Chemical auxiliary-free polymerization yielding non-linear PEG for protein-resistant application

Abstract

Current polymerization methods require the use of initiators or catalysts as auxiliaries to produce free radicals and then facilitate the chain propagation. However, these residual auxiliaries may affect polymer functions and cause toxicity, therefore, a time-consuming purification process is essential. Recently, synchrotron light has been established for generation of radicals in water without catalysts and initiators for polymerization. In this study, we used intensive synchrotron light as a potential tool to synthesize non-linear polyethylene glycol (NLPEG) which exhibits excellent protein-resistant property as compared to linear PEG (LPEG) on the surface. This novel synthesis concept provides an opportunity to produce NLPEG in a very short time (5 min) without employing various chemicals (pollutants) and with minimum environmental concern.

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1. Introduction

Polymerization is a well-known process in the chemical industry which is used to prepare polymers that can be further manufactured into high-performance polymer-based materials. The process of polymerization is not spontaneous, and initiators that can produce active species to bring about chain growth must be used. Almost all of the current polymerization processes are based on the generation of free radicals from initiators in the presence of external forces such as using catalysts to facilitate a cascade of new bond formations between repeating monomers.¹

Many efforts from both academic and industrial scientists have focused on the development of various catalysts to improve the activation efficiency of initiators.^2–4 However, such catalyst residues can make polymers less functional and more toxic. Additionally, the current polymerization strategies threaten global ecosystems because of the consumption of large amounts of organic solvents and reagents. This consumption prevents polymerization processes from meeting the requirements of green chemistry and limits the use of polymers in biological applications. It is essential to develop a novel approach to surpass these conventional polymerizations for the preparation of truly biocompatible polymers. In our previous studies, a fast and one-pot synthetic strategy based on synchrotron light (4–30 keV, 10⁵ Gy s⁻¹), which is capable of generating free radicals from water in the absence of catalysts and initiators,⁵ have been demonstrated to synthesize polyethyleneimine-based polymers for siRNA delivery.^6,7 Such one-pot synthetic strategy is not only simple but also shortens the reaction time from several days (traditional protocol is a stepwise polymerization) down to several minutes. Here, we re-emphasize that the simple and chemical auxiliary-free strategy can also be used for the preparation of PEG with a non-linear structure.

PEG is commonly used as a coating material to overcome nonspecific protein adsorption by forming an impenetrable barrier on the surface of bulk-scale implants and nanoparticle payloads. Such a barrier decreases the accumulation of blood proteins, which can cause adverse side effects such as thrombosis and severe inflammation.^8–21 Additionally, the PEG can also prolong the blood circulation time of nanoscale biomaterials and reduce the uptake of nanoparticles by the reticuloendothelial system, thus improving tumor-targeting efficiency.^22–25 The efficiency of the protein resistance of PEG depends strongly on the chain-packing density, the thickness of the PEG layer and the structures of the PEG.^19,26–40 Presumably, surface decoration with high density, thick PEG layer or PEG brush (one of NLPEGs was constructed from LPEG by several chemical reactions) could provide more hydrogen bonds for water molecules as compared to low-density coating coverage or LPEG.²⁸ In conventional protocols, a stepwise polymerization process and subsequent grafting reactions are performed with large amounts of catalysts and organic solvents to prepare LPEG, and then LPEG is converted into NLPEG with comb-like or star-like structures.^41–43 To decrease the environmental impact and improve biocompatibility, it is essential to develop a clean approach to fit these requirements.

In this study, the PEG monomer, ethylene glycol, was dissolved in water, and then the solution was irradiated with synchrotron light for approximately 5 min to generate NLPEG with monodispersive molecular weights. The power of synchrotron light can induce the radiolysis of water to generate radical intermediates⁴⁴ for facilitating subsequent polymerization; the possible mechanism of this process is presented in Scheme 1. The NLPEGs contained CH and CH₂ in a ratio of approximately 1 : 12 ± 3 and the molecular mass varied from 2 to 25 kDa depending on the concentration of the monomer and reaction time. Importantly, our NLPEGs were prepared by one-step synthesis, no safety concerns for any part of the reaction process, and not associated with bovine serum albumin (BSA, a common protein). This novel strategy demonstrated that synchrotron light is a powerful tool to simply prepare NLPEGs in a simple process without using extra chemical auxiliaries. The process not only has a minimal environment impact but also truly improves the biocompatibility of the final polymers.

