Bio-based elastomer nanoparticles with controllable biodegradability

Abstract

Recently, biodegradable polymers and nanoscale polymers have become hot topics in advanced polymer research. In this manuscript, we report novel electron beam irradiation vulcanized soft polyester nanoparticles through a three-step method. Some dicarboxylic acids (e.g., succinic acid, sebacic acid, itaconic acid) and diols (e.g., 1,3-propanediol and 1,4-butanediol), which can also be derived from biomass resources, were firstly synthesized into the aliphatic unsaturated polyester by using melt polycondensation, which was followed with polyester emulsification and radiation crosslinking to obtain the elastic polyester nanoparticles. Interestingly, the crosslinked soft polyester nanoparticles with controllable gel content, controllable biodegradability (exhibiting a weight loss ratio of 52.3% within 5 days in the presence of lipase) and low glass transition temperature of about −55 °C are potential candidates to meet the requirements not only for drug delivery carriers but also for modifying some biodegradable brittle polymers which are widely used in pharmaceutical sciences, biotechnology, and biomedicine.

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1 Introduction

Recently, research into nanoscale particles has become a hot topic in the field of advanced materials.^1–4 The small size, large specific surface area (defined as the total surface area of a solid material per unit of mass) and special structure of these nanoscale particles play an important role in pharmaceutical sciences, biotechnology, and biomedicine.^5–8 Different types of polymer carriers, such as polymeric micelles,⁹ polymersomes,¹⁰ polymer capsules,¹¹ and polymer replica particles,¹² are expected to revolutionize diagnostics and the delivery of therapeutics.^13–16

Generally, polymer nano- and microspheres with intricate internal domain structures have been prepared from emulsion copolymerization,¹⁷ self-assembly of block copolymers,¹⁸ and the sol–gel process.^19,20 Discher and co-workers also found that cross-linking nanopolymer particles with resultant increased stiffness led to rapid phagocytic clearance via the immune system.²¹

Using some synthetic rubber latex (nitrile–butadiene latex, styrene–butadiene latex) and electron beam irradiation technology, our previous work has prepared some nano- and micro elastomer particles with controllable crosslinking density. The irradiation-vulcanized elastomer particles has the effects on toughening some brittle polymer while improving the crystallization behavior and heat resistance of some polymer.^22–24 However, the conventional synthetic rubber latex derived from fossil resource are not biodegradable, thus the resultant crosslinked elastomer particles are not biodegradable. Therefore, how to fabricate the crosslinked nanopolymer particles which can be biodegradable is more significative.

In this manuscript, we report on the fabrication of a novel biodegradable elastomer nanoparticles (BEN) with controllable biodegradability. Firstly, biodegradable unsaturated polyester (BUP) was firstly synthesized by melt polycondensation using some dicarboxylic acids (e.g., succinic acid, sebacic acid, itaconic acid) and diols (e.g., 1,3-propanediol and 1,4-butanediol) which can be selected from bio-derived monomers. Secondly, the BUP was emulsified into biodegradable unsaturated polyester emulsion (BUPE), followed with radiation crosslinking to obtain the BEN. This three-step strategy has advantages to control the final products with well-defined particle size, controllable gel content and biodegradability.

On the one hand, the resultant biodegradable crosslinked polyester elastomer nanoparticles is the potential candidate to meet the requirements for certain biomedical applications, such as drug delivery systems. On the other hand, the biodegradable elastomer particles are suitable for the modification of biodegradable brittle polymers, such as polylactic acid (PLA)^25–28 and polyhydroxyalkanoate (PHA),^29,30 which are widely used as tissue engineering scaffolds, sutures and clips.

2 Experimental section

2.1 Materials

Itaconic acid (IA) with a molecular weight of 130.10 g mol⁻¹ and a concentration of 98% was supplied by Qingdao Langyatai Biochemical Technology Co., Ltd., China; 1,3-propanediol (PDO) with a molecular weight of 76.09 g mol⁻¹ and a concentration of 98% was supplied by Shanghai Aladdin Biochemical Technology Co., Ltd., China; 1,4-butanediol (BDO) with a molecular weight of 90.12 g mol⁻¹ and a concentration of not less than 99.8% was supplied by Tianjin Bodi Chemical Industry Co., Ltd., China; succinic acid (SU) with a molecular weight of 118.09 g mol⁻¹ and a concentration of not less than 99.5% was supplied by Tianjin Bodi Chemical Industry Co., Ltd., China; sebacic acid (SA) with a molecular weight of 202.25 g mol⁻¹ and a concentration of not less than 99% was supplied by Tianjin Basifu Chemical Co., Ltd., China; para-toluenesulfonic acid with a molecular weight of 202.25 g mol⁻¹ and a concentration of not less than 99.0% was supplied by Sinopharm Chemical Reagent Co., Ltd., China; polyethylene glycol monooleate was supplied by Jiangsu Haian Petrochemical Company, China. Above chemical reagents except for IA and PDO were of analytically pure grade. Lipase, 30 000 U, was supplied by Xinyu Food Additives Co., Ltd., China.

2.2 Fabrication of the BEN

2.2.1 Synthesis of the BUP. Fig. 1(A) displays the esterification schematic of the BUP macromolecules while Fig. 1(B) showing the formation diagrams of clustered BUP macromolecules from monomers. Monomers of IA, SA, SU, PDO and BDO (with the mole ratio of 1 : 2:2 : 2.75 : 2.75) were placed in a reactor which was blanketed with nitrogen gas for protection, and slowly heated to 180 °C until the monomers melted. The unsaturated polyester prepolymer was prepared by the polycondensation reaction for 1 h, and the water produced during the preparation of prepolymer was removed with help of nitrogen gas. Then the catalyst (para-toluenesulfonic acid, with weight ratio of 0.05%), polymerization inhibitor (hydroquinone, with weight ratio of 0.01%), and stabilizer (phosphorous acid, with weight ratio of 0.01%) were subsequently added into above polyester prepolymer. After decompressing the pressure of the reactor below 5000 Pa and increasing the temperature of polyester prepolymer from 180 °C to 220 °C, the esterification reaction retained for 4 h, and the finished product (BUP) was obtained. Molecular weight and molecular weight distribution of the BUP was measured by a gel permeation chromatograph (GPC, HLC-8320, manufactured by Tosoh Corp., Japan).

Fig. 1 Schematic diagrams of the BUP formation. (A) Esterification schematic of the BUP macromolecules. (B) Schematic diagrams of clustered BUP macromolecules.

2.2.2 Preparation of the BUPE. Hydrophile lipophilic balance (HLB) of a surfactant is a measure of the degree to which it is hydrophilic or lipophilic, determined by calculating values for the different regions of the molecule. Having the similar HLB values with complex emulsifier, the BUP molecules are easy to form the BUPE with uniform colloid size. Firstly, sodium oleate was mixed with poly(ethylene glycol) monooleate to form the complex emulsifier which has the same hydrophile lipophilic balance (HLB) value of the BUP. Then the BUP was added to the complex emulsifier at 30 °C. Finally, the BUPE was obtained by mechanical stirring with stirring rate of 10 000 rpm in 30 min and ultrasonic processing in 5 min with frequency of 32 kHz.

2.2.3 Fabrication of the BEN. BUPE was irradiation-vulcanized using an electron-beam accelerator (DD-3.0, supplied by Shanghai Institute of Applied Physics, Chinese Academy of Sciences, China) with a electron beam energy of 2.2 MeV, and the scanning was performed over the conveyor at room temperature. A dose (12 kGy) was given in each pass, and radiation doses used for various BUPEs were 12, 24, 36, and 48 kGy, respectively. Subsequently, the BENs were obtained by spray-drying the irradiation-vulcanized biodegradable polyester emulsion (BPE).

2.3 Characterization and testing methods

2.3.1 Measurement of the acid value of BUP. Measurement of acid values, an important parameter to assessing the condensation reaction or degradation of polyester, were performed according to GB/T 2895-2008. 2 g of BUP was weighed and dissolved in the mixed solvent of toluene and ethanol with a volume ratio of 2 : 1. The mixed solution was titrated by the ethanol solution of potassium hydroxide until the indicator, thymol blue, turned blue. Thus, the acid value can be calculated from the results of titration.

2.3.2 Molecular structure analysis of the BUP. 2 g of BUP was weighed and dissolved in 5 mL of trichloromethane. The mixed solution was then added to 50 mL of cooling methanol for precipitation and filtration. Purified BUP was prepared by washing the precipitates by methanol and then left to dry.

¹H-NMR characterization of purified BUP was performed by a nuclear magnetic resonance (NMR) spectrometer (AVANCE, bought from Bruker Corp., Germany) at ambient temperature with a testing frequency of 300 Hz. Deuterated chloroform and tetramethylsilane (TMS) were used as the solvent and internal standard, respectively.

IR spectra of purified BUP was performed by a ATR-IR analyser (VERTEX 70, from the Bruker Company, Germany) at room temperature, and the scanning wavenumbers were ranged from 4000 cm⁻¹ to 600 cm⁻¹.

