Hydroxyl end-capped macromers of N-vinyl-2-pyrrolidinone as precursors of amphiphilic block copolymers

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EUROPEAN POLYMER JOURNAL

European Polymer Journal 43 (2007) 4628–4638

www.elsevier.com/locate/europolj

Hydroxyl end-capped macromers of N-vinyl-2-pyrrolidinone as precursors of amphiphilic block copolymers Irene Bartolozzi a, Roberto Solaro a, Etienne Schacht b, Emo Chiellini

a,*

a

Laboratory of Bioactive Polymeric Materials for Biomedical and Environmental Applications, INSTM-UdR, Department of Chemistry and Industrial Chemistry, University of Pisa, via Risorgimento 35, 56126 Pisa, Italy b Department of Organic Chemistry, Polymer Chemistry and Biomaterials Research Group, University of Gent, Krijgslaan, 281-S4, 9000 Gent, Belgium Received 9 July 2007; received in revised form 2 August 2007; accepted 3 August 2007 Available online 30 August 2007

Abstract Telechelic N-vinyl-2-pyrrolidinone (NVP) oligomers terminated by hydroxyl groups were prepared by radical polymerization in the presence of functional chain transfer agents. Then hydroxy-terminated poly(NVP) was used as initiator in the ring opening polymerization of e-caprolactone (e-CL). Experiments were performed either under basic conditions or by using SnOct2 or ZnEt2 as catalyst. The resulting amphiphilic AB-type block copolymers were thoroughly characterized by spectroscopic and thermal techniques. These data and fractionation in protic solvents indicated that the copolymerization products are constituted by a mixture of copolymers with a wide composition range. The water-soluble copolymer fractions formed micelles and nanoaggregates that showed an appreciable capacity of loading piroxicam, a hydrophobic non-steroidal anti-inflammatory drug. Contact angle measurements, atomic force microscopy, and surface plasmon resonance measurements indicated that the surface of films prepared from the insoluble fractions does not have antiopsonizing properties in spite of their high hydrophilicity.  2007 Published by Elsevier Ltd. Keywords: Functionalized PVP oligomers; Ring opening polymerization; Amphiphilic block copolymers; Micelles

1. Introduction Amphiphilic block copolymers are constituted by alternated hydrophilic and hydrophobic segments and are known to self-assemble in aqueous environment into polymeric micelles with mesoscopic size * Corresponding author. Tel.: +39 050 2210 301; fax: +39 050 28436. E-mail addresses: [email protected] (I. Bartolozzi), [email protected] (R. Solaro), [email protected] (E. Schacht), [email protected] (E. Chiellini).

0014-3057/$ - see front matter  2007 Published by Elsevier Ltd. doi:10.1016/j.eurpolymj.2007.08.011

and fairly narrow size dispersity [1]. Polymeric micelles are characterized by a unique core-shell architecture, where the hydrophobic segments are segregated from the aqueous environment to form an inner core surrounded by a fence of hydrophilic segments. The core segregation from aqueous environment is the driving force for micellization and it is due to a combination of hydrophobic and electrostatic interactions, including hydrogen bonding. Polymeric micelles prepared from amphiphilic block copolymers are very appealing as drug carriers because of their capacity to solubilize poorly

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water-soluble drugs in the inner hydrophobic core and to transport them in the biological environment, protecting them from inactivation and preventing their sudden release within body compartments. The small size of micelles is optimal to avoid physical clearance by filtration in the lungs and in the spleen and excretion through the kidneys, thus making them suitable as long-circulating drug carriers [2]. The absolute majority of long-circulating polymeric drug carriers uses poly(ethylene glycol) (PEG) as hydrophilic and sterically protecting polymer for its well known characteristics of hydrophilicity, flexibility, low toxicity, immunogenicity, and antigenicity [3]. However, polymers with similar characteristics have been used as alternative materials, such as poly(N-vinyl-2-pyrrolidinone) (PVP), polyacrylamide, and poly(vinyl alcohol) [4]. The replacement of PEG by PVP as hydrophilic segment in drug delivery systems has drawn attention since Torchilin proposed PVP as steric protector for liposomes [5]. Indeed, the unique feature of PVP has been attractive in the chemical, pharmaceutical, and material fields because of the combination of properties including the solubility in water and organic solvents, very low toxicity, good biocompatibility and high complexation ability [6–10]. PVP has the capacity to interact with a wide variety of hydrophilic and hydrophobic pharmaceutical agents and to enhance the solubility of several poorly water-soluble molecules by hydrophobic interactions. The cryoprotective properties of PVP prevent the aggregation of nanoparticles after freeze-drying, which can instead occur with PEG. This unique behaviour was already exploited by using PVP as a biocompatible cryoprotectant for a wide variety of cells and a lyoprotectant for proteins [11]. PCL is extensively used as hydrophobic segment in amphiphilic block copolymers, since it is a highly hydrophobic and crystalline polyester, and a suitable component of micelle inner core that favours the loading of hydrophobic drugs. End-functionalized N-vinyl-2-pyrrolidinone (NVP) oligomers can be used as hydrophilic macroinitiators in the ring opening polymerization of cyclic monomers such as lactones or lactides in order to prepare block copolymers with well-characterized segment lengths and narrow polydispersity [6,12]. The degradation rate and consequently the drug release rate can be tuned according to the composition of the block copolymer and its final hydrophilic/hydrophobic ratio.