Scheme 1 Possible proposed polymerization mechanism of NLPEGs after irradiation with synchrotron light.

2. Results and discussion

Synchrotron light generates free radicals for polymerization applications. A simple procedure was used in this study, wherein an ethylene glycol monomer was introduced into DI water before being irradiated with synchrotron light. After being irradiated for about 5 min, the excess monomers were removed by freeze drying, resulting in a pale-yellow product. The structure of the product was further identified by various technologies including FT-IR, ¹H NMR and ¹³C NMR in combination with distortionless enhancement by polarization transfer spectroscopy (DEPT, three angles—45, 90 and 135°—were used, denoted as DEPT-45, DEPT-90 and DEPT-135, respectively) and an inverse gated (IG) ¹³C NMR experiment.

First, presented in Fig. 1 is a typical transmission spectrum of our pale-yellow product that was prepared in a KBr pallet. The broad band appearing at 3124 cm⁻¹ can be assigned to the stretching mode of the hydroxyl group, which was expected to originate from one of the terminal groups in the new product. The peaks at 2939 and 2883 cm⁻¹ can be assigned to ν_a(CH₂) and ν_s(CH₂), respectively, illustrating the presence of methylene groups in the pale-yellow product. In addition, two bands at 1041 and 1081 cm⁻¹ can be attributed to ν_str(CO), indicating the presence of C–O bonds in the new compound.

Fig. 1 Transmission IR spectrum of NLPEG. Characteristic assignments: O–H stretches of NLPEG were located at 3124 cm⁻¹ and C–H bending vibration was located at 1460 cm⁻¹. The adsorption at 1041 cm⁻¹ and 1081 cm⁻¹ can be attributed to the C–O stretch from the different side chain and main chain, respectively.

Second, the ¹H NMR spectra of the new product (Fig. S1)† exhibit multiple peaks from 3.6 to 3.8 ppm, which can be assigned to the protons on the carbon atoms adjacent to the oxygen atoms. This assignment illustrates that the structure of the products is very similar to those of ethylene glycol and commercial LPEG, except that the splitting pattern has a large difference. Third, ¹³C NMR in combination with DEPT is a well-established method to distinguish methylene carbon (CH₂) from methine carbon (CH). Fig. 2 depicts a discernible difference between commercial LPEG and our PEG-like product. (1) The DEPT-45 spectrum of our product (Fig. 2b, upper panel) exhibits two peaks at 62.4 and 71.4 ppm, in contrast with only one peak at 71.5 ppm in commercial LPEG (Fig. 2a, upper panel). This observation indicates that the PEG-like product has two kinds of carbon atoms adjacent to oxygen atoms. (2) The DEPT-90 spectrum (Fig. 2b, middle panel) depicts a single peak at 71.4 ppm, which is not observed in the commercial LPEG (Fig. 2a, middle panel), indicating that the structure of the new product contains a tertiary carbon-oxide motif (CH–O) but no similar signal from the commercial LPEG. (3) The DEPT-135 spectrum (Fig. 2b, lower panel) shows two signals in the up and down directions that can only be observed from the new product and not from the commercial LPEGs, confirming that the structure of the new product was non-linear and comprised of CH–O and CH₂–O groups. On the contrary, none of these CH–O signals was observed in commercial LPEG.

Fig. 2 ¹³C NMR combined with DEPT spectra of (a) commercial LPEGs and (b) our NLPEGs.

In order to further verify the ratio of CH/CH₂ in the NLPEG, an inverse gated (IG) ¹³C NMR experiment was performed.⁴⁵ The IG ¹³C NMR spectrum (Fig. S2)† depicts the integration of CH and CH₂ signals from the NLPEG. The ratios of CH/CH₂ were estimated to be approximately 1 : 12 ± 3 and the degrees of branching (DB) ranged from 0.118 to 0.182; the former parameters were dependent on the radiation intensity during the reaction. This finding re-confirmed that the new product is a typical non-linear PEG polymer. In addition, the weight-averaged molecular mass (M_w) was estimated by size-exclusion chromatography coupled with a multi-angle light scattering (SEC-MALS), the M_w of the NLPEGs was 2 and 25 kDa depending on the reaction time, and the polydispersity index (PDI) ranged from 1.09 ± 0.48 to 1.45 ± 0.29 and was dependent on the reaction time during the irradiation of synchrotron light (2 kDa for 5 min and 25 kDa for 10 min). The M_w can be gradually increased by prolonging the reaction to 10 min; however, after 20 min, a gel-like polymer was formed. Moreover, the M_w and PDI were not affected by being stirred or not during the light irradiation.

