Thermoresponsive Polymers for Nuclear Medicine: Which Polymer Is

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Thermoresponsive polymers for nuclear medicine: Which polymer is the best? Ondrej Sedlacek, Peter Cernoch, Jan Kucka, Rafal Konefal, Petr Stepanek, Miroslav Vetrik, Timothy P. Lodge, and Martin Hruby Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.6b01527 • Publication Date (Web): 28 May 2016 Downloaded from http://pubs.acs.org on May 31, 2016

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Article type: Full Paper

Thermoresponsive polymers for nuclear medicine: Which polymer is the best? Ondřej Sedláčeka, Peter Černocha, Jan Kučkaa, Rafał Konefala, Petr Štěpáneka, Miroslav Vetríka, Timothy P. Lodgeb and Martin Hrubýa* a

Institute of Macromolecular Chemistry, v.v.i., Academy of Sciences of the Czech Republic, Heyrovsky Sq. 2, 162 06 Prague 6, Czech Republic; b

Department of Chemistry, University of Minnesota, 207 Pleasant Street SE, Minneapolis, Minnesota 55455, U.S.A *-corresponding author, e-mail: [email protected], Tel.: +420 296 809 130, Fax.: +420 296 809 410. Keywords: Polymeric Materials, Self-Assembly, Stimuli-Responsive Materials, Biomedical Applications

Graphical abstract

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Abstract Thermoresponsive polymers showing cloud point temperatures (CPT) in aqueous solutions are very promising for the construction of various systems in biomedical field. In many of these applications these polymers get in contact with ionizing radiation, e.g., if they are used as carriers for radiopharmaceuticals or during radiation sterilization. Despite this fact, radiosensitivity of these polymers is largely overlooked to date. In this work, we describe the effect of electron beam ionizing radiation on the physico-chemical and phase separation properties of selected thermoresponsive polymers with CPT between room and body temperature. Stability of the polymers to radiation (doses 0 – 20 kGy) in aqueous solutions increased in the order poly(N-vinylcaprolactam) (PVCL, the least stable) 1.5 kGy.h–1), polymer solution was heated above 4 ACS Paragon Plus Environment

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the CPT due to the irradiation, an opaque gel with heterogeneous microporous structure was formed. The same author reported synthesis of PNIPAM hydrogels by 150 kGy of γ irradiation above the CPT of PNIPAM (18 wt% aqueous solution). The obtained hydrogel was opaque and non-porous. This hydrogel showed lower crosslink density and elastic modulus compared to that prepared by chemical crosslinking.20 There are also reports describing radiation polymerization of monomers, which are however not relevant for polymeric delivery systems that are already radiolabeled for application into the body.21 The effect of radioactivity on polymer carriers may be very important, because the radiation may change the molecular weight of the polymer, change its CPT due to radiodegradation, and create byproducts, which all may drastically change the biological behavior of the system.22 Especially during the storage of relatively concentrated aqueous solutions of radiolabeled polymer systems the dose delivered to the sample is very high, e.g., a solution containing 1 GBq.mL–1 of 90Y receives a maximal radiation dose of 32 kGy per day, assuming complete capture of radiation in the sample (i.e., an infinite volume of sample).15 Furthermore, the typical irradiation dose used for sterilization (mainly γ, but sometimes also β- rays) is 25 kGy.23 This paper provides the first direct comparison of radiation effects on the thermoresponsive polymers most commonly used for the construction of biomedical systems. We find very significant changes of polymer properties upon β- irradiation, and quite different responses among the various polymers. This will have strong consequences especially for the design of polymer radiotherapeutics typically bearing β--emitters, for the design of polymer PET radiodiagnostics bearing positron emitters (due to similarity of positrons and electrons in radiochemical effects and because also the common PET radionuclide used for nanoradiodiagnostics and nanoradiotheranostics is 64Cu, which also shows less probable, yet


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far not negligible β- emission) as well as for the construction of polymer systems intended to be sterilized with β- radiation.

2. Results and discussion Thermoresponsive polymers and their molecular weights (Figure 1 and Table 1) were selected to match the typical cases which are described in literature for biomedical uses.24, 25 A Microtron source of accelerated electrons was used to homogenously deliver highly defined dose of radiation to the sample.26 The samples were irradiated in their diluted aqueous solution to simulate their typical use. The concentration (c = 5 mg.mL) of irradiated polymers were below their initial critical overlap concentration c*, where the polymer chains behave like individual non-interacting coils.

Mw Polymer [kDa]

a

Ða

c* [mg.mL-1] b

CPT [°C] c

PNIPAM

22.0

1.13

50.7

30

DFP

18.1

1.07

59.1

22

POX

11.0

1.04

87.4

30

PVCL

339

1.29

5.93

32

Table 1: Characteristics of polymers prior to irradiation. a Mass-averaged molecular weight Mw and polydispersity Ð of the polymers were determined by SEC;

b

critical overlap

concentration of the polymers estimated based on SEC measurement at 20°C; c measured at cpolymer = 5 mg.mL–1. The first series of experiments were performed to analyze the macroscopic effects seen on the polymer solution (Table 2). Irradiation was performed at temperatures both below the CPT at 2 °C (where all polymers are molecularly soluble) and above the CPT for all polymers at 60°C (where all polymers are phase separated even after radiochemical hydrophilization during irradiation, see below). To see the effect of radiation on the phase separation of the 6 ACS Paragon Plus Environment

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irradiated polymer samples, these were allowed to equilibrate at 20 °C after irradiation. When irradiated below the CPT, PNIPAM and POX remained soluble up to a dose of 20 kGy. In contrast, DFP was soluble only up to 5 kGy, and formed a precipitate above this dose, while PVCL forms hydrogel from 5 kGy above. The formation of hydrogel can be ascribed to the higher c/c* ratio of PVCL and thus the better overlapping of its polymer chains leading to higher degree of intramolecular crosslinking. After irradiation in the phase separated state (above the CPT), POX remained soluble after subsequent cooling, whereas PNIPAM irradiated with doses above 5 kGy remained cloudy even after cooling. DFP and PVCL were soluble up to doses of 2 or 5 kGy, respectively, and formed cloudy solutions above 5 or 10 kGy. Macroscopic precipitates are formed at higher doses - 10 kGy (DFP) or 20 kGy (PVCL). In this case, formation of precipitates, rather than hydrogels, can be explained by already collapsed polymer chain structures in the phase separated state leading to the reduced mobility of chain, which impedes the formation of hydrogel. As a result, even doses which are highly relevant to the synthesis and storage of polymer radiopharmaceuticals, as well as radiation sterilization (typical doses 10 - 25 kGy) of thermosensitive polymer systems cause macroscopic effects on polymers.

(B)

Dose (kGy)

20

Polymer

0

1

2

5

10

20

S

S

PNIPAM

S

S

S

C

C

C

S

P

P

DFP

S

S

S

C

P

P

S

S

S

S

POX

S

S

S

S

S

S

S

H

H

H

PVCL

S

S

S

S

C

P

(A)

Dose (kGy)

Polymer

0

1

2

5

10

PNIPAM

S

S

S

S

DFP

S

S

S

POX

S

S

PVCL

S

S


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Table 2: Macroscopic appearance (at 20 °C) of polymer samples after irradiation (cpolymer = 5 mg.mL–1). (A) Polymers irradiated at 2 °C; (B) polymers irradiated at 60 °C. S - Transparent solution; C - cloudy solution; P - precipitate; H - Hydrogel. More quantitatively, we examined the changes in molecular weight (mass-average Mw) and dispersity Ð of the polymers upon irradiation both below and above the CPT (Figure 2). For particular SEC traces, and Mw and Ð of polymer samples see Figures S1-S4 and Tables S1-S4).

Missing points in Figure 2 are cases where hydrogels, macroscopic

precipitates or other inhomogeneities formed, or where Mw is close to exclusion limit of the SEC column, preventing accurate determination of Mw and/or Ð. In the case of polymers where the SEC signal was beyond the column exclusion limit only Mw from the static light scattering is provided (this is the case of PVCL irradiated with 1 and 2 kGy).

