Thermoresponsive polymer system based on poly(N-vinylcaprolactam) intended for local radiotherapy applications

Applied Radiation and Isotopes 98 (2015) 7–12

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Thermoresponsive polymer system based on poly(N-vinylcaprolactam) intended for local radiotherapy applications Peter Černoch n, Zulfiya Černochová, Jan Kučka, Martin Hrubý, Svetlana Petrova, Petr Štěpánek Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic v.v.i., Heyrovského Sq. 2, 16206 Prague 6, Czech Republic

H I G H L I G H T S


New radiolabeled thermoresponsive polymer system for injectable brachytherapy is described.
Two ways of introducing radiolabelable moiety into poly(N-vinylcaprolactam) are demonstrated.
The method allows preparation of a stable product carrying 125I.

art ic l e i nf o

a b s t r a c t

Article history: Received 7 July 2014 Received in revised form 29 December 2014 Accepted 5 January 2015 Available online 6 January 2015

Brachytherapy represents effective local therapy of unresectable solid tumors with very few side effects. Radiolabeled thermoresponsive polymers offer almost noninvasive approach to brachytherapy applications. A radioiodinated, water-soluble, thermosensitive poly(N-vinylcaprolactam) (PVCL) polymer was prepared using two approaches. The direct copolymerization with N-methacryloyl-L-tyrosinamide, as well as end-capping of carboxy-terminated PVCL homopolymer with tyramine, were used. In both cases the product was successfully radiolabeled with 125I. The obtained polymers demonstrate cloud-point temperature (TC) values in the range of 33–35 °C in all the studied solvent systems (water, PBS (pH 7.4) and physiological saline solution). Above the cloud point temperature, the molecularly dissolved polymer is macroprecipitated from the solution. The TC values close to the human body temperature of this biocompatible poly(N-vinylcaprolactam) polymer makes it a promising material intended for local therapy of solid tumors. & 2015 Elsevier Ltd. All rights reserved.

Keywords: Poly(N-vinylcaprolactam) Tumor Brachytherapy Thermosensitive polymer

1. Introduction Biocompatible and biodegradable polymers continuously attract attention to be used in medical applications. An important role in this field is played by water-soluble thermosensitive (co) polymers (Aseyev et al., 2011) characterized by lower critical solution temperature (LCST) or upper critical solution temperature (UCST), which separates solubility regions at different temperatures. Well-known biocompatible polymer systems based on N-isopropylacrylamide or 2-isopropyl-2-oxazoline monomer units show LCST close to human body temperature (Adams and Schubert, 2007; Diehl et al., 2010; Huber et al., 2008; Nam et al., 2002; Schild, 1992). This means that below the body temperature, the polymer is fully soluble in water-based media, while above this n

Corresponding author. E-mail address: [email protected] (P. Černoch).

http://dx.doi.org/10.1016/j.apradiso.2015.01.005 0969-8043/& 2015 Elsevier Ltd. All rights reserved.

temperature it precipitates. In a series of previous works (Hruby et al., 2011, 2009a, 2009b) of Hruby et al. it has been shown that thermosensitive polymers carrying isotopes of 125I or 131I have strong potential to be used as radionuclide delivery and/or treatment systems. The main promising area of application is the field of local radiotherapy of solid tumors, brachytherapy, traditionally based on surgical application of active material into the tumor. Brachytherapy keeps the active dose high while the impact of the radiation on the whole body is relatively low. After the treatment, the active material is, again surgically, removed. The importance of biodegradable and thermosensitive polymer systems in this field appears in the possibility to replace the surgical stress on the patient by a simple injection of active solution to the treatment site. The polymer, which at ambient temperatures below LCST is well soluble in water-based media, immediately precipitates at body temperature to a stable polymer at the desired location. Because of biodegradability of the chosen material, it is gradually released from the body so that no (surgical) removal procedure is