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However, the synthesis of end-functionalized PVP oligomers with controlled molecular weight and polydispersity has been very challenging so far. In fact, NVP cannot easily undergo living/controlled polymerization, as it does not form stabilized radicals. In the past, ingenuous alternative routes were explored to overcome this issue [13]. There are some very recent reports referring to the living radical polymerization of NVP. Gnanou et al. [14] and Okamoto et al. [15] demonstrated the reversible addition-fragmentation chain transfer (RAFT) polymerization of NVP with xanthates as chaintransfer agents. Hadjichristidis and his group reported both nitroxide-mediated radical polymerization (NMRP) and RAFT polymerization techniques for the controllable living polymerization of NVP [16]. In order to prepare PVP block copolymers, they combined anionic polymerization and NMRP [17]. These systems produced PVP with relatively broad molecular weight distributions. Noticeably, Yamago et al. [18] recently reported highly controlled living radical polymerization of NVP. Based on organostibine-mediated living radical polymerization, PVP with expected numberaverage molecular weight was prepared. ATRP has been applied to a wide variety of functional monomers. In contrast, the controllable polymerization of NVP has not yet been obtained. Matyjaszewski [19] reported the ATRP of NVP using Me4Cyclam as a ligand, but it is difficult to introduce end-functionalities by this method. Only very recently Meng et al. [20] disclosed the controlled synthesis of telechelic PVP by atom transfer radical polymerization. On the other hand, the synthesis of telechelic oligomers can be accomplished by exploiting the action of functionalized chain transfer agents in radical polymerization processes [21-23]. In particular, Ranucci et al. [24,25] prepared hydroxy-terminated PVP oligomers by radical polymerization of NVP in the presence of 2-isopropoxyethanol. More recently, Luo et al. [6] reported that molecular weight, polydispersity, and extent of functionalization of PVP prepared by radical polymerization can be controlled by using isopropyl alcohol and 2-mercaptoethanol as chain transfer agents. In the framework of our continuing interest in polymeric materials for biomedical applications, [26–35] we report here the synthesis of telechelic PVP oligomers carrying hydroxyl end-groups by free radical polymerization in presence of suitable chain transfer agents. The resulting PVP oligomers

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were then used for the preparation of amphiphilic PVP/PCL block copolymers by ring opening polymerization. Chemical composition, molecular weight, spectroscopic characteristics, thermal and surface properties of the polymeric products, as well as self-organization in aqueous solution and drug loading properties were investigated. 2. Experimental methods All commercial products were purchased from Aldrich and used as received, unless otherwise stated. Solvents were distilled before use. AIBN was recrystallized from acetone. N-Vinyl-2-pyrrolidinone (NVP) and e-caprolactone (e-CL) were distilled under reduced pressure. 2.1. Synthesis of mono hydroxy-terminated NVP oligomers (PVPOH) NVP (15.3 g, 0.14 mol), AIBN (153 mg, 0.9 mmol) and 590 mL of 2-isopropoxyethanol (IPE) were submitted to three freeze-pump-thaw cycles and kept under stirring at 70 C for 24 h under dry nitrogen atmosphere. The mixture was partially evaporated and then poured into excess diethyl ether. The coagulated material was extracted with diethyl ether in a Soxhlet and the solid residue was dried under vacuum (97% yield) and stored over P2O5 under anhydrous atmosphere. FT-IR (cast film): m ¼ 3500 (m O–H), 2957 (m CH), 1673 (m C@O lactame ring), 1423 (d CH), 1288 (m C–N), and 750 cm1 (q CH2). 1 H NMR (CDCl3): d = 3.7 (CHCH2), 3.2 (CHCH2), 2.4 (CH2C@O), 2.0 (CH2CH2C@O), 1.7 (CH2CH) and 1.1 ppm (CCH3). 2.2. Ring opening polymerization of e-caprolactone initiated by PVPOH Ring opening polymerization of e-caprolactone initiated by hydroxy-terminated PVP was carried out according to standard procedures. Typical experiments are presented by following. 2.2.1. Run PK1 PVPOH (1.16 g, 0.31 mmol) was dried by azeotropic distillation of toluene and then dissolved in 10 mL THF at 80 C. After cooling, the resulting solution was added to a suspension of KH (55 mg, 0.4 mmol) in 5 mL THF at 0 C. The mixture was kept under stirring for 1 h at 0 C and for 4 h at

room temperature, then a solution of e-CL (1.0 g, 9 mmol) in 5 mL THF was added. The reaction was stopped after 24 h by addition of acetic acid. The solution was poured into excess diethyl ether and the coagulated polymer was dried under vacuum to give 1.36 g (63% yield) of white powder product. FT-IR (cast film): m ¼ 2945 (m C–H), 1722 (m C@O ester), 1664 (m C@O lactame ring), 1421– 1367 (d CH), 1290 (m C–N), 1242 and 1188 cm1. 1 H NMR (CDCl3): d = 4.05 (t, OCH2CH2, PCL), 3.72 (s, CHCH2, PVP), 3.64 (t, CH2CH2OH, PCL), 3.24 (CH2CH2N, PVP), 2.30 (CH2CH2C@O, PCL), 2.05 (CH2CH2CH2, PVP), 1.65 (OCH2CH2, O@CCH2CH2, PCL), 1.38 (CH2CH2CH2, PCL), and 1.14 ppm (CCH3). 2.2.2. Run PS3 PVPOH (0.5 g, 0.13 mmol) was dissolved in e-CL (0.5 g, 4.5 mmol) under dry nitrogen atmosphere and then 1%-wt of SnOct2 were added. After three freeze-pumping-thaw cycles, the vial was sealed under vacuum and heated at 140 C for 24 h. The pale yellow, highly viscous product was dissolved in dichloromethane and the resulting solution was poured into excess diethyl ether. The coagulated polymer was dried under vacuum to give 0.95 g (95% yield) of white powder product. 2.2.3. Run PZ3 A solution of PVPOH (1.0 g, 0.24 mmol), e-CL (1.0 g, 9 mmol), and 242 lL (242 lmol) of ZnEt2 (1 M n-hexane solution) in 6 mL of CH2Cl2 was stirred at room temperature for 24 h under dry nitrogen atmosphere. The reaction was quenched with acetic acid and the solution was poured into excess diethyl ether. The coagulated polymer was dried under vacuum to give 1.76 g (88% yield) of white powder product. 2.3. Methods 2.3.1. Copolymer fractionation Polymers were fractionated by stirring a weighed amount in the selected solvent for 8 h. The suspension was filtered through a sintered glass filter and both the solid residue and the filtrate were dried under vacuum to constant weight and characterized. 2.3.2. Micelle preparation Micelles were prepared and loaded with piroxicam by dissolving a known amount of polymer

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and piroxicam (20% by weight) in dichloromethane. The two solutions were mixed under stirring for 10 min and then the solvent was evaporated by flushing argon. Deionized water was added and the opalescent yellowish solution was dialyzed (cut-off 1000 Da) against water for 48 h. The dialyzed solution was lyophilized. Dichloromethane solutions containing about 0.025 mg/mL of piroxicam-loaded micelles were analyzed by UV spectroscopy at 326 nm, by using a calibration curve obtained with 0.01–0.09 mM piroxicam standard solutions. 2.4. Characterizations

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on the polymer surface and the contact angle was determined by using the imaging software provided by the supplier (SCA 20, version 2.1.5 build 16). At least six measurements were made on each sample. Atomic force microscopy (AFM) was performed with a Digital Instrument Nanoscope III. Surface plasmon resonance (SPR) measurements were performed using a Biacore-X instrument. The experiments were carried out in phosphate buffer (10 mM, pH 7.4) at a flow rate of 20 lL/min on polymer films spin coated on gold. Protein solutions in the concentration range of 0.1–0.5 mg/mL were flown over the surfaces. After each measurement, the surface was washed with pure phosphate buffer.

