Introduction

Owing to the evolving plastic disposal problem, modern technology is in constant need of new, environmentally friendly polymeric materials with varying properties over those demonstrated by many homopolymers. Block copolymers combine two or more polymer segments into a main chain that reflect the thermal, mechanical and physicochemical properties of each related segment [1, 2]. In many polymerization methods, macro initiators are used to synthesize block (or block/graft) copolymers [3,4,5,6,7,8,9,10]. Free radical macroinitiators are frequently prepared from macromolecules containing azo and/or peroxide groups. Typically, 2,2’-azobis cyanopentanoyl chloride is reacted with the hydroxyl termini of the polymers (e.g., polyethylene glycol (PEG), polytetrahydrofuran diol, poly(dimethyl siloxane) diol and polycaprolactone diol) to produce macroazo initiators that can be used in the synthetic process [11,12,13,14,15,16,17,18]. Another type of macroazo initiator involves the reaction of PEG with 2,2’-azobis isobutyronitrile under dry hydrogen chloride to produce a PEG-azo ester [19, 20].

Anionic polymerization has garnered a significant role in the synthesis of well-defined polymers. This technique allows excellent molecular weight control by regulating the ratios of monomer and initiator throughout the polymerization process [21,22,23,24]. Recent progress in controlled/living radical polymerization (CLRP) has provided a powerful tool for the controlled synthesis of well-defined polymers. CLRP covers a myriad of polymerization techniques including nitroxide-mediated radical polymerization (NMP) [25,26,27], atom transfer radical polymerization (ATRP) [28,29,30,31], and reversible addition fragmentation chain transfer (RAFT) polymerization [32,33,34,35,36,37,38,39,40], among others.

Biopolymers are gaining importance for biomedical and industrial applications in view of their biorenewability, biodegradability and other environmental benefits. Among the known biopolymers, microbial polyesters can be obtained from bacteria when gown on renewable resources such as sugar, and/or aliphatic carboxylic acids under favorable growth conditions. Poly(3-hydroxyalkanoate)s, (PHA)s, are accumulated in bacterial cells as a result of a metabolic stress causing unbalanced growth due to a limited supply of an essential nutrient and an excess of utilizable carbon. These polymers are promising materials for many different applications due to their biocompatibility and biodegradability [41,42,43,44,45,46,47,48,49]. Improvements in the mechanical, thermal, and hydrophilic properties of PHA biopolymers have been previously reported [50,51,52,53,54,55,56,57,58,59,60,61,62,63]. One interesting class of PHA with enhanced mechanical properties is the copolymer composed of 3-hydroxybutyric acid (3HB) and 3-hydroxyhexanoic acid (3HHx). 3HHx-containing PHA copolymers are typically prepared by bacterial fermentation using bacteria such as wild-type Aeromonas hydrophila, and Pseudomonas species resulting in copolymers with 3HHx units comprising as much as 12% of the total polymer composition [64,65,66]. By varying the amount of 3HHx subunits within the mature copolymer, the melting transition (Tm) and glass transition (Tg) temperatures can be controlled. For example, Doi et al. synthesized PHBHHx with a Tm of 112.7 °C and a Tg of 1.6oC while those of PHB are around 170 °C and 3oC, respectively [67]. DMAEMA was chosen as the monomer for the copolymerization initiated by the chemically synthesized PHBHHx macroazo initiator. PDMAEMA is an interesting polymer because it is a thermosensitive polymer in aqueous solution at neutral pH and temperatures up to about 50˚C [68,69,70].

Great efforts have been undertaken to synthesize polymeric derivatives of microbial polyesters for use in medical applications such as drug delivery systems and tissue engineering. Previous reports have documented the synthesis of amphiphilic and thermoresponsive copolymers of PHB for this purpose [56, 71, 72] however, to our knowledge, there are no reports detailing the synthesis of PHBHHx amphiphilic and thermoresponsive block copolymers using PHBHHx macroazo initiator with DMAEMA.

In this work, we report a new macroazo initiator based on PHBHHx and 2,2’ azobis cyanopentanoic acid (PHBHHx-AI). While PHBHHx derivatives are known, they are only minimally described in the literature and until now, specific PHBHHx free radical macroinitiators have not been published. To our knowledge, this is the first report of this type of free radical macro intermediate. Specifically, free radical polymerization of DMAEMA was initiated by this macroazo initiator to obtain a PHBHHx-b-PDMAEMA block copolymer. Polymerization kinetics were evaluated and the synthesized block copolymers were characterized by physicochemical methods.

Experimental

Materials

PHBHHx samples containing 9 mol% of 3-hydroxyhexanoic acid (PHBHHx-9) was supplied by Procter & Gamble Company (Cincinnati, OH, USA). Al2O3 (≥ 99.9%), DMAEMA (98%), dimethyl formamide (99.8%), chloroform (≥ 99.9%), 4,4’-azobis cyanopentanoic acid (AI-COOH), and dichloromethane (≥ 99.9%) were supplied from Sigma-Aldrich (St. Louis, MO, USA) and passed through Al2O3 before use. Carbon disulfide (≥ 98%), methyl amino ethanol (mae) (≥ 98%), 4, 4’-azobis cyanopentanoic acid (98%), N, N′-dicyclohexylcarbodiimid (DCC; 99%), dimethyl amino pyridine (DMAP; 99%), stannous 2-ethyl hexanoate (Sn-oct; ≥92.5%), and all other chemicals were purchased from Sigma-Aldrich.

Synthesis of Hydroxylated PHBHHx Using Methyl Amino Ethanol (mae), (PHBHHx-mae-9)

Hydroxylated PHBHHx was obtained by the reaction of PHBHHx with mae according to the slightly modifed procedure reported in the cited reference [73]. Briefly, a mixture of 15.6 g of PHBHHx-9, 4.13 g of mae (or dea depending on the desired product) and 0.29 g of Sn-oct in 250 mL of CHCl3 was stirred at room temperature for 24 h. Then, the mixture was refluxed for 1 h. The solvent was distilled under atmospheric conditions at between 90 °C and 105oC. The product was washed with excess methanol and filtered. The crude product was dried under vacuum at 40 °C. For further purification, the obtained polymer was dissolved in 100 mL of CHCl3 and filtered from any undissolved residue. The polymer solution was poured into excess methanol. The obtained polymer was filtered and dried under vacuum at 40 °C for 24 h. Yield was 9.3 g. Characteristic FTIR signals were at 1567 cm− 1 corresponding to the amide carbonyl, 3301 cm− 1 attributed to the primary hydroxyl groups of dea, 1721 cm− 1 which belongs to the ester carbonyl of PHB. The characteristic chemical shifts of the PHBHHx-mae sample in 1 H NMR spectrum were observedat 1.3 ppm for –CH3, 2.4–2.6 ppm for –CH2-COO–, 2.8 ppm for –N-CH2–, 3.3 ppm for –N-CH3, 3.5–3.8 ppm for –CH2-OH, 4.1 ppm for –CH-OH and 5.1–5.3 ppm for –CH-O–.

Synthesis of PHBHHx Macroazo Initiator (PHBHHx-mae-AI-9)

The synthesis of the PHBHHx macroazo initiator was carried out by the reaction between PHBHHx-mae-OH (8.15 g) and 2,2’-azobis cyanopentanoic acid (0.70 g) in the presence of DCC (0.28 g) and DMAP (0.20 g) in CH2Cl2 (20 mL). After the mixture was stirred at room temperature for 48 h the precipitated side product formed in the excess methanol was filtered through the solution. The solvent was evaporated and the obtained PHBHHx macroazo initiator was dried under vacuum at room temperature giving a yield of 7.0 g.

Free Radical Polymerization of DMAEMA Initiated by (PHBHHx-AI-9) at 70oC in DMF

A solution of PHBHHx-AI-9 (0.40 g), DMAEMA (2.50 g) and DMF (2.50 mL) was reacted at 70oC for from 10 to 65 min under argon. The obtained polymer solution was poured into excess petroleum benzene to isolate the PHBHHx-b-PDMAEMA block copolymer which was dried under vacuum at 40oC for 24 h.

Characterization

1 H NMR spectra of the synthesized products were taken at 25 °C with an Agilent NMR 600 MHz NMR (Agilent, Santa Clara, CA, USA) spectrometer equipped with a 3 mm broadband probe. Acquisition parameters included a 45° hard pulse angle, a sweep width of 14 ppm, 1.7 s acquisition time, 0.1 s pulse delay and continuous WALTZ – 16 broadband 1 H decoupling. Up to 2000 scans were collected per sample, corresponding to ~ 1 h of collection time. FT- IR spectra of the polymer samples were recorded using a Perkin-Elmer FT-IR Spectrometer 100. Size exclusion chromatography (SEC) was used to determine the molecular weights of the reacted polymers. A calibration curve was generated with five polystyrene (PS) standards of molecular weight 2960, 8450, 50,400, 200,000 and 696,500 Da with low polydispersity. A gel permeation chromatography instrument was used which included a Viscotek GPC max auto sampler system, consisting of a pump, three ViscoGEL GPC columns (G2000H HR, G3000H HR and G4000H HR), and a Viscotek differential refractive index (RI) detector. CHCl3 (or THF) was used as the mobile phase with a flow rate of 1.0 mL/min at 30 °C. The RI detector was calibrated with polystyrene standards having narrow molecular weight distributions. Data were analyzed using Viscotek OmniSEC Omni – 01 software.

Differential scanning calorimetry (DSC) was used in the thermal analysis of the obtained polymers. The DSC analysis was carried out under nitrogen using a TA Q2000 DSC instrument that was calibrated using indium (Tm = 156.6 °C) and a Q600 Simultaneous DSC-TGA (SDT) series thermal analysis system. DSC measures the temperatures and heat flows associated with thermal transitions in the polymer samples obtained. The dried polymer samples were heated from − 60 to 120 °C under a nitrogen atmosphere. All melting endotherms (Tm) were reported as peak temperatures while all glass transition temperatures (Tg) were reported as midpoint temperatures. Thermogravimetric analysis (TGA) is used to determine the decomposition temperature (Td) characteristics of the polymers by measuring weight loss under a nitrogen atmosphere over time. In these analyses the obtained polymers were heated from 20 to 600 °C at a rate of 10 °C/min and the results were determined based on the 1st derivative of each curve.

Dynamic light scattering (DLS) was used to determine micelle formation within the synthesized polymers. In order to prepare micelles, 10 mg of the PHBHHx-PDMAEMA was dissolved in 1 mL of DMF under stirring for 2 h. Then, 10 mL of Milli-Q ultra-purified water was added dropwise to the solution under stirring. After 3 h of stirring at room temperature, the DMF was removed by dialysis against water for 3 days. DLS measurements were performed at 633 nm on a Malvern Instrument Zetasizer Nano ZS provided with a He-Ne laser source. Measurements were conducted at an angle of 173° at 25 °C. The cumulative method was used to analyze the autocorrelation functions to determine diameters and distributions.

Results and Discussion

PHBHHx-9 was chosen as the precursor for the macroazo initiator. The PHBHHx chain originally contains one OH group and one –COOH terminal group. The carboxylic acid terminus was converted into a hydroxyl group by the reaction with methyl amino ethanol to obtain dihydroxyl terminated PHBHHx. The hydroxyl ends were reacted with the carboxylic acid terminated reagent, 4,4’-azobis cyanopentanoic acid, to obtain the PHBHHx-macroazo initiator (PHBHHx-AI-9). The reaction scheme can be seen in Fig. 1. An excess amount of azobis cyanopentanoic acid was used in this reaction to prepare the azo terminated PHBHHx derivative.

Fig. 1
figure 1

Chemical reactions for the synthesis of PHBHHx-macroazo initiator

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Free Radical Polymerization of DMAEMA Initiated by PHBHHx-AI

A series of PHBHHx-b-PDMAEMA block copolymers were obtained by varying the polymerization times from between 10 and 65 min at 70oC in DMF solution. The individual copolymers were coded as PHDM-AI-0, -1, -2, -3, and − 4. The results and the reaction conditions can be seen in Table 1.

Table 1 Free radical polymerization of DMAