An injectable PEG-based hydrogel synthesized by strain-promoted alkyne–azide cycloaddition for use as an embolic agent

Abstract

With PEG as a macromolecular initiator, cyclooctyne and azide functionalized PEGs were conveniently prepared by the ring-opening polymerization of cyclooctyne-bearing epoxy monomer and azide-bearing cyclocarbonate monomer, respectively. Via the strain-promoted alkyne–azide cycloaddition (SPAAC) reaction of cyclooctyne and azide groups, the two PEG polymers formed a hydrogel in a few minutes upon simply mixing under physiological conditions. The formation, degradation, and biocompatibility of the hydrogel were investigated in vitro and in vivo. Injection of a mixture of the gel precursors into the auricular central artery of rabbits blocked the fast-flow of the vessel rapidly without any invasive operation, and the vessel flow restored spontaneously by gel degradation in two days. This kind of injectable hydrogel would be useful for staunching wounds and blocking blood vessels temporarily.

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Introduction

Though the Cu-catalyzed azide–alkyne click reaction has a number of advantages,^1–4 the copper catalyst has certain biological toxicity in certain cases.^5–7 Copper-free click reactions, such as the thiol-ene reaction,^8,9 Diels–Alder reaction,^10–12 and strain-promoted alkyne–azide cycloaddition (SPAAC) reaction^13–15 were developed in order to perform benign bio-orthogonal reactions in vivo without Cu catalysts. Bertozzi^16–18 et al. proved that the ring tension of cyclooctyne can accelerate the rate of copper-free azide–alkyne [3 + 2] cycloaddition.¹⁹ Injectable hydrogels formed via the sol–gel phase transition caused by solvent exchange, UV-irradiation,²⁰ ionic cross-linkage, pH change,^21,22 and temperature modulation,²³ are widely applied in tissue engineering,^24,25 medical devices, embolization, etc.^26–29 Poly(ethylene glycol) (PEG) hydrogels formed by thiol-ene photo-click chemistry have great biocompatibility proved by biological experiments.³⁰ The direct encapsulation of cells within click hydrogels was achieved by a robust synthetic strategy where macromolecular precursors react through a copper-free click chemistry.³¹ PEG-based microgels for bioorthogonal encapsulation and pH-controlled release of living cells were constructed by SPAAC.³² Though the biocompatibility and applications of SPAAC-crosslinked hydrogels have been well demonstrated by cell culturing in vitro in the literature, related in vivo studies are rare.³³ Herein, we report a convenient synthesis of multi-cyclooctyne and multi-azide functionalized PEGs by the ring-opening polymerization of cyclooctyne-bearing epoxy and azide-bearing cyclocarbonate monomer, respectively, with PEG as a macromolecular initiator (Scheme 1). By this ring-opening polymerization approach, the number of the functional groups would be conveniently adjustable. As injectable hydrogel precursors, these functionalized PEGs react with each other forming biocompatible and biodegradable hydrogel in vivo rapidly. The hydrogel was tested as embolic agents in blood vessel of rabbit ears to block the blood temporarily and the obstructed vessel restore in 2 days due to the biodegradation of the hydrogel.

Scheme 1 Synthesis of gel precursors CO-PEG and azide-PEG and a schematic presentation for the formation of the hydrogel.

Experimental section

Chemicals and materials

THF was distilled over Na–K alloy in the presence of benzophenone before use. Dichloromethane was dried over CaH₂ and distilled prior to use. Poly(ethylene glycol) (PEG4000, M_n = 4000, PDI = 1.06, Sigma-Aldrich) was purified by precipitation from petroleum ether prior to use. Potassium naphthalenide (0.89 M solution in THF) was prepared by mixing naphthalene (3.0 g) and potassium (0.92 g) in THF (26 mL) in a flask. Stannous octoate [Sn(Oct)₂, 95%] was purchased from Aldrich, purified by distillation under reduced pressure prior to use. 2,2-Bis(azidomethyl)trimethylene carbonate (5)³⁴ and 8,8-dibromobicyclo[5.1.0]octane³⁵ were synthesized as described in the literature. Other reagents were purchased from Sinopharm Chemical Reagent in China and used as received.

Characterizations

NMR spectra were obtained using a Mercury VX-300 spectrometer in CDCl₃ or D₂O. Chemical shifts are calibrated using residual solvents signals (CDCl₃: δ (H) = 7.26, δ (C) = 77.16) or using tetramethylsilane (TMS) as a reference. Mass spectra were recorded on ESI-TOF Mariner spectrometer (Perspective Biosystem). FT-IR spectra were obtained by a Perkin-Elmer spectrometer. The experiments involving live animals were performed in compliance with the guidelines for the care and use of animals established by Wuhan University. The experimental protocol of the study was approved by the Ethics Committee of Wuhan University.

Synthesis of 2-(2-bromocyclooct-2-enyloxy)ethanol (2)

To a dry 100 mL flask equipped with an stirrer was added 8,8-dibromobicyclo[5.1.0]octane (1) (5.00 g, 18.7 mmol), ethanediol (10.0 g, 161 mmol), AgClO₄ (11.6 g, 56 mmol, 3 eq.) and 50 mL of anhydrous dichloromethane under Ar atmosphere. The reaction was stirring at 0 °C in dark for 2 h and then terminated by the adding of HCl (100 mL, 1 M). The insoluble silver salt was filtered off. The organic layer was separated and washed with 50 mL of saturated sodium bicarbonate solution and saturated NaCl solution, successively. The organic layer was concentrated and purified by silica gel chromatography with 10–20% EtOAc in petroleum ether as an eluent to yield 3.23 g (70% yield) of light yellow oil (R_f = 0.25, EtOAc : petroleum ether 1 : 5). ¹H NMR (CDCl₃, 300 MHz, TMS): δ 6.20 (dd, 2H, J = 3.3, 11.4 Hz), 3.90 (q, 1H, J = 4.8 Hz), 3.78 (s, 1H), 3.64 (s, 1H, J = 4.8 Hz), 3.46 (s, 1H, J = 4.8 Hz), 2.74 (m, 1H), 2.73 (dq, 1H, J = 4.8, 12.0 Hz), 2.30 (m, 1H), 2.06–1.83 (m, 4H), 1.75 (m, 1H), 1.49 (app dq, 1H, J = 3.6, 12.6 Hz), 1.30 (m, 1H), 0.82 (m, 1H); ¹³C-NMR (CDCl₃): δ 134.8, 127.7, 76.3, 70.2.

Synthesis of 2-(cyclooct-2-ynyloxy)ethanol (3)

A solution of 2-(2-bromocyclooct-2-enyloxy)ethanol (2) (3.00 g, 12.1 mmol) and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) (5.50 g, 36.3 mmol) in 10 mL of anhydrous DMSO in a 100 mL round-bottom flask was stirred at 60 °C for 2 h. More DBU (18.4 g, 121 mmol) was added and the solution was stirred at 60 °C overnight. Dichloromethane (20 mL) and hydrochloric (50 mL, 1 M) were added to the reaction mixture to neutralize the basic solution. The mixture was transferred to a separatory funnel and the organic layer was separated. The organic layer was washed with 50 mL of saturated sodium bicarbonate solution and saturated sodium chloride solution, successively. The organic layer was concentrated and purified by silica gel chromatography with 5–15% EtOAc in petroleum ether as an eluent to yield 1.47 g (72% yield) of light yellow oil (R_f = 0.25 on silica TLC plate, petroleum ether containing 12% of EtOAc). FT-IR: 2205 cm⁻¹ (C≡C); ¹H NMR (CDCl₃, 300 MHz, TMS): δ 4.22 (q, 1H, J = 5.4 Hz), 3.73 (m, 1H), 3.69–3.64 (m, 2H, J = 5.4 Hz), 3.46 (dq, 1H, J = 6 Hz), 2.30–2.26 (m, 1H), 2.22–2.13 (m, 2H), 2.08–2.03 (m, 1H), 1.97–1.92 (m, 1H), 1.89–1.83 (m, 2H), 1.73–1.62 (m, 2H), 1.49–1.39 (m, 1H); ¹³C-NMR (CDCl₃): δ 100.6, 92.8, 73.0, 70.7, 62.0, 42.5, 34.5, 29.9, 26.5, 20.9; ESI MS: m/z = 203.1 [M + Cl]⁻.

Synthesis of 2-((2-(cyclooct-2-ynyloxy)ethoxy)methyl)oxirane (4)

A mixture of 3 (1.00 g, 5.95 mmol) and NaH (0.36 g, 60% suspension in oil) in 50 mL of anhydrous THF was stirred for 4 h in a 100 mL round-bottom flask under argon. Then, epichlorohydrin (5.51 g, 59.5 mmol) was added. The reaction mixture was stirred for 2 h at room temperature and at refluxing temperature overnight. After being cooled to room temperature, the reaction mixture was filtered to remove insoluble salts. The filtrate was concentrated and purified by silica gel chromatography with 10–15% EtOAc in petroleum ether as an eluent to yield 0.98 g (73% yield) of light yellow oil (R_f = 0.3 on silica TLC plate, petroleum ether containing 15% of EtOAc). FT-IR: 2205 cm⁻¹ (C≡C), 910 cm⁻¹ (CHOCH₂); ¹H NMR (CDCl₃, 300 MHz, TMS): δ 4.25–4.20 (t, 1H), 3.82–3.64 (m, 4H), 3.53–3.39 (m, 2H), 3.16 (d, 1H), 2.79 (t, 1H), 2.61 (t, 1H), 2.30–2.26 (m, 1H), 2.22–2.13 (m, 2H), 2.08–2.03 (m, 1H), 1.97–1.92 (m, 1H), 1.89–1.83 (m, 2H), 1.73–1.62 (m, 2H), 1.49–1.39 (m, 1H); ¹³C-NMR (CDCl₃): δ 99.8, 92.5, 72.5, 71.7, 71.6, 70.3, 68.2, 61.3, 45.5, 44.0, 42.0, 34.0, 29.5, 26.1, 20.4; ESI MS: m/z = 259.1 [M + Cl]⁻.

Synthesis of the cyclooctyne-modified poly(ethylene glycol) CO-PEG

In a 100 mL dry flask was added dry PEG4000 (2.00 g, 0.500 mmol) and 5 mL of anhydrous THF under inert atmosphere. Potassium naphthalenide (3.0 mL of 0.89 M solution in THF) was added via a syringe. After the mixture was stirred for 15 min while the dark green color persisted, 4 (900 mg, 4.02 mmol) was added via a syringe. The reaction solution was stirred at room temperature overnight and then the reaction was terminated by adding HCl (0.80 mL, 12 M) drop by drop. The mixture was evaporated to dryness in vacuum. The residue was dissolved in 5 mL of THF, and the undissolved inorganic salt was removed by filtration. The copolymer was precipitated from the filtrate by adding diethyl ether. The precipitate was collected by filtration, dissolved in THF and precipitated again by adding diethyl ether. A light yellow precipitate was collected by filtration and dried under vacuum at 40 °C for 48 h, affording 1.23 g of white powder.

Synthesis of the azide-modified poly(ethylene glycol) azide-PEG

PEG4000 (2.0 g, 0.50 mmol) was dried in vacuum for 8 h at 120 °C in a 25 mL flask. The flask was charged with Ar. 2,2-Bis(azidomethyl)trimethylene carbonate (5) and Sn(Oct)₂ were added in the flask at a [PEG4000] : [5] : [catalyst] molar ratio of 1 : 8 : 0.008. The mixture was stirred at 120 °C for 24 h under Ar atmosphere, and the reaction was terminated by cooling down to room temperature. The crude product was dissolved in 5 mL of chloroform. Trace of precipitate was removed by filtration, and the copolymer was precipitated from the filtrate by adding diethyl ether. The white precipitate was collected by filtration, redissolved in chloroform and precipitated by adding cold methanol. The precipitate was collected and dried under vacuum at 40 °C for 48 h, affording 1.56 g of white powder. FT-IR (KBr): 2108 cm⁻¹ (N₃), 1750 cm⁻¹ (C=O); ¹H-NMR (CDCl₃, 300 MHz, TMS): δ 4.10 (br s), 3.85–3.50 (m), 3.48 (br s).