1

H NMR spectra were recorded on 10% (w/v) solutions in CDCl3 or in D2O at 25 C, by using a Bruker Avance-500 spectrometer (500 MHz). FT-IR spectra were recorded on cast films by using a Perkin–Elmer Spectrum One spectrometer. UV–Vis measurements were carried out at 25 C in 10 mm quartz cells with a UVIKON XL (Biotek instruments) spectrophotometer. Thermal gravimetric analysis (TGA) measurements were carried out on 10-12 mg samples in the 30–700 C range under nitrogen atmosphere at 10 C/min heating rate, using a Mettler TG 50 thermobalance. Differential scanning calorimetry (DSC) analyses were performed under nitrogen atmosphere using a Mettler DSC 30. Glass transition temperatures were measured at the inflection point of the thermograms relevant to the second heating cycle. Size exclusion chromatography (SEC) analyses were performed in chloroform by using a Jasco PU-1580 HPLC pump equipped with two 300 · 7.5 PL Mixed-D columns, Jasco 830-RI refractive index detector and Perkin–Elmer LC-75 spectrophotometric detector. Monodispersed polystyrene samples were used as calibration standards. Dynamic light scattering (DLS) measurements were carried out with a Malvern 4800 instrument equipped with an air-cooled Ar laser operating at 488 nm and an Avalanche photodiode detector. All measurements were performed at 90 angle. The measured values were averaged over three sets of six measurements. Static contact angle measurements were performed on polymer films obtained by spin coating of 2% chloroform solutions on glass slides by using an OCA 20 from Dataphysics. For each measurement, a 3 lL droplet of Milli-Q water was placed

3. Results and discussion 3.1. Synthesis of telechelic PVP oligomers PVP oligomers have found application in the biomedical field as sterically protective molecules as well as hydrophilic segments in amphiphilic block copolymers. The introduction of different functional groups would allow for a great versatility of this polymer, which could be used, for example, as macroinitiator in ring opening polymerizations of lactides, lactones or N-carboxy anhydrides as well as difunctionalized segments for the synthesis of multiblock copolymers. However, the synthesis of PVP oligomers with controlled molecular weight and functionalization degree is still a challenge. The synthesis of mono and ditelechelic PVP oligomers was carried out by radical polymerization of NVP in presence of functionalized chain transfer agents. Mercaptanes and disulfides are known to be very efficient chain transfer agents and they can afford oligomers functionalized at one or both chain ends [24]. However, the molecular weight of the reaction product decreases on increasing the concentration of the chain transfer agent. Moreover, because of mercaptane high reactivity, most of the chain transfer agent is normally consumed during the first polymerization stages, thus affording a mixture of functionalized and non-functionalized oligomers of different chain lengths. To overcome these drawbacks, less reactive chain transfer agents have been used instead of thiols, for instance molecules containing tertiary hydrogen atoms. In this case, very large excess has to be used to attain a significant decrease of the polymer molecular weight. Mono hydroxy-terminated NVP oligomer (PVPOH) was prepared according to a literature

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procedure [25] by free radical polymerization of NVP in the presence of a chain transfer agent. The reaction was carried out at 70 C for 24 h by using AIBN as free radical initiator and 2-isopropoxyethanol (IPE) as both chain transfer agent and polymerization solvent (Scheme 1). The use of a large excess of IPE almost suppresses the competing chain transfer and chain termination reactions, thus favouring the formation of telechelic oligomers. Moreover, the low chain transfer constant of IPE towards NVP (CT = 3.29 · 104) allows for a better control of the target molecular weight and relevant narrow polydispersity. The FT-IR spectrum of the polymerization product displayed diagnostic bands at 1673 and 1288 cm1, attributed to the C@O stretching (Amide I) and C–N stretching in the lactame ring, respectively. In addition to the typical PVP broad signals, the 1H NMR spectrum of the polymer presented a small signal at 1.13 ppm that was attributed to the two methyl groups of 2-isopropoxyethyl chain end. When a polymer sample was reacted with trichloroacetyl isocyanate (TAIC) shift reagent (Scheme 2), a new triplet attributable to the methylene group in a-position to the urethane group appeared in the 1H NMR spectrum at 4.25 ppm. The relative intensity of this signal indicated that only about 50% of the polymeric chains contained an hydroxyl end group [36]. A number average molecular weight of 7430 was calculated from the relative intensity of the 1H NMR signals at 3.24 and 4.25 ppm. On the other hand, SEC analysis showed a number average molecular weight of 3700 and a dispersity index of 1.73. These results suggest that the adopted conditions did not afford a full control of polymer chain functionalization.

H

O

N

O

OH

n

AIBN N O

O

OH

Scheme 1. Synthesis of hydroxy-terminated telechelic N-vinyl-2pyrrolidinone oligomers.