Figure 2: Effect of electron-beam irradiation on Mw and Ð of thermosensitive polymers in aqueous solutions (cpolymer = 5 mg.mL-1). (A) Polymers irradiated at 2 °C; (B) polymers irradiated at 60 °C. The polymers differ greatly in their sensitivity to radiation, with the order PVCL (the most sensitive) >> DFP > PNIPAM >> POX (the least sensitive). In fact, POX remained almost intact up to a dose of 10 kGy, with only a modest effect at 20 kGy. For the other polymers the effects are very strong. Generally molecular weight and dispersity increased 8 ACS Paragon Plus Environment

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with increasing radiation dose, such that the polymer crosslinking definitively prevails over degradative cleavage of polymer chains. The trends are evident for irradiation both below and above the CPT, being slightly stronger in the latter case. In short, doses as low as 1 kGy cause significant increase of molecular weight and dispersity due to the crosslinking for most polymers. Irradiation may also cause radiochemical reactions leading to hydrophilization of the polymer chains (e.g., introduction of hydroxy- or carboxy-groups, scission of side-groups).27 Hydrophilization of thermoresponsive polymers leads to an increase in the CPT.28 In case that CPT reaches a value above body temperature, the biological efficacy of such system may be compromised. On the other hand, we have shown above a strong increase in molecular weight upon irradiation.

For thermoresponsive polymers, an increase in molecular weight can

decrease the CPT, i.e. the effect is opposite to that of polymer chain hydrophilization.29 It is therefore important to understand which of these effects prevails. We studied the properties of irradiated polymer samples over a range of relevant temperatures from 20 °C (room temperature) to 45 °C (the practical limit for externally implemented hyperthermia in vivo is ca. 44 °C). 2.1 Poly(N-isopropyl acrylamide) (PNIPAM) Irradiation of PNIPAM below the CPT (at 2 °C, Figure 3A) definitively leads to its hydrophilization, with even a dose of 1 kGy being enough to increase CPT by ca. 2 °C. The doses of 10 and 20 kGy cause complete loss of thermoresponsive behavior within the studied temperature range.


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Figure 3: Effect of electron-beam irradiation on transmittance of PNIPAM in aqueous solution (cpolymer = 5 mg.mL-1). (A) Polymers irradiated at 2°C; (B) polymers irradiated at 60°C. The predominant radiolysis mechanism of amides is their dissociative deamidation.27 In the case of PNIPAM and DFP, it is possible that some polymeric units were converted into acrylamides. This mechanism is supported by NMR spectroscopy of irradiated PNIPAM polymer (Figure 4). The 1H-NMR spectrum confirmed partial radiolytic degradation of Nisopropylamide group to the primary amide group by the appearance of signal f (δ = 3.37 ppm) related to the -NH2 amide group. The signals of main chains of resulting copolymer (d, d’, e, e’) overlapped each other at δ ≈ 1.25-2.5 ppm. Moreover, the spectrum shows the side chain signals of PNIPAM: broad amide peak c at δ ≈ 5.8 - 7.5 ppm, methyl a (δ = 1.11 ppm) and methylene b group at δ = 3.97 ppm. The broadness of the signals arises from the high crosslinking of polymers and thus their reduced mobility affecting the NMR relaxation. The same degradation pathway was confirmed by

13

C-NMR spectra, where at δ = 178.44 ppm

appears a signal c’ related to the carbonyl carbon of a primary amide.30 From the

13

C NMR

spectra, the fraction of NIPAM monomeric units converted to acrylamide monomeric units was calculated (7%) as a ratio of integral intensity of carbonyl signals c' to c + c' (Figure 4B). This also excludes the possibility of degradation of N-isopropylamide group to a free carboxylic acid.31 The spectrum shows also the characteristic signals of PNIPAM main chain 10 ACS Paragon Plus Environment

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d, e overlapping each other at δ ≈ 36 ppm and signals of side chain: a, b, c at δ = 22.7, 41.5 and 174.36 ppm, respectively.