2.3.3 The molecular weight and molecular weight distribution of BUP. The molecular weight and molecular weight distribution of BUP were measured using the gel permeation chromatograph (HLC-8320, manufactured by Tosoh Corp., Japan). Tetrahydrofuran was used as the mobile phase, and the flow rate was 0.35 mL min⁻¹.

2.3.4 DSC analysis. The DSC curves were measured to study the thermoanalysis of BENs by a differential scanning calorimeter (204F1, bought from Netzsch Corp., Germany). With a blanket of nitrogen air for protection, the BUP was heated at a heating rate of 10 K min⁻¹ until the temperature reached 150 °C where it was left for an additional 5 min. BUP and BEN were then cooled at a cooling rate of 10 K min⁻¹ until the temperature reached −100 °C where it was left for 5 min. The polyester was finally heated to 150 °C at a heating rate of 10 K min⁻¹.

2.3.5 Measurements of colloid sizes of BUPE. The colloid sizes of BUPE were measured using a laser particle analyzer (Nano-ZS90, manufactured by Malvern Co., Ltd., UK). BUPE was diluted and placed into a cuvette at 25 °C for 2 min, and the colloid sizes were then measured. BUPE was placed onto the copper grid. After drying, the morphology of the BUPE was observed using a transmission electron microscope (TEM, JEM-2100, manufactured by JEOL Corp., Japan).

2.3.6 Determinations of gel content of BEN. Gel is a part of the polymer which can not be dissolved by the good solvent. Polymer gel content is generally used to characterize the polymer crosslinking degree. The gel content of the BEN was measured using a Soxhlet extractor. Approximately 1 g of the irradiation-vulcanized polyester elastomer particles was weighed. We denote this mass as W_s. The sample was wrapped in a filter paper and steel mesh. We denote the elastomer particles total mass as W_a. The wrapped sample was placed in the Soxhlet extractor and extracted via toluene reflux at a refluxing temperature of 80 °C for 24 h. The sample was then collected, washed, and dried until a constant weight was achieved, which we denote as W_b. Finally, the gel content of the irradiation-vulcanized elastomer particles was calculated using eqn (1).

Gel content (100%) = [(W_a − W_b)/W_s] × 100% (1)

2.3.7 Biodegradation performance test of the BEN. About 0.5 g of BEN was weighed, which is denoted as W_p, and placed into a tube. Approximately 0.01 g of lipase was added and weighed, and the total mass (sample, tube and lipase) is denoted as W_o. An appropriate amount of mixed phosphate buffer solution (about 8 mL) with a pH value of 6.8 was added into the sampling tube. This solution was then placed into a thermostatic water bath at a constant temperature of 37 °C. The tube was taken out every 24 h. After centrifugal separation, the tube was dried in a vacuum drying oven to a constant weight, which is denoted as W_t. Finally, the mass loss rate was calculated using eqn (2).

Weight loss ratio (100%) = [(W₀ − W_t)/W_p] × 100% (2)

About 0.01 g of lipase and a specific amount of mixed phosphate buffer solution with pH = 6.8 were weighed and added into the sampling tube to repeat the tests, which ensures that similar biodegradation conditions is supplied for biodegradation tests of remainder BENs in following biodegradation tests.

3 Results and discussion

3.1 Structural analysis of the BUP macromolecule

Fig. 2 shows the ¹H-NMR spectrum of the BUP with the number-average molecular weight of 2300 and the weight-average molecular weight of 5736 (Table 1). The peaks at 5.74 and 6.34 ppm are the resonance absorption peaks of H in C=CH₂ bond of itaconic acid, thereby suggesting the existence of double-bond in polyester. The peaks at 1.30, 1.71, and 2.30 ppm are the resonance absorption peaks of H in H3, H2, and H1 of methylene in sebacic acid, whereas the peaks at 2.63, 4.13, 1.97, and 3.69 ppm are the resonance absorption peaks of H6, H4, H5, and H7 of methylene in succinate, propanediol alginate, and butanediol, respectively. The ¹H-NMR spectrum indicates that BUP was produced using itaconic acid, succinic acid and sebacic acid.

Fig. 2 ¹H-NMR spectrum of the BUP.

Table 1 The molecular weight and molecular weight distribution of BUP

Sample	Ratio of IA/SA/SU (mol%)	Mn	Mw	Mz + 1	PDI	[η] (g mL^−1)
BUP	0.2/0.4/0.4	2300	5736	21 738	2.49	2.069

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3.2 Effects of BUP weight concentration on the colloid size distribution of BUPE

Fig. 3 presents the relationship between the BUP weight concentration and the emulsion colloid sizes in BUPE (with the wt/wt of emulsifiers to BUP is 18%). With increased weight concentration of BUP, the viscosity of the emulsion increases and a good interface layer of the surfactant can hardly be formed. Moreover, the probability of collision and aggregation of the oil phases in the emulsion increases, thereby leading to a large and non-uniformly distributed colloid size.