Differential scanning calorimetry (DSC) analysis of PVPOH showed a glass transition at about 177 C with DCp = 0.35 J g1 K1. No melting peak was detected in agreement with PVP amorphous structure. TGA analysis of PVPOH exhibited a degradation step with maximum decomposition rate at about 174 C with 7% weight loss tentatively attributed to end-group degradation. The main decomposition step was at about 437 C with 89% weight loss, and the residue at 700 C was 4%. A commercial sample of high molecular weight PVP (PVPK30) showed a single decomposition step at 446 C and 3% residue at 700 C. 3.2. Ring opening polymerization of e-caprolactone initiated by PVPOH At first, PVPOH potassium salt was used as macroinitiator for the ring opening polymerization (ROP) of e-CL. Indeed, the coordination-insertion mechanism of the ring opening polymerization catalyzed by metal catalysts such as stannous octoate (SnOct2) was claimed to be ineffective when using PVPOH as initiator, because of its low nucleophilicity [24]. In this case, potassium PVP-hydroxylate can be utilized to enhance the PVPOH reactivity in anionic ROP (Scheme 3). The reaction was performed in THF at room temperature for 24 h under anhydrous conditions to give a white solid with 65% yield (Table 1, PK1). FT-IR and 1H NMR spectra of the polymerization product presented signals attributable to both e-CL and NVP units. SEC analysis showed a molecular weight of 6200 and a fairly narrow polydispersity index (1.83). The chemical composition of the copolymerization product was evaluated from the relative intensities of 1H NMR signals at 3.24 and 4.05 ppm, attributed to the PVP ring methylene group in a-position to the nitrogen atom and to PCL methylene in a-position to the ester oxygen, respectively. The copolymer content of e-CL units was much lower than that expected on the basis of the feed, namely 23% instead of the 50%.

O H2 C P P

OH 1.13 ppm

+

O

C OCN

C Cl

Cl Cl

H2 C P

O

C O

4.25 ppm

C N H

C

Cl Cl

Cl

Scheme 2. End-functionalization of telechelic PVPOH with trichloroacetyl isocyanate.

I. Bartolozzi et al. / European Polymer Journal 43 (2007) 4628–4638 H

O n

N

O

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O OH + m

O

O

KH, THF, r. t. SnOct2, bulk, 140ºC

H

O n

N ZnEt2, CH2Cl2 r.t.

O

O

H

m

O

Scheme 3. Ring opening polymerization of e-CL.

Table 1 Ring opening polymerization of e-caprolactone initiated by telechelic PVPOHa Runa

PK1 PS4 PS1 PS2 PS3 PZ1 PZ2

Feed

Polymeric product

Yield (%)

e-CL/PVPOH molar ratio

e-CLb (%-mol)

e-CLb,c (%-mol)

Mnd (Da)

Mw/Mnd (%)

33 104 69 35 23 33 8

50 76 67 51 41 50 17

23 63 71 58 33 52 3

6200 5800 8200 7300 6400 4900 3800

1.83 1.98 2.83 1.75 3.61 1.16 1.47

65 93 99 75 67 75 55

a

PVPOH (Mn 3700, Mw/Mn = 1.73) as macromonomer, PK1: in THF at room temperature for 24 h by using PVPOK as initiator; PS1– PS4: in bulk at 140 C for 18 h in the presence of 1%-wt SnOct2; PZ1 and PZ2: in CH2Cl2 solution at room temperature for 24 h in the presence of ZnEt2, PVPOH/ZnEt2 molar ratio = 1. b Referred to the overall content of e-CL and NVP monomeric units. c Evaluated by 1H NMR. d Evaluated by SEC.

These data suggest that the PVPOH potassium salt is not very efficient as initiator and that unwanted side reactions also took place. Hence, the PVPOH-initiated ring opening polymerization of e-CL catalyzed by SnOct2 was evaluated. Experiments were carried out in bulk at 140 C for 24 h under strict anhydrous conditions. Different PVPOH/e-CL feed ratios were used to modulate the hydrophilic/hydrophobic balance of the final products (PS1–PS4). The polymerization products were recovered in rather high yields. In all cases, 1 H NMR characterization showed that the polymer compositions substantially agree with those of the relevant feed mixtures. However, it must be pointed out that 1H NMR analysis does not allow for clearly differentiating block copolymers from homopolymers mixtures. Indeed, the shift of the signal relevant to the methylene group in aposition of the PVP hydroxyl end-group from 3.6 ppm to 4.2 ppm, due to the ester bond formation following the reaction with e-CL, could be diagnostic of a blocky structure, but unfortunately,