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needed. Biodegradation may be due to chemical reaction or may be physical due to shifting the equilibrium precipitated phase–liquid phase towards dissolution by continuous washing out of the liquid phase in the tissue. We have in fact shown that thermoresponsive polymers (if not tailored to contain biodegradable structures (Hruby et al., 2009a, 2009b)) are “degradable” only physically, i.e. they can only be redissolved in biological media and then eliminated from the organism by the kidneys and bile “as-is” without deiodination and with no observable redeposition in healthy organs (Hruby et al., 2011). The physical degradability and activity of the system can be tuned by variation of the molecularweight and type of the chosen polymer, carrier of the radionuclide, radionuclide type and dose. Apart from the well-known and widely studied thermosensitive polymers such as poly(N-isopropylacrylamide) (PNIPAM) and poly(2-isopropyl-2-oxazoline) (PIPOX), there exists a whole group of N-vinyl lactams (Chen et al., 2010; Chua and Kelland, 2012; Lai et al., 2012) allowing the synthesis of thermosensitive materials that are known to have excellent biocompatibility (well-recognized especially for FDAapproved poly(N-vinyl pyrrolidone)) superior to other biocompatible polymers (Sedlacek et al., 2012). Among them an important role is played by poly(N-vinyl caprolactam) (PVCL), a biocompatible polymer demonstrating LCST close to human body temperature (Lau and Wu, 1999; Spěváček et al., 2012). The main difference of PVCL compared to other studied thermoresponsive polymers is it inability to form hydrogen bonds as a donor (unlike e.g., PNIMAM), which results in lower interaction with biomolecules, higher expected bioacceptability, and no tendency to crystallization (which may cause PIPOX to phase separate partly irreversibly). It is also not poly(ethylene oxide)-based, which would result in formation anti-PEO antibodies. From a theoretical scope of view, we compared stimuli-responsive and biocompatible polymers in our recent critical review (Sedlacek et al., 2012). Although there exist studies dealing with the importance of the PVCL-based polymers (c.f., Aseyev et al., 2011; Kermagoret et al., 2013; Kudyshkin et al., 2004; Lau and Wu, 1999; Maeda et al., 2002; Spěváček et al., 2012; Vihola, 2007; Yerriswamy et al., 2010), according to our best knowledge, this is the first time that PVCL was studied as a radionuclide carrier for potential radiotherapy applications. As it was shown in the publication of Kucka et al. (2010), in vivo experiments confirmed that 131I covalently bound to thermosensitive copolymer of N-methacryloyl tyrosinamide (MTAM) and N-isopropylacrylamide represents an effective system for local radiotherapy. Therefore the copolymer of MTAM and PVCL (PVCL– MTAM) is a natural choice. However, N-vinyl lactams generally do not copolymerize with methacrylamides into random copolymers, but due to monomer reactivity ratios rather into multiblock copolymers with less molecular uniformity. Therefore an indirect approach, the coupling of polymer chains terminated with a reactive end group with a suitable radiolabelable moiety, was used in this case. For this purpose we have selected coupling of carboxy-terminated PVCL (PVCL-COOH) with tyramine (TA) via N ,N ′-disuccinimidyl carbonate route (Ghosh et al., 1992; Morpurgo et al., 1999) to prepare tyramine functionalized PVCL (PVCL–TA). During the experiments it was found that the routinely used standard procedure of iodination (Koyama et al., 1993) was not sufficiently effective due to iodine-complexing properties of PVCL so we developed an alternative approach giving stable products. Phase separation was also studied showing favorable properties for the intended use.

2. Experimental 2.1. Chemicals N-Vinylcaprolactam (VCL), 3-mercaptopropanoic acid (MPA), 2,2′-Azobis(2-methylpropionitrile) (AIBN), benzene, L-tyrosinamide (TAM), tyramine (TA), phosphate buffered saline-pH 7.4 (PBS), tris(hydroxymethyl)aminoethane (TRIS), L-ascorbic acid, chloramine T, N,N′-disuccinimidyl carbonate (DSC) and triethylamine (TEA) were supplied by Sigma-Aldrich Ltd. (Prague, Czech republic) and were used as received. Methacryloyl chloride (Sigma-Aldrich) was freshly distilled before used. MilliQs water was used to prepare solutions and to conduct reactions in water medium. Tetrahydrofuran (THF), diethyl ether, dimethylformamide (DMF), acetonitrile (AN) and methanol were obtained from Lachner, Czech Republic. DMF and AN were kept over molecular sieve (3 Å), the other solvents were used as received. The PD-10 desalting columns (Sephadex G-25) were received from Amersham Biosciences, Sweden. Sephadex LH-20 was obtained from Sigma. Na125I (20 ml, 370 MBq) was received from Lacomed Ltd. (Řež, Czech Republic). 2.2. Instrumental methods 1