The NLPEGs were further examined by elemental analysis, and only three elements including C, O, H could be detected, re-confirming the aforementioned characterization by NMR. The weight ratio of C/O in the NLPEG was roughly estimated to be 1.35, which is less than 1.72 in commercial LPEG. This analysis re-confirms that our NLPEG has a completely different structure in comparison with the commercial LPEG. The repeating units, including the x, y, z and n of our NLPEGs, were estimated and listed in Table 1. First, to estimate these parameters including x, y, z and n of the NLPEG with 2 kDa, the x value (Table 1, column 5) was denoted either 1 or 2 to allow the n value to be maximized (Table 1, column 9), regardless whether the ratio of CH/CH₂ (Table 1, column 3) was an odd or even number. Second, the sum of y and z was an integer (Table 1, column 8) and the possible values of y and z were estimated to be in a narrow range, as shown in columns 6 and 7. The estimation could also be used for NLPEG with 25 kDa, all parameters were like that with 2 kDa, but the n number (Table 1, column 10) had a significant increase. The detailed calculation was described in the ESI.† To separate the y value into two conditions, y ≠ 0 and y = 0, the comb-like structures of NLPEG in are presented in Chart 1.

Chart 1 Proposed conformations of our NLPEG based on the predictions in Table 1. For simplification, these conformations only show that two conditions at a ratio of CH/CH₂ were 1 : 9 and 1 : 10, respectively.

Table 1 Various estimated parameters of the prepared NLPEG

M w	Reaction time	Ratios of CH/CH2	Degree of branching	Repeat units (x, y, z, n)^a
				x	y	z	y + z	n 1	n 2
a The x, y, z and n are integers: the calculations of these parameters were based on the ratios of CH/CH2 and that the x and n had supposed minimum and maximum value, respectively. For illustrative purposes, the x, y, z and n values were estimated as follows with Mw = 2 kDa as an example: with a ratio of CH/CH2 = 1 : 12, y + z = 11x/2, thus, if x = 1, then y + z = 5.5 (non-integer, being culling) and n = 3 (maximum), and if x = 2, then y + z = 11 and n = 3. With a ratio of CH/CH2 = 1 : 13, y + z = 6x; thus, if x = 1, then y + z = 6 and n = 6 (maximum) and so on. The values of n1 (in column 9) and n2 (in column 10) were presented from the NLPEG with 2 kDa and 25 kDa, respectively.
2 kDa (25 kDa)	5 min (10 min)	1 : 9	0.182	1	0∼3	4∼1	4	8	106
		1 : 10	0.167	2	0∼7	9∼2	9	4	50
		1 : 11	0.154	1	0∼4	5∼1	5	7	89
		1 : 12	0.143	2	0∼9	11∼2	11	3	43
		1 : 13	0.133	1	0∼5	6∼1	6	6	77
		1 : 14	0.125	2	0∼11	13∼2	13	3	37
		1 : 15	0.118	1	0∼6	7∼1	7	5	68

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We attempted to resolve these conformations using atomic force microscopy (AFM), but the resolution of AFM was limited due to the NLPEG being hardly adsorbed on mica substrate for the tip scanning. Instead, the SEC-MALS, a well-known technology to provide information on the radius of gyration (r_g) and the molecular mass,⁴⁶ was employed as another alternative tool to characterize the conformation of the NLPEG. Fig. 3a (blue line) depicts a conformation plot of log r_gvs. log M_w for our NLPEG and two other well-defined polymers, including the commercial LPEG (Fig. 3a, black line) and sphere-like dendrimers (Fig. 3a, red line). Their slopes from S₁ to S₃ were 0.67 ± 0.01, 0.99 ± 0.02 and 0.3 ± 0.09, respectively. According to previous reports,^46,47 the slope values are 0.33, 1 and ∼0.5–0.6, which can be correlated to three kinds of structures—spheres, rigid rods and random coils, respectively. As expectation, the two model compounds were 0.3 ± 0.09 (very close to 0.33) and 0.99 ± 0.02 (very close to 1) respectively, indicating that their conformations are spherical (dendrimers) and rod-like (LPEGs). In this study, the slope value (0.67 ± 0.01) of our NLPEGs was approximately equal to 0.5∼0.6, indicating that its conformation is close to a random coil. Additionally, the results demonstrate that our NLPEGs is well-dispersed in water, rather than aggregative. This observation may illustrate that the repeating units of our NLPEG have an anisotropic growth.