this signal is too close to the strong PCL peak at 4.08 ppm and in most cases, it was not detectable. The number average molecular weight values determined by SEC were lower than those determined by 1H NMR, suggesting that the copolymerization products might be constituted by mixtures of PVP–PCL copolymer, PCL homopolymer, and unreacted PVPOH. Finally, ZnEt2 was used as alternative catalyst. Indeed, this compound is less toxic and hence more suitable than SnOct2 for the preparation of materials to be used in biomedical applications [37]. Experiments performed in dichloromethane at room temperature for 24 h under anhydrous conditions afforded the expected polymerization products in medium-high yields (PZ1–PZ2). 3.2.1. Fractionation of the polymeric products The copolymerization products are constituted by rather complex mixtures and fractionation in water, ethanol, or isopropanol allowed for the separation of NVP and e-CL enriched soluble

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Table 2 Fractionation by different solvents of PVP–PCL copolymerization products Sample

PS1 PS1 PS2 PS3 PS4 PZ1 a b

Mna (Da) 8200 8200 7300 6400 5800 4900

e-CLb (%-mol)

71 71 58 33 63 52

Solvent

H2O iPrOH H2O EtOH EtOH H2O

Soluble fraction

Insoluble fraction

Weight (%)

Mna (Da)

e-CLb (%-mol)

Weight (%)

Mna (Da)

e-CLb (%-mol)

2 27 20 61 62 49

ND 3700 6100 4300 5200 5400

9 33 4 17 23 7

98 73 80 39 38 51

8200 11000 8600 11200 nd 4900

83 90 68 73 86 90

Evaluated by SEC; nd = not determined. Content of e-CL repeating units as determined by 1H NMR.

residue was left at 700 C. By taking into account the close correspondence between the observed weight losses and the copolymer content of e-CL and NVP structural units, the two degradation steps were attributed to the decomposition of PCL and PVP blocks, respectively [38]. In some cases (samples PS1, PS3, and PS4), the presence of a further decomposition step at about 510–540 C indicates the occurrence of a more complex degradation pattern. DSC analysis of the copolymers and their soluble and insoluble fractions, showed an endothermal transition between 60 and 70 C, attributed to the melting of PCL crystalline phase. Samples containing more than 70% PVP units presented a glass transition at 160–180 C (Table 4). The observed

and insoluble fractions, respectively (Table 2). Soluble fractions contained about 70–90% of NVP structural units whereas insoluble fractions were mainly constituted (70–90%) by e-CL units, as determined by 1H NMR. As expected, isopropanol afforded soluble (insoluble) fractions having larger (lower) e-CL content than the corresponding water fractions. SEC analysis of the recovered fractions showed monomodal molecular weight distributions. 3.2.2. Thermal analysis of block copolymers Thermogravimetric analysis of the copolymers displayed two main decomposition steps with maximum weight loss rate at about 300–360 C and 420–440 C (Table 3). In all cases, 5–10% weight Table 3 TGA of fractionated PVP–PCL block copolymers Sample

e-CL (%-wt)

Td1 (C)

Dw1 (%)

Td2 (C)

Dw2 (%)

Td3 (C)

Dw3 (%)

R700 (%)

PVPOH PK1 PS1sa PS1ib PS2 PS2se PS2if PS3 PS3sc PS3id PS4 PS4sc PZ1 PZ1se PZ1if

0 24 33 92 58 4 69 33 17 74 64 23 53 7 92

174 355 313 333 354 327 321 331 330 348 310 347 285 323 323

7 29 32 87 48 7 66 37 19 87 65 18 52 10 90

437 442 420 429 441 436 434 430 438 434 419 439 434 437 428

89 63 61 10 34 84 28 49 74 10 25 73 34 81 7

– – 535 – – – – 535 – – 511 538 – – –

– – 7 – – – – 8 – – 6 4 – – –

4 8 1 3 13 9 6 3 7 3 1 5 8 9 3

a b c d e f

Isopropanol soluble fraction. Isopropanol insoluble fraction. Ethanol soluble fraction. Ethanol insoluble fraction. Water soluble fraction. Water insoluble fraction.