H NMR spectra (300 MHz) were obtained using a Bruker Avance DPX 300 NMR spectrometer with THF-d8 as the solvents at 25 °C. The chemical shifts are relative to TMS using hexamethyldisiloxane (HMDSO, 0.05 from TMS in 1H NMR spectra) as internal standard. Gel permeation chromatography (GPC) was performed in dimethylformamide (DMF) as a mobile phase at 25 °C on PL gel MIXED-B-LS column using Pump Deltachrom (Watrex Corp., Prague, Czech Republic) HPLC system equipped with evaporative light scattering detector PL ELS-1000 (Varian, Inc., Palo Alto, CA, U.S.A.). The system was calibrated to polystyrene standards. Dynamic light scattering experiments were conducted using a Flex02-20 autocorrelator (Correlator.com, USA) based setup with 22 mW He–Ne laser (wavelength λ ¼ 632.8 nm) and PMT detector at scattering angle 90°. The polymer (either PVCL–MTAM or PVCL– TA) was dissolved in water, PBS or physiological saline solution respectively at concentrations 1 mg/ml to minimize influence of multiple scattering around LCST. The samples were filtered prior to measurement through 0.45 mm PVDF syringe filter into a 100  10 mm glass tube. The temperature scans were performed with the heating step of 0.2 °C followed by 10 min of stabilization. The measured intensity correlation functions g2(t) were analyzed using the REPES algorithm (Jakeš, 1995) resulting in the distribution of relaxation times A(τ). The hydrodynamic radius (RH) of the nanoparticles was calculated using the relation: RH ¼(kBTq2τ)/ (6πη) where kB is the Boltzmann constant, T is the absolute temperature, q is the scattering vector, η is the viscosity of the solvent and τ is the mean relaxation time related to the diffusion of the nanoparticles. 2.3. Synthetic procedures N-Methacryloyl-L-tyrosinamide (MTAM) was synthesized according to the literature (Lee et al., 1990). Briefly, to a 25 ml flask with a magnetic stirrer 13 ml of water and 447 mg (2.48 mmol) of TAM were placed. The solution was cooled to 0 °C and while stirring 120 ml (1.23 mmol) of freshly distilled methacryloyl chloride was added. The solution was stirred at ambient temperature for 5 h. The white precipitate was filtered off, washed thoroughly on filter with water and dried in vacuum. 1H NMR (THF-d8) δ: 1.79 (3H, –CH3 in methacryloyl group), 2.50 (2H, –NH2 in amidic group), 6.53–6.55 (2H, aromatic CH, meta to aromatic –OH).

P. Černoch et al. / Applied Radiation and Isotopes 98 (2015) 7–12

Elemental analysis: Calc: C 62.89, H 6.50, N 11.28. Found: C 62.79, H 6.52, N 10.89. Poly(N-vinylcaprolactam-co-N-methacryloyl tyrosinamide) (PVCL– MTAM, scheme in Fig. 1). The VCL (5 g, 35.9 mmol), MPA (0.381 g, 3.59 mmol), AIBN (25.5 mg, 0.18 mmol), MTAM (25 mg, 0.1 mmol) and benzene (25 ml) were placed into a 50 ml round flask with a magnetic stirrer. The flask was sealed and inertized with argon. Polymerization was carried out for 21 h at 63 °C. The solution was evaporated in vacuo, redissolved in 25 ml of THF and reprecipitated into 300 ml of diethyl ether. The precipitate was filtered-off, redissolved in THF and reprecipitated into diethyl ether. The product was dried in vacuo. Yield 3.5 g (70%). GPC: weight-average molecular weight (Mw)/number-average molecular weight (Mn)¼14,140/5352. To remove traces of low-molecular weight impurities, a column chromatography-based purification was realized: The PVCL– MTAM (110 mg) was dissolved in methanol (4 ml). The solution was passed through LH-20 column (30  200 mm) with methanol as the mobile phase. Fractions of 10 ml each were collected and evaporated. Majority of the product was recovered from the frac-

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tion No. 4 and was used in further experiments. The content of Ltyrosinamide determined spectroscopically at λ ¼280 nm in methanol solution was 0.45 nmol/g. Carboxy-terminated poly(N-vinylcaprolactam) homopolymer (PVCL-COOH, scheme in Fig. 2). The VCL (5 g, 35.9 mmol), MPA (0.381 g, 3.59 mmol), AIBN (25.5 mg, 0.18 mmol) and benzene (25 mL) were placed into a 50 ml round flask with a magnetic stirrer. The flask was sealed and inertized with argon. The polymerization proceeded at 60 °C for 24 h. The polymer was two times precipitated from diethyl ether as in the case of PVCL–MTAM (see above). Yield 1.2 g (24%). GPC: Mw/Mn ¼7344/4264. Elemental analysis: C 65.48, H 9.45, N 9.45, S 0.41. Sulfur-content based data: amount of –COOH groups¼0.0128 mmol/100 mg. The related sulfur-based molecular weight (M) calculated using equation M¼(100MS)/S%, where MS is molecular weight of sulfur and S% is percentage of sulfur determined by elemental analysis, is 7812. Activation of PVCL-COOH with DSC (PVCL–DSC). The PVCL-COOH (150 mg, 0.021 mmol) was dissolved in a mixture of 10 ml AN and 6 ml DMF. DSC (27 mg, 0.11 mmol) was dissolved in 3 ml AN and

Fig. 1. Scheme of preparation and radiolabeling of PVCL–MTAM.

Fig. 2. Scheme of preparation and radiolabeling of PVCL–TA system.

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dropwise added into the solution of PVCL-COOH during 10 min. TEA (15 mL, 0.11 mmol) was added at once into the mixture and the solution was stirred at room temperature for 20 h. The solution volume was reduced in vacuo and precipitated into diethylether. The product (yield 56 mg, 38%) was filtered off and dried in vacuo. Coupling of PVCL–DSC with TA (PVCL–TA). The solution of TA (2.1 mg, 0.047 mmol) in DMF (2 mL) was added into the solution of PVCL–DSC (56 mg, 0.008 mmol) dissolved in DMF (2 mL). TEA (7 ml) was added and the mixture was stirred for 48 h. The final solution was evaporated in vacuo, redissolved in methanol (2 mL) and passed through Sephadex LH-20 column (20  130 mm) using methanol as the mobile phase. Fractions of 5 mL were taken. Polymer corresponded to the highest molecular weight fraction (No. 3, yield 32 mg, 57%) was used in further radiolabeling experiments. The content of L-tyrosinamide, determined spectroscopically at λ ¼ 280 nm in methanol solution was 29 mmol/g. 2.4. Radiolabeling The purified and lyophilized polymer (PVCL–MTAM, 2.1 mg) was dissolved in 200 ml of PBS buffer, pH 7.4. The solution was mixed with Na125I (1.0 ml, 22.25 MBq) and chloramine-T (10 ml, 10 mg/ml). The mixture was incubated for 60 min at 20 °C under continuous shaking. The reaction was quenched by adding of 100 ml of ascorbic acid (25 mg/ml, pH of the stock solution was adjusted to 7.4 with sodium hydroxide) and 10 ml of TRIS (100 mg/ ml) and incubated for next 15 min. The labeled polymer was separated from the low-molecular-weight fractions using PD-10 desalting column and PBS as a mobile phase, taking 10 fractions of 1.5 ml each. Radioactivity was measured with a Bqmetr 4 (Empos Ltd., Prague, Czech Republic) ionization chamber. Yield of the labeling procedure was calculated as an activity of the high-molecular-weight fraction related to the total activity applied to the studied sample (Nos. 3 and 4). Stability studies were carried out by incubation in PBS buffer; briefly: The solution of polymer in PBS (fraction No. 3, 1 mL, 5.4 MBq) was mixed with aqueous solution of ascorbic acid (200 mL, 25 mg/mL, pH adjusted to 7.4 with sodium hydroxide) and incubated during 1 week. After 1, 2 and 7 days samples 400 mL were taken from the incubated solution and fractionated using PD10 column. Activities of individual fractions were measured and evaluated relatively to the activity of the whole part. Procedures of radiolabeling and examination of PVCL–TA were identical to the described techniques used at PVCL–MTAM system.