Figure 4: NMR spectrum of PNIPAM irradiated with a dose of 5 kGy at 2°C. Spectra measured in CDCl3 at 47°C. (A) 1H-NMR spectrum; (B) 13C-NMR spectrum. For PNIPAM irradiated well above the CPT (at 60 °C, Figure 3B) the effect is similar. However, after receiving higher doses of radiation (10 and 20 kGy) solutions remained turbid even at low temperatures (Table 2). This difference can be explained by the formation of microgel-like particles as a result of the crosslinking of the aggregates. This can be confirmed by the large hydrodynamic radius as shown in Figure S5. The size of polymers is very important for their biological behavior, e.g., for renal elimination, which is the main elimination route for polymers present in the bloodstream. Accordingly, the effect of irradiation on the polymer hydrodynamic radius in solution was studied by dynamic light scattering (DLS). The hydrodynamic radius of PNIPAM samples irradiated below the CPT (Figure S5A) increases slightly with increasing radiation dose due to hydrophilization, which increases chain solvation and polymer coil expansion. The hydrodynamic radii of PNIPAM samples irradiated above the CPT (Figure S5B) show a similar trend, but the analysis is complicated by macroscopic phase separation at higher radiation doses (Table 2 and Figure 4).


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2.2 Poly[N-(2,2-difluoroethyl)acrylamide] (DFP) For the solutions of DFP irradiated at temperatures below the CPT (Figure 5A) the effect of hydrophilization and the attendant increase in CPT was similar to PNIPAM, but even more pronounced; 1 kGy caused an increase in the CPT of about 4 °C and starting from 5 kGy there was gradual loss of thermoresponsive behavior within the studied temperature range. Samples irradiated by doses 10 kGy and higher showed no CPT within the studied temperature range. Upon irradiation the phase separation also becomes less sharp and extends over a wider range of temperatures. On the other hand, when DFP is irradiated at temperatures above the CPT (Figure 5B), the decrease in CPT due to crosslinking-caused increase in molecular weight (Figure 2B) prevails. There is some turbidity at lower temperatures for samples irradiated with 5 kGy, but the turbidity measurement was impossible at 10 and 20 kGy due to macroscopic phase separation (Table 2).

Figure 5: Effect of electron-beam irradiation on transmittance of DFP in aqueous solution (cpolymer = 5 mg.mL-1). (A) Polymers irradiated at 2°C; (B) polymers irradiated at 60°C.

The hydrodynamic radius of DFP samples irradiated below the CPT (Figure S6A) shows that DFP forms clusters with a hydrodynamic radius of ca. 90 nm, which are decomposed to smaller fragments upon irradiation, presumably due to the radiochemical 12 ACS Paragon Plus Environment

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hydrophilization as confirmed by the CPT change. The effect is similar for irradiation above CPT with smaller doses; however at higher doses a macroscopic precipitate is formed (Table 2). Therefore, at higher irradiation doses, crosslinking prevails (as confirmed by determination of molecular weight, see Figure 2) and the hydrodynamic radius increases again. 2.3 Poly(2-isopropyl-2-oxazoline-co-2-n-butyl-2-oxazoline) (POX) Poly(2-isopropyl-2-oxazoline-co-2-n-butyl-2-oxazoline) (POX) proved to be nearly completely radioresistant up to the highest doses applied, both when irradiated below and above the CPT. There is some minor increase in molecular weight (Figure 2) at the highest irradiation doses leading to a slight decrease in the CPT (Figure 6), but the decrease in CPT is within 2 °C. Therefore, there is no evidence of polymer hydrophilization. The hydrodynamic radius also does not change significantly with irradiation (Figure S7). Therefore, POX seems to be the best polymer for nuclear medicine applications from the perspective of radiation stability. This radiation stability of POX is most plausibly explained by the fact, that the most stable and therefore the most probable radical to be formed is the tertiary one. This sterically demanding radical recombines less likely with another tertiary macroradical of another chain which would cause crosslinking. On the other hand, e.g., for PVCL (see below), the sterically much less demanding secondary radical is formed, considerably increasing the probability of intramolecular chain crosslinking.