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Table 4 DSC of PVP–PCL block copolymers Sample

NVP units (%-mol)

Tm (C)

DHm (J g1)

Tg (C)

DCp (J g1 K1)

PVPOH PCL-diol PK1 PS2 PS2ia PZ1 PZ1ia PZ1sb

100 0 77 42 32 44 10 93

– 61 62 64 64 61 50 –

– 75.2 5.4 33.0 40.0 35.0 75.4 –

177 55 159 – – – – 161

0.36 0.05 0.02 – – – – 0.29

a b

Water insoluble fraction. Water soluble fraction.

thermal behavior indicates that the copolymers are phase separated.

ume) and 300–600 nm aggregates (80–90%) (Table 5). The influence of temperature and concentration on the aggregates formation was investigated on the water-soluble fraction of PZ1 sample (PZ1s). By increasing the temperature from 25 to 37 C, the size of both micelles and aggregates slightly decreased from 47 ± 1 to 39 ± 5 nm and from 401 ± 6 to 370 ± 27 nm, respectively. Correspondingly, the percentage of the smaller population increased from 17% to 30%. Dilution from 5.0 to 0.625 g/L did not appreciably affect the size of either micelles or aggregates, whereas the amount of aggregates decreased from 70% to 50%. The

3.2.3. Preparation of block copolymer micelles and drug loading Micelle formation by the water-soluble fractions of NVP/e-CL block copolymers was investigated at 25 C in water at 5 mg/mL concentration. PVPOH solution, tested as reference material, displayed an average particle size of 8 nm, which corresponds to the unimer dimension. Dynamic light scattering (DLS) measurements on two water-soluble samples displayed a bimodal size distribution constituted by 40–50 nm micelles (10–20% by volTable 5 Characterization of polymeric micelles by dynamic light scattering Samplea

PVPOH PS2s PZ1s PZ1s PZ1s PZ1s PZ1s a

PCL (%-mol)

0 4 7 7 7 7 7

Conc. (g/L)

5.0 5.0 5.0 5.0 2.5 1.25 0.625

Temp. (C)

25 25 25 37 37 37 37

Micelles Volume (%)

Size (nm ± r)

Volume (%)

Size (nm ± r)

100 11 17 30 39 29 48

8±1 42 ± 10 47 ± 1 39 ± 5 42 ± 1 43 ± 6 35 ± 8

– 89 83 70 61 71 52

– 620 ± 23 400 ± 6 370 ± 27 340 ± 6 375 ± 42 380 ± 44

Water-soluble fractions.

Table 6 DLS measurements of piroxicam loaded micelles (0.5% w/v) Samplea

PCL (%-mol)

Loaded piroxicam (wt-%)

PS2s PS2sPir PZ1s PZ1sPir

4 4 7 7

0 2 0 9

a

Water-soluble fraction.

Micelles

Size (nm ± r)

Volume (%)

Size (nm ± r)

Volume (%)

11 55 29 85

42 ± 10 920 ± 244 43 ± 6 320 ± 122

89 45 71 15

620 ± 23 13,500 ± 3500 375 ± 42 nd

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Table 7 Static contact angle measurements of block copolymer films Sample

NVP (%-mol)

Contact angle (degree ± r)

Glass PCL PS1ia PS2ia PZ1ia

– 0 17 32 10

59.6 ± 3.6 72.3 ± 0.9 70.0 ± 0.8 23.6 ± 6.3 11.5 ± 3.3

a

Fraction insoluble in water.

reported behaviour indicates that the water-soluble copolymer fractions can form stable micelles in water, in spite of their very low PCL content and at the same time aggregates are formed, as already reported in the literature [4]. In agreement, 1H NMR spectra of the water-soluble fractions recorded in D2O showed a sharp decrease of PCL signals as compared to spectra in CDCl3. This effect can be attributed to an increase of relaxation times, due to PCL segregation out of the aqueous environment into a structure (micelle core) with low chain mobility. In order to investigate their potential application as drug delivery systems, PZ1s and PS2s micelles were loaded with piroxicam, a hydrophobic non-steroidal anti-inflammatory drug. Depending on the experimental conditions, the amount of entrapped drug ranged from 2% to 9%, as evaluated from the UV absorbance at 326 nm. However, dynamic

Fig. 1. AFM topography of the surface of PS2i thin film.

light scattering highlighted the formation of large aggregates (Table 6). 3.2.4. Surface characterization of copolymer thin films Thin films of the water-insoluble fractions of PZ1, PS1 and PS2 samples were prepared on glass slides by spin coating of 1%-wt chloroform solutions. Films were characterized by low contact

Fig. 2. Influence of protein concentration on BSA adsorption by polymer film surface.

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angles (11–24) indicating a remarkable hydrophilicity in spite of their large content (68–90%) of eCL units (Table 7). On the other hand, optical microscopy and AFM measurements highlighted the presence of PCL crystallite structures on the film surface, confirming the phase separation between amorphous PVP and crystalline PCL (Fig. 1). This behavior can be assigned to the reorganization of the film surface: when exposed to water, the hydrophilic PVP segments are reoriented towards the aqueous phase as driven by the interfacial energy minimization. Surface plasmon resonance (SPR) experiments were carried out to evaluate the interaction between the surface and proteins. In particular, bovine serum albumin (BSA) was chosen as model protein. The influence of BSA concentration on the adsorption kinetics and layer thickness was evaluated by increasing the protein concentration from 0.1 to 0.5 mg/mL. A bare golden sensor was always taken as reference. Signal saturation was reached at 0.5 mg/mL concentration, probably because of the formation of an adsorbed albumin monolayer on the polymeric film (Fig. 2). Comparison of SPR experiments carried out on PCL and PS2i films did not display any significant reduction of the extent of BSA adsorption, indicating that the amount of PVP exposed on the film surface is not enough to appreciably modify the bioadhesive properties of the polymer surface. 4. Conclusions Telechelic PVP oligomers carrying reactive end groups can be obtained by free radical polymerization of NVP in presence of suitable chain transfer agents. In particular, hydroxy-terminated PVP can be prepared in high yield by free radical polymerization of NVP in presence of 2-isopropoxyethanol. However, control over the degree of chain-end functionalization and molecular weight is not optimal. As consequence, block copolymers obtained by ROP of e-CL initiated by PVPOH and catalyzed by either SnOct2 or ZnEt2 are constituted by a complex mixture of water-soluble and water-insoluble fractions. Water-soluble fractions of the copolymers in aqueous solution form micelles and nanoaggregates susceptible to be loaded with hydrophobic drugs such as piroxicam. In spite of the hydrophilicity of the films obtained from the copolymer insoluble fractions, the presence of PCL crystalline domains on the film surface

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clearly demonstrates that PCL and PVP blocks are phase segregated. On the other hand, the surface hydrophilicity does not help preventing albumin adhesion, very likely because of phase separation. Acknowledgements This work was performed with the partial financial support by the Italian Ministry of University and Research (MIUR). The authors wish to acknowledge Dr. Veska Toncheva for helping with DLS measurements and Dr. Peter Dubruel for helping with SPR measurements. References [1] Kataoka K, Harada A, Nagasaki Y. Block copolymer micelles for drug delivery: design, characterization and biological significance. Adv Drug Deliver Rev 2001;47:113–31. [2] Allen C, Maysinger D, Eisenberg A. Nano-engineering block copolymer aggregates for drug delivery. Coll Surf B: Biointerf 1999;16:3–27. [3] Peracchia MT, Gref R, Minamitake Y, Domb A, Lotan N, Langer R. PEG-Coated nanospheres from amphiphilic diblock and multiblock copolymers: investigation of their drug encapsulation and release characteristics. J Control Release 1997;46:223–31. [4] Kim SY, Shin IG, Lee YM, Cho CS, Sung YK. Methoxy PEG and e-caprolactone amphiphilic block copolymeric micelle containing indomethacin II Micelle formation and drug release behaviors. J Control Release 1998;51:13–22. [5] Torchilin VP, Trubetskoy VS, Whietman KR, Caliceti P, Ferruti P, Veronese FM. New synthetic amphiphilic polymers for steric protection of liposomes in vivo. J Pharm Sci 1995;84:1049–53. [6] Luo L, Ranger M, Lessard DG, Le Garrec D, Gori S, Leroux JC, et al. Novel amphiphilic diblock copolymer of low molecular weight poly(N-vinylpyrrolidone)-blockpoly(D, L-lactide): synthesis, characterization and micellization. Macromolecules 2004;37:4008–13. [7] Haaf F, Sanner A, Straub F. Polymers of N-vinylpyrrolidone: synthesis, characterization and uses. Polym J 1985;17:143–52. [8] D’souza AJM, Schowen RL, Topp EM. Polyvinylpyrrolidone-drug conjugate: synthesis and release mechanism. J Control Release 2003;94:91–100. [9] Lou XJ, Panaro NJ, Wilding P, Fortina P, Kricka LJ. Increased amplification efficiency of microchip-based PCR by dynamic surface passivation. Biotechniques 2004;36:248–52. [10] Zhang L, Liang Y, Meng L, Lu X, Liu Y. Preparation and PCR-amplification properties of a novel amphiphilic poly(N-vinylpyrrolidone) (PVP) copolymer. Chem Biodivers 2007;4:163–74. [11] Benahmed A, Ranger M, Leroux JC. Novel polymeric micelles based on the amphiphilic diblock copolymer poly(N-vinyl-2-pyrrolidone)-block-poly(D, L-lactide). Pharm Res 2001;18:323–8.

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