendigestion of aggregated polymer was attempted [19]. Thisindicated that the hydrophobic Gly-Phe-Phe was likely tobe hidden in the interior of mesoglobule [19], while thehydrophilic Gly-Arg-Lys-Phe-Gly-dansyl sequence westudied formed the particles’ corona accessible to the ac-tion of enzymes.

4. Summary

Herein, a procedure for the preparation of the syntheticconjugate PNIPAM-Gly-Arg-Lys-Phe-Gly-dansyl was pre-sented. Due to the hydrophilic character of the pentapep-tide linked to the polymer chain, the bioconjugateexhibited a higher phase transition temperature than thecorresponding homopolymer. DLS measurements showedthat the bioconjugate chains were capable of formingmesoglobules of small sizes by abrupt heating of the bio-conjugate solution. For the studied PNIPAM-Gly-Arg-Lys-Phe-Gly-dansyl conjugate the cleavage of peptide partby trypsine-catalyzed hydrolysis occured when bioconju-gate was dissolved below its TCP and when was aggregatedto mesoglobules. It indicated that peptides formed the out-er layer of mesoglobule what made them fully available forenzyme. The introduction of arginine or lysine into the bio-conjugate structure ensured the possibility of cleaving thepeptide segment from the polymeric anchor, which couldbe useful for peptide release.

Acknowledgements

The authors acknowledge the Ministry of Science andHigher Education Grants NN 209 144136 for financial sup-port. B. Robak, R. Trzcinska and D. Szweda gratefullyacknowledge the European Social Fund within the RFSD 2project for financial support.

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