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Figure 6: Effect of electron-beam irradiation on transmittance of POX in aqueous solution (cpolymer = 5 mg.mL–1). (A) Polymers irradiated at 2 °C; (B) polymers irradiated at 60 °C.

2.4 Poly(N-vinylcaprolactam) (PVCL) In contrast to POX, PVCL is the polymer most sensitive to irradiation; extensive radiation crosslinking occurs even at the lowest dose applied (1 kGy, Figure 2) leading to a slight decrease of the CPT when the polymer is irradiated both below (Figure 7A) and above the CPT (Figure 7B). Extensive crosslinking is confirmed by steep increase in molecular weight upon irradiation (see Figure 2). The increase in molecular weight generally decreases the CPT until higher molecular weights where the CPT gradually becomes molecular weightindependent. For PVCL, the dependence of CPT on molecular weight is shifted to higher molecular weights compared to other thermoresponsive polymers considered so the PVCL utilized within our study still lies in the area of molecular weight where the CPT is molecular weight-dependent32-34. When irradiated at 2 °C, PVCL formed an opaque hydrogel with doses above 5 kGy (Figure 8) preventing turbidity measurement. Opacity of this hydrogel is presumably caused by heterogeneity in crosslinking, as is often observed for lower concentration hydrogels. However, when irradiated above the CPT, just a precipitate was formed (Table 2). The effect of irradiation on the hydrodynamic radii is shown in Figure S8, 14 ACS Paragon Plus Environment

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i.e., a strong increase in hydrodynamic radius after irradiation even with low radiation doses, supporting the observation of rapid radiation crosslinking. From this vantage point PVCL seems to be inappropriate for the construction of thermoresponsive biomedical systems that should show some robustness against ionizing radiation. However, the newly discovered preparation of PVCL hydrogels by electron-beam irradiation could possibly be further utilized in the construction of advanced thermo-responsive PVCL-based biomaterials.

Figure 7: Effect of electron-beam irradiation on transmittance of PVCL in aqueous solution (cpolymer = 5 mg.mL–1). (A) Polymers irradiated at 2 °C; (B) polymers irradiated at 60 °C.


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Figure 8: Hydrogel formed by irradiation of PVCL solution (cpolymer = 5 mg.mL–1) with 20 kGy at 2 °C.

3. Conclusions Polymer sensitivity to β- radiation doses (0 – 20 kGy) decreases in the order PVCL (the most sensitive) >> DFP > PNIPAM >> POX (the least sensitive, in fact nearly radioresistant within the range of radiation doses studied). Even low doses of radiation (1 kGy), which are highly relevant to storage of radiopharmaceuticals or sterilization of polymers, cause significant changes in the structure of most of the studied polymers, which may severely influence biological behavior of the delivery using these polymers. The most prominent changes include a strong increase in molecular weight due to crosslinking (seen for all polymers except for POX, where this effect is weak) leading in some cases to formation of hydrogels (PVCL) or precipitates. The chain hydrophilization led to significant increases in the cloud point temperature of PNIPAM and DFP. In the case of PVCL the cloud point temperature decreases with increasing radiation dose, due to an increase in molecular weight as a result of crosslinking. From the scope of view of radioresistance, POX is the most suitable polymer for the construction of delivery systems that will be exposed to radiation, PVCL is the least suitable, and PNIPAM and DFP are suitable only for low radiation demands. In the case of the last two, radiation-induced cloud point temperature changes must be taken into account in design of delivery systems.