Fig. 3 Effect of BUP weight concentration of BUPE on the colloid size distribution.

To sum up, the appropriate BUP weight concentration and the amount of surfactant in BUPE are significant in producing the polyester emulsions with small and uniformly distributed colloid sizes.

3.3 Formation of the electron beam irradiated BEN

During the electron beam irradiation process of the BUPE, the C=C bonds of BUP macromolecules will be broken and the polyester macromolecules free radicals occurred, thus leading to the crosslinking of BUP macromolecules in the colloid particles (Fig. 4), and the vulcanized BEN is obtained. Fig. 5 shows TEM images of some BENs. The particle sizes of BENs can be controlled with uniform distribution, and the particle sizes also can be controlled within the range of 100 nm.

Fig. 4 Schematic diagrams of the crosslinked BEN from BUP macromolecules.

Fig. 5 TEM image of some BENs.

Fig. 6 is some DSC curves of various BENs irradiated with different radiation doses. With the increasing of radiation dose, the glass transition temperature (T_g) of various BENs increases from −55.2 °C to −53.4 °C within the dose range of 48 kGy, and the corresponding gel content of BEN increases to 62% (Fig. 7) because the electron beam irradiation induces the generation of polyester macromolecular free radicals that are easily cross-linked with each other to form the three-dimensional network. Therefore, with the radiation dose increasing, the cross-linking density and gel content of the BEN increase.

Fig. 6 DSC curves of some BENs irradiated with different radiation dose.

Fig. 7 Gel contents of various BENs.

3.4 Biodegradation performance of the BEN

In the lipase buffer solution, the bio-degradation of polyester is mainly introduced by the breaking of the ester bond.³¹ In this study, Fig. 8 illustrates the biodegradation schematic of the vulcanized BEN, and we focused on the biodegradation mechanism of the BEN and the relevant factors.

Fig. 8 Biodegradation diagrams of the BEN particles.

Fig. 9(a) presents the degradation curves of BEN samples with different gel contents, and the degree of degradation increases steadily with increasing degradation time. The BEN with the lowest cross-linking density exhibits a weight loss ratio of 52.3% within 5 d in the presence of lipase. With increasing cross-linking density of the BEN, the degradation rate gradually decreases. By controlling the number of double-bonds in the BUP molecules and the radiation dose, we can control the cross-linking density of the polyester elastomer as well as the degradation rate. Fig. 9(b) presents the acid values of the centrifugal supernatant liquid of various BEN degraded for 1 and 5 d. With increasing degradation time, the acid value of the degraded supernatant liquid of BEN increases. However, acid value of the degraded supernatant liquid decreases with increasing gel content of the polyester elastomer. These results suggest that the amount of terminal hydroxylic groups in the polyester degradation system increases with increasing degradation rate. In other words, with the catalytic action of lipase, the ester bond in the polyester macromolecular chain is easily broken, thereby leading to the generation of new acids and alcohols.

Fig. 9 Biodegradation of BEN samples. (a) Weight loss ratios of various BENs; (b) acid values of the centrifugal supernatant liquid of various degraded BENs.

The infrared spectra of the BEN before and after the degradation are shown in Fig. 10. Using the absorption peak of –CH₂ (2858 cm⁻¹) as the internal standard, it is obvious that the absorption peak area of hydroxylic group (around 3500 cm⁻¹) significantly increases after the polyester is degraded. The polyester molecular chains vary in length after degradation and exhibit a broad absorption peak. The results indicate that the number of the terminal hydroxylic groups of the polyester increases after the degradation, which is accompanied by the generation of hydroxy and carboxyl groups.

Fig. 10 Infrared spectra of BENs before and after biodegradation.

4 Conclusions

We successfully prepared the BEN with controllable particle sizes and degradation performance by melting polycondensation, polyester emulsification and radiation vulcanization. The BEN exhibited controllable degradability to a particular extent, which decreased with increasing cross-linking density. It is worth noting that the resulted BEN is a potential candidate to meet the requirements for both biomedical applications in drug delivery carrier and modifying brittle biodegradable polymers.

Acknowledgements

This research is supported by National Basic Research Program of China (973 Program) (Grant No. 2015CB654706), National Natural Science Foundation of China (Grant No. 51373085) and Collaborative Innovation Center of Green Tyres & Rubber (2014GTR0006).

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