3. Results and discussion Preparation of the polymers: Two polymers carrying radioiodinable groups were synthesized with a good yield by free radical polymerization of N-vinylcaprolactam either by direct copolymerization with methacryloyl tyrosinamide (yield 70%) or by functionalization of carboxy-terminated PVCL copolymer (yield 24%). The telechelic copolymers are less convenient to synthesize, however they are molecularly uniform according to distribution of phenolic moieties per chain that is statistical for random copolymers. With respect to the content of phenolic moiety determined in both copolymers, the route using functionalization of carboxyterminated PVCL appears more favorable compared to the direct copolymerization of VCL and MTAM. (The direct copolymerization of acrylic (MTAM) and vinylic (VCL) monomers is less favorable in this case probably due to monomer reactivity ratio.) According to sulfur content in PVCL-COOH, Mn is 7812 while according to GPC it is 4264; this means carboxy terminus is on 55% of chains, which is fully sufficient to obtain acceptable radioiodine loading. However, we should note that for the intended activities tens to hundreds of

MBq per mg of polymer, only one chain per several thousand would be radiolabeled and the remaining chains serve as adjuvants. The molecular weight of the polymers (Mw is 7–14 kDa) is kept well below renal threshold (which is ca 30–50 kDa (Armstrong et al., 2004; Duncan, 2003; Fox et al., 2009)) which should assure fast elimination of the polymers via renal route after redissolution of the depot (“physical degradation”). DLS experiments: According to DLS measurements, in the region below TC the solutions exhibit bimodal character with faster and slower dynamic processes characterized by corresponding smaller and bigger RH values, respectively. The DLS results are summarized in Table 1 for PVCL–MTAM system and in Table 2 for PVCL–TA system. We assign the presence of molecularly dissolved polymer to the species with small RH values (Teraoka, 2002) (defined as RH1) and the supramolecular clusters to the species with bigger RH values (defined as RH2). The size related to the molecularly dissolved polymer is similar in all solvents. Compared to this the size of clusters fluctuates, which may be caused by variation in concentration and the type of salts in the solutions. An example of intensity-based distribution of hydrodynamic radii and a derived volume-based distribution for PVCL–MTAM system in water is shown in Fig. 3. Amplitudes of the small and big components were similar in all systems. It is therefore clear that the majority of the dissolved polymer (499%) is in the molecularly dissolved form. In all solvents at TC and higher temperatures we have observed disappearing contribution of the molecularly dissolved polymer (RH1) and a rapid increase of RH2 of the supramolecular clusters finally leading to a macroscopic precipitation of PVCL. The effect is caused by increasing hydrophobicity of the polymer chains when temperature approaches TC. Noticeable is also the decrease of TC in PBS and NaCl solutions compared to water solutions, caused probably by a salting-out effect. Radiolabeling of PVCL–MTAM with 125I was based on the chloramine T method (Koyama et al., 1993). Stability of various synthetic polymers radioiodinated via copolymerized tyrosinamide (e.g., poly[N-(2-hydroxypropyl)methacrylamide] (Kissel et al., 2001) or poly(N-isopropyl acrylamide) (Hruby et al., 2011; Kucka et al., 2010)) was tested in vivo in an animal model and it was found that radioiodination is stable against deiodination with enzymes such as dehalogenase with even no free iodide found in, e.g., thyroid. Therefore, we have chosen incubation in PBS as the measure of stability based on the previously published data. As this study was performed with the radiolabeled polymer, so the Table 1 Summary of DLS experiments in solutions of the PVCL–MTAM (1 mg/ml) in corresponding solvents-water, PBS pH 7.4 and physiological solution. TC – cloud point temperature, RH – Hydrodynamic radius at 25 °C (RH1 – single molecules, smaller size peak; RH2 – larger multimolecular aggregates). Solvent

TC (°C)

RH1 (nm)

RH2 (nm)

Water PBS Physiological sol.