Formation and characterization of the hydrogel

The formation of the hydrogel was tested by mixing the solutions of azide-PEG and CO-PEG at various concentrations (5 wt%, 10 wt%, and 15 wt% in PBS, pH 7.4) in a 5 mL glass tube at 37 °C. The two polymers were mixed at various mass ratios (4 : 1, 2 : 1, 1 : 1, 1 : 2, or 1 : 4), with a fixed total volume of 1.0 mL. A tube-inversion method was used to determine the gelation time. As an example, the gel with a total concentration of 10 wt% at a CO-PEG/azide-PEG ratio of 2 : 1 had a gelation time of 5 minutes.

The morphology of the hydrogel was observed with a Zeiss SIGMA field-emission scanning electron microscope. The samples were freeze-dried under vacuum and then conductively coated with gold.

Oscillatory rheology

The formation of hydrogels was monitored through the evolution of storage (G′) and loss (G′′) moduli with the reaction time at 37 °C by dynamic rheological measurements (strain of 1% and frequency of 1 rad s⁻¹). Gelation time was defined as the cross point of G′ and G′′ curves.

Determination of in vitro cytotoxicity by MTT assay

The cytotoxicity of CO-PEG and azide-PEG was evaluated by MTT assay on L929 cells. L929 cells were seeded in 96-well plates at an initial density of 5000 cells per well in 100 mL of RPMI 1640 complete medium. The cells were allowed to grow for 24 h. The two block polymers solutions were added to the media. Each dosage was replicated in 4 wells. Treated cells were incubated at 37 °C under a humidified atmosphere of 95% air and 5% CO₂ for 24 h. MTT reagent (20 μL in PBS, 5 mg mL⁻¹) was added to each well, and the cells were incubated for 4 h at 37 °C. The liquid in each well was removed and 150 mL of DMSO was added to each well to dissolve the crystals. The absorbance at 570 nm in each well was recorded using a Multiskan Go spectrophotometer (Thermo Scientific). Cell viability was calculated according to the following equation: cell viability (%) = (OD_sample − OD_blank)/(OD_control − OD_blank) × 100, where OD_sample is the absorbance of the solution of the cells cultured with the medium only.

In vivo hydrogel formation and biocompatibility studies

A mixed solution of azide-PEG/CO-PEG (0.5 mL, 15 wt% in pH 7.4 PBS) was injected subcutaneously at 37 °C in Sprague-Dawley rats. To observe the gelation of the injected solution, one of the animals was anesthetized 15 min after the injection. The skin of the injection site was carefully incised and the in situ formed gels were observed and photographed. For 3 sequential days after the injection, two rats each day were sacrificed. At sacrifice, the hydrogel and surrounding tissues were isolated, fixed in 4% paraformaldehyde. Then they were embedded in paraffin and sectioned to a thickness of 4 μm. The sections were stained with hematoxylin–eosin (H&E) reagent and examined under an inverted fluorescence microscope. The remained rats were kept for 2 weeks to observe the effects of the hydrogels.

Vessel embolization

New Zealand rabbits (1.8–2.5 kg) were purchased from The Experimental Animal Center of Wuhan University. A mixed solution of azide-PEG/CO-PEG (0.25 mL, 15 wt% in pH 7.4 PBS) was injected to the auricular central artery of the rabbits. For comparison, a solution of PEG4000 (15 wt% in pH 7.4 PBS) was used as a control. After the injection, the rabbits were maintained for 7 days under normal conditions. The changes in color and shape of their ears were inspected and recorded by photography.

Results and discussion

Scheme 1 shows the preparation of the hydrogel. 2-(2-Bromocyclooct-2-enyloxy)ethanol (2) was prepared in 70% yield through the ring opening of 8,8-dibromobicyclo[5.1.0]octane (1) with ethylene glycol in the presence of silver perchlorate in darkness. Elimination of HBr induced by 8-diazabicyclo[5,4,0]-7-undecene (DBU) converted 2 to 3-(hydroxyethoxy)cyclooctyne (3) in 72.3% yield. The reaction of 3 with NaH and epichlorohydrin (ECH) afforded 2-((2-(cyclooct-2-ynyloxy)ethoxy)methyl)oxirane (4) in 73% yield. Cyclooctyne-modified PEG (CO-PEG) was synthesized by anionic ring-opening polymerization of 4 with PEG (PEG4000, M_n = 4000) as a macromolecular initiator which was pretreated with potassium naphthalenide (K-Naph). By this ring-opening polymerization approach, the number of cyclooctynyl groups attached to the PEG chain would be adjusted by simply changing the feed ratio of 4/PEG. In addition, cyclooctyne derivative 4 might be useful in further research for attaching cyclooctynyl groups to biomolecules owing to the high reactivity of the oxirane group with amino groups. On the other hand, azide-modified PEG (azide-PEG) was synthesized by the ring-opening polymerization of 2,2-bis(azidomethyl)trimethylene carbonate (5) with PEG4000 as a macromolecular initiator and Sn(Oct)₂ as a catalyst by a procedure similar to the previous work.¹⁸ Based on the ¹H NMR spectra (Fig. 1), the average degrees of polymerization of cyclooctyne-oxirane 4 and diazidocarbonate 5 of the obtained copolymers are 3.4 and 4.2, respectively. Therefore, each CO-PEG macromolecule contains 3.4 cyclooctyne groups and each azide-PEG contains 8.4 azide groups on average. Once being mixed in an aqueous solution, the two linear copolymer precursors CO-PEG and azide-PEG cross-link to form a hydrogel via the SPAAC click reaction.

Fig. 1 ¹H NMR spectra of gel precursors azide-PEG and CO-PEG (300 MHz, CDCl₃).

The cytotoxicity of precursors CO-PEG and azide-PEG was evaluated on the basis of MTT assay on L929 cells (Fig. 2). In the tested concentration range of up to 2 mg L⁻¹, the cell viability was elevated slightly by the presence of both precursor polymers, in comparison with that in the blank medium. It suggests that the two gel precursors have excellent cytocompatibility or even enhance the cell proliferation.

Fig. 2 Relative cell viability of L929 cells at 24 h after the addition of the polymers.

Solutions of azide-PEG and CO-PEG in PBS (pH = 7.4) at a concentration of 15 wt% were mixed to form hydrogels at 37 °C (Fig. 3a). The volume of CO-PEG solution was twice of the azide-PEG solution. After the hydrogels were shaped, the resultant hydrogels were immersed in double-distilled water to remove any residual chemicals and unreacted polymers. After vacuum freeze drying, the gel ratio, i.e. the mass of dried gel divided by the total mass of the two precursors, was measured to be >85%.

Fig. 3 Photographs of the hydrogel and its precursor polymer solutions (a), curves of dynamic rheological measurements (b), SEM pictures of the surface (c) and internal (d) of the lyophilized hydrogel.

The formation of hydrogels was monitored through the evolution of storage (G′) and loss (G′′) moduli with the reaction time at 37 °C by dynamic rheological measurements (strain of 1% and frequency of 1 rad s⁻¹). It is generally considered the gelation point when G′ reaches and surpasses G′′. The gel with a total concentration of 10 wt% at a CO-PEG/azide-PEG ratio of 2 : 1 had a gelation time of 5 minutes (Fig. 3b). Morphology of the lyophilized hydrogel was observed by SEM. Both surface (Fig. 3c) and internal (Fig. 3d) pictures of the hydrogel show a porous morphology.

The FT-IR spectra of the dried hydrogel and the gel precursors are shown in Fig. 4. Precursors azide-PEG and CO-PEG showed characteristic absorptions at 2105 and 2207 cm⁻¹ for the azide and alkyne groups, respectively. These absorptions disappeared in the spectrum of the hydrogel, indicating the completeness of the alkyne–azide reaction.

Fig. 4 FT-IR spectra of azide-PEG, CO-PEG and the dried hydrogel.

The swelling and degradation of the hydrogel was evaluated by monitoring its mass change as a function of time in PBS (pH = 7.4) at 37 °C. The hydrogel swelled and reached a peak value of 600% in 50 min, and then began its mass loss because of degradation that completed in 72 h. The degradation could be attributed to the hydrolysis of the carbonate bonds that links the azide groups to the PEG chains. Though water-insoluble polycarbonates degrade very slow in a neutral medium,³⁶ water-swollen polycarbonate was reported to degrade completely in vitro in 16 h in PBS buffer.³⁷ The degradation of polycarbonates can be accelerated by enzymes in vivo.^36,38

In situ formation and in vivo biocompatibility of the hydrogel were investigated by subcutaneously injection of a mixed solution of precursors azide-PEG and CO-PEG (0.5 mL, 15 wt% in pH 7.4 PBS) in Sprague-Dawley rats (Fig. 5). Fifteen minutes after the injection, a bead of hydrogel was found at the injection site of the rat (Fig. 5a), proving in situ formation of the hydrogel rapidly through the click reaction. The fate and histological effects of the hydrogel were observed by hematoxylin and eosin stain (H&E stain), with typical photographs showing in Fig. 5. One day after the injection (Fig. 5b), the hydrogel presented as a bulky block. Inflammatory cells such as macrophages and lymphocytes were clearly viewable in the tissue surrounding the hydrogel. Two days after injection (Fig. 5c), the hydrogel collapsed to smaller pieces, and the amount of inflammatory cells apparently decreased than the first day. Three days after injection (Fig. 5d), most hydrogel pieces disappeared. Though lymphocytes still existed, macrophages disappeared and scar tissue emerged, indicating a relief of the inflammatory reaction. No weight loss, diarrhea, anorexia, skin ulceration or toxic death occurred on the test rats in 2 weeks, suggesting a high systematic biocompatibility of the hydrogel.

Fig. 5 Hydrogel formed in situ (a) and images of H&E staining of surrounding tissues 1 day (b), 2 days (c), and 3 days (d) after the injection of azide-PEG/CO-PEG solution (L, M, and ST represent lymphocytes, macrophages, and scar tissue, respectively).

To evaluate the properties of the injectable hydrogel as an embolic agent, 0.2 mL of azide-PEG/CO-PEG mixed solution (15 wt%) were injected to the auricular central artery of healthy New Zealand rabbits. A solution of PEG4000 was used as a control for comparison. After the injections into the auricular central artery, rabbits were maintained for 7 days under normal conditions. The changes in color and shape of the rabbit ears around the injection sites were inspected and photographed. As shown in Fig. 6, after the injection of azide-PEG/CO-PEG, the vessel rapidly became pale and protuberant in 15 min (Fig. 6b), indicating that the blood flow was blocked at the injection position. The embolization decreased with time (Fig. 6c and d) and the vessel restored to the normal within 48 h (Fig. 6e) after the injection, which may be due to the degradation of the hydrogel. In contrast, there was no apparent change in the control group after the PEG4000 injection (Fig. 6f). The in vivo formed hydrogels would act like a thrombus in the vessels that largely impeded the blood flow in a similar mechanism as embolotherapy. The hydrogel could be used in local operation to blocking blood vessels temporarily and the vessel could restore in appropriate days without any invasive operation.

Fig. 6 Photographs of rabbit ears: before injection (a); 15 min (b), 12 h (c), 24 h (d), and 48 h (e) after the injection of 0.2 mL of azide-PEG/CO-PEG mixture solution; 15 min (f) after the injection of 0.2 mL of PEG4000 solution into the auricular central artery.

Conclusion

Cyclooctyne and azide functionalized PEGs were conveniently prepared by the ring-opening polymerization of cyclooctyne-bearing epoxy or azide-bearing cyclocarbonate monomer, respectively, with PEG as a macromolecular initiator. Via the SPAAC click reaction of cyclooctyne and azide groups, the two polymers formed hydrogel in a few minutes upon simply mixing under physiological conditions. In vivo experiments revealed good biocompatibility and biodegradability of the hydrogel. Injection of a mixture of the gel precursors into the auricular central artery of rabbits blocked the fast-flow vessel rapidly without any invasive operation, and the vessel flow restored spontaneously by gel degradation in two days. This kind of injectable hydrogels would be useful as biomaterials such as embolic agents and stanching materials.

Acknowledgements

This work was financially supported by the National Natural Science Foundation of China (21374084) and National Basic Research Program of China (2011CB606202).

Notes and references

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