4. Experimental Section Chemicals: The starting polymers PNIPAM,35 DFP,36 POX (15 mol % 2-butyl-2oxazoline monomeric units according 1H-NMR spectra)37 and PVCL6 were synthesized according to the references, see Table 1. Thiol-containing RAFT end groups of PNIPAM and


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DFP were removed by heating with excess of dibenzoyl peroxide as described earlier.36 All other chemical were purchased from Sigma-Aldrich Ltd. (Prague) and were used as received. Irradiation of polymer samples: In a typical case, the polymer (25 mg) was dissolved in milliQ water (5 mL) in a glass ampoule. The samples were then degassed, covered with argon atmosphere and sealed. Irradiation was performed with MT 25 Microtron instrument (NPI, ASCR v.v.i., Prague) with RF pulse repetition rate of 423 Hz, pulse length 3.5 µs and dose rate 0.5 kGy.min–1. The absorbed dose was measured by a TN34045 ionizing chamber (PTW Freiburg) and by a Keithley 617 programmable electrometer. The polymer samples were irradiated in a rotated target immersed in the thermostatic bath set to either 2 °C or 60 °C. Characterization of polymers: The molecular weights of the polymers were determined by size exclusion chromatography (SEC) using an HPLC Ultimate 3000 system (Dionex, USA) equipped with a GPC column (TSKgel SuperAW3000 150 x 6 mm, 4 µm for POX samples; TSKgel G4000SWxl 300 x 7.8 mm, 8 µm for PNIPAM, DFP and PVCL samples). Three detectors, UV/VIS, refractive index (RI) Optilab®-rEX and multiangle light scattering (MALS) DAWN EOS (Wyatt Technology Co., USA) were employed, and a methanol and sodium acetate buffer (0.3 M, pH 6.5) mixture (80:20 vol. %, flow rate of 0.5 mL/min) was the mobile phase. The apparent chain overlap concentration (c*) was calculated from molecular weight and gyration radius (Rg) assuming non-interacting close-packed hard spheres as described earlier.38 The Rg values of polymers were estimated according to the reference.39 1H NMR and

13

C NMR spectra (600.2 and 150.9 MHz,

respectively) were obtained using a Bruker Avance III 600 NMR spectrometer with CDCl3 as the solvent at 320 K. The chemical shifts are relative to the TMS signal using hexamethyldisiloxane (HMDSO, 0.05 and 2.0 ppm from TMS in 1H NMR and

13

C NMR

spectra) as internal standard. Before the measurement, the irradiated samples were purified by


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chromatography on Sephadex PD-10 column with MilliQ water as an eluent followed by lyophilisation. Dynamic light scattering (DLS) experiments: DLS experiments were conducted using a Flex02-20 autocorrelator (Correlator.com, USA) based setup with 22 mW He–Ne laser (632.8 nm) and PMT detector at a scattering angle 90°. The polymer samples were filtered prior to measurement through 0.45 µm PVDF syringe filter into a 10 mm glass tube. The measured intensity correlation functions g2(t) were analyzed using the REPES algorithm resulting in the distribution of relaxation times A(τ).40 The hydrodynamic radius (Rh) of the nanoparticles was calculated using the relation: Rh=(kBTq2τ)/ (6πη), where kB is the Boltzmann constant, T is the absolute temperature, q is the scattering vector, τ is the mean relaxation time related to the diffusion of the nanoparticles and η is the viscosity of the solvent. Turbidity measurements: The cloud point temperature (CPT) was determined by measuring the change in transmittance of polymer solution as a function of temperature. Transmittance was measured at ߣ = 450 nm, with a UV/VIS spectrophotometer (Thermo Scientific Evolution 220, equipped with a Thermo Scientific Single Cell Peltier element). Temperature was varied with the step 1 °C.min–1.

Supporting Information Supporting Information is available.

Acknowledgements The authors acknowledge the ﬁnancial support from Ministry of Education, Youth and Sports of the Czech Republic (grant # KONTAKT LH14079), Czech Grant Foundation (grant # 1603156S) and Ministry of Health of the Czech Republic (grant # 15-25781a). Partial support from the National Science Foundation through the University of Minnesota MRSEC (DMR18 ACS Paragon Plus Environment

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1420013) is also acknowledged (T.P.L.). The authors are grateful to Dr. David Chvátil and Dr. Pavel Krist from Microtron Laboratory, Nuclear Physics Institute of the ASCR, v. v. i., Rez, Czech Republic for assistance in irradiation experiments.

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