35.0 33.2 33.5

9.3 70.4 9.3 70.4 9.3 70.3

85.0 7 8.9 95.8 7 23.4 66.27 6.4

Table 2 Summary of DLS experiments in solutions of the PVCL–TA (1 mg/ml) in corresponding solvents-water, PBS pH 7.4 and physiological solution. TC – cloud point temperature, RH – Hydrodynamic radius at 25 °C (RH1 – single molecules, smaller size peak; RH2 – larger multimolecular aggregates). Solvent

TC (°C)

RH1 (nm)

RH2 (nm)

Water PBS Physiological sol.

36.0 33.0 33.0

107 4.8 6.3 7 0.4 6.17 0.9

937 3.7 1327 7.9 1247 10.5

P. Černoch et al. / Applied Radiation and Isotopes 98 (2015) 7–12

Fig. 3. Normalized intensity and volume based distributions of hydrodynamic radii (RH) observed in  0.1 w/v % solution of PVCL–MTAM in water. Data measured at scattering angle 90° and temperature 25 °C.

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covalent iodination of the phenolic core, the yield of radiolabeling was in both studied cases significantly lower (yield 47% for PVCL– MTAM and 38% for PVCL–TA, see Figs. 4 and 5) compared, for example, to the formerly studied PNIPAM polymers (Hruby et al., 2005), which gave a yield of 77%. The stability of the radioiodinated PVCL–MTAM and PVCL–TA systems was proved during a 1-week stability test. In both cases, we found that after the first day, the activity of the high molecular weight fraction dropped to about 77% of the original value, which indicates the presence of an additional portion of adsorbed iodine. However, during the rest of the time period, the activity was kept constant (4 99%) and no additional low molecular weight fractions were released. An addition of ascorbic acid and a single passing of the reaction mixture through a PD-10 desalting column (a procedure established in our laboratory to remove non-covalently bound iodine residues with low molecular weight) is therefore not sufficient to break the complex and to reduce iodine to a water-soluble iodide. However, we have found that the residues of the non-covalently bound 125I can be removed by simultaneously adding tris(hydroxymethyl)aminoethane and ascorbic acid to the system, followed by two fold passing through the desalination column (ascorbic acid is reducing agent reducing strongly PVCL-complexed iodine species to iodide while tris(hydroxymethyl)aminoethane is complexing iodine species in the same way as PVCL does and thus shifts recomplexation equilibrium towards purified polymer). This allows preparation of a stable product carrying 125I in acceptable yield.

4. Conclusions Fig. 4. Activity bound in individual fractions of PVCL–MTAM after radiolabeling.

We have described and characterized two thermosensitive polymer systems based on N-vinyl caprolactam, carrying a radiolabelable moiety and intended as a biocompatible material for local radiotherapy. The polymer systems display TC in water, PBS and physiological solution in the range of 33–35 °C. The sufficient binding capacity for radioisotope was proved using covalently bound 125I.

Acknowledgment Financial support provided by the Ministry of Industry and Trade of the Czech Republic (Grant # MPO TIP FR-TI4/625), the Academy of Sciences of the Czech Republic (Grant # M200501201) and the Ministry of Education, Youth and Sports (Grant # MSMT KONTAKT II LH14079) is gratefully acknowledged. Fig. 5. Activity bound in individual fractions of radiolabeled PVCL–TA.

stability against radiolysis is inherently included in it. During preliminary experiments with radiolabeled PVCL–MTAM we have observed continuous release of substantial amount radioiodine from the labeled polymer, which is not acceptable in practical applications. As the chemical structure of N-vinyl caprolactam (VCL) is similar to N-vinyl pyrrolidone, a well-known complexant of iodine (the complex is used as a local disinfectant) (Ignatova et al., 2008), we can suppose a strong physical interaction between iodine and PVCL causing this effect. In the case of PVCL chain with a radiolabelable moiety, e.g. MTAM, part of the iodine is therefore covalently bound in the MTAM molecule, while the other part (ca. 40% as estimated from activity of separated low-molecularweight compounds; plausibly mixture of inorganic radioiodine species as iodine monochloride, iodine and iodide) probably forms a physical PVCL–iodine complex. Because of the formation of physical complexes between PVCL and free iodine that shield

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