Fig. 3 (a) Conformation plot obtained by fitting the raw data, including S1 (blue line), S2 (black line) and S3 (red line) for NLPEG, commercial LPEG and sphere-like dendrimers, respectively. (b) Lyophilized samples of NLPEG (1), molecular weight 0.6 kDa (2), 1 kDa (3), 1.5 kDa (4) and 6 kDa (5) of commercial LPEGs. (c) The experiment of protein resistance was performed by adsorption of FITC conjugated BSA on SiN wafer with pre-grafted various polymers from left to right, including: SiN alone, PDMS, LPEG (1 kDa), LPEG (6 kDa) and our NLPEG (2 kDa). To compare the images, the fluorescence intensity of our NLPEG is closer to the background intensity (the black area of each picture).

Moreover, we found that our NLPEGs (M_w = 2 kDa) had liquid-like morphology (Fig. 3b, vial 1). However, very similar morphology is only observed from the LPEG with M_w smaller than 0.6 kDa (Fig. 3b, vial 2). Furthermore, the protein repellent property of our NLPEG was verified by a hydrophilic silicon nitride wafer (SiN) which was various pre-modified polymers including polydimethylsiloxane (PDMS), two sets of LPEG and NLPEG. Fig. 3c shows several images with a strong fluorescence signal from FITC conjugated BSA on these samples including the SiN-alone (image 1), SiN/PDMS (image 2), SiN/LPEG (1 kD) (image 3) and SiN/LPEG (6 kD) (image 4). Note that the chain overlapping of coating LPEG (6 kDa) may be lower than that of coating LPEG (1 kDa) leading to decrease of protein-resistant efficiency, which is consistent with a previous report.⁴⁸ In contrast, a weak fluorescence signal can only be observed from SiN/NLPEG (image 5). It is noteworthy that our NLPEG coating has excellent BSA-resistant property as well as that of commercial star-PEG coating (Fig. S4).† To test a lysozyme (14.6 kDa) with lower M_w than BSA (66 kDa), Fig. S5 shows that our NLPEG can repel lysozyme as well as those of LPEG and commercial star-PEG.† This result illustrates that our NLPEG has an excellent protein-resistant property as compared to LPEG.

Next, our NLPEGs were further examined for toxicity by the cell culture. Fig. 4 shows a comparison of biocompatibility between our NLPEG, a commercial LPEG and an as-prepared star-PEG (the synthetic method was given in ref. 33). The significant toxicity (Fig. 4e, column 4 and column 9) was caused by the as-prepared star-PEG prepared by other methods while the residual chemicals have not been removed for the following cell culture. On the contrary, our NLPEG (Fig. 4e, column 5 and 10) as well as the commercial LPEG (Fig. 4e, column 3 and 6) were non-toxic even at high dosage (100 μg mL⁻¹). To monitor the cell morphology and proliferation, no subtle change can be observed from Fig. 4a–4d. This indicated that our synthesis strategy to prepare the NLPEG did not produce any toxic products after the reaction, even no further purification.

Fig. 4 The cell morphology and proliferation were examined after treatment for 72 h in the absence or presence of commercial LPEG, as-prepared star-PEG and our NLPEG. (a)-(b) H460 and (c)-(d) A549 presents both cells without any treatment and with 100 μg ml⁻¹ NLPEG treatment, respectively. (e) Analysis of a cell proliferation assay from two kinds of lung cancer cell lines in the various PEG treatments. The data shown in column 1/6 and 2/7 present positive and negative control experiments, respectively.

3. Experimental

3.1 Synthesis of NLPEG

Ethylene glycol was purchased from Sigma-Aldrich and no additional purification was performed prior to use. To synthesize the NLPEGs, 100 μL of ethylene glycol was added to 5 mL of water (18 MΩ cm⁻¹) and then this reactant solution was irradiated with synchrotron X-rays (4–30 keV, 10⁵ Gy s⁻¹) at room temperature for 5 min. The final products were lyophilized to remove residual monomers.

3.2 Instruments and measurements

The NLPEGs were characterized using FT-IR (Jasco FT/IR-4200), ¹H NMR and ¹³C NMR (Varian 400 MR). All NMR spectra were obtained from the lyophilized sample which had been redissolved in deuterium oxide. The IR spectrum of NLPEG was obtained with a KBr-pelleted sample. The molecular weight and conformation of the NLPEGs were determined by multi-angle light scattering (MALS). A MALS (miniDAWN, Wyatt) module was coupled to an HPLC system (Agilent 1100 Infinity LC) with a size exclusion column (Biosep-SEC-S2000, Phenomenex). The NLPEGs solution was injected with 1× phosphate buffered saline as eluent at a 1 mL min⁻¹ flow rate.

3.3 Evaluation of anti-protein adhesion

All chips were cut from a silicon nitride wafer (SiN), cleaned by piranha solution (concentrated H₂SO₄/30% H₂O₂ = 1 : 1, v/v) at room temperature for 30 min and rinsed thoroughly with Millipore-Q water prior to use. The PDMS coating layer was made by the deposition of pre-mixed PDMS gels on the clean SiN, which was composed of Sylgard 184 polymer base and its curing agent in a ratio of 10 : 1, (w/w). The coating PDMS chip was degassed in vacuum and curried at 65 °C for 1 h. All PEGs coating layers such as LPEG and our NLPEG were fabricated by the deposition of PEG aqua (1 mg/10 μL) on SiN and heated at 100 °C in an oven for 42 h.⁴⁹ All coating samples were further cleaned by sonication for 5 min in a water bath to remove the non-conjugated polymers. The surfaces were dried and stored at room temperature. Protein resistance was performed by immersing the chips in a 0.5 mg ml⁻¹ of FITC-BSA solution at 37 °C for 1 h and then washing the chips with PBS solution. The degree of protein resistance was analyzed in an image system (FluorChem FC2, Alpha Innotech) with excitation at 470 nm and emission at 525 nm for detecting FITC-BSA on surfaces.

3.4 Evaluation of Bio-safety for the NLPEG

The in vitro biocompatibility of the NLPEGs was evaluated by using a cell proliferation assay toward A549 cell and H460 cell. A549 cell (from BCRC, the Bioresource Collection and Research Center, Taiwan) were cultured in F12K Nutrient Mixture and H460 cells (from BCRC) in RPMI 1640 medium (both media were supplemented with 10% fetal bovine serum) under 5% CO₂ atmosphere at 37 °C. These cell lines were inoculated with 1 × 10⁵ cells/well into a 6-well cell-culture plate and cultivated for 24 h at 37 °C. Then the culture medium was changed to different treatment (Arabinosylcytosine, AraC, a nucleotide analogue which can inhibit nuclear DNA synthesis, 10 μg mL⁻¹ as negative control, no treatment as positive control, commercial LPEG(1 kD) 100 μg mL⁻¹, as prepared star-PEG (catalyst was still inside) 100 μg mL⁻¹ and NLPEG 100 μg mL⁻¹, respectively) in a culture medium (three replicates). The tests were performed for 3 d without changing the medium. The proliferation assay was administered with Trypsin/EDTA solution to determine the number of cells growing in the absence or presence of NLPEGs.

4. Conclusion

In conclusion, we reported a simple and chemical auxiliary-free strategy by using synchrotron light for the synthesis of a novel NLPEG. The intensive light allows an anisotropic chain-propagation of a single monomer via an efficient radical reaction within 5 min. The resulting PEG had non-linear conformation to act as excellent protein-resistant material and none of prerequisite purifications need be used in the following biological experiments unlike other PEGs from the multiple-step synthesis. This simple, chemical auxiliary-free polymerization strategy does not employ or produce any toxic chemicals and is environmentally friendly, which makes this process potentially useful for future biological applications.

Acknowledgements

This work was supported by grants from the National Health Research Institutes of Taiwan (NHRI-NM-100-PP-10) and the National Science Council (NSC-98-2113-M-400-002). Dr Chih-Te Chien contributed.

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Footnote

† Electronic supplementary information (ESI) was available from ¹H NMR spectra and detailed structure analysis. See DOI: 10.1039/c2ra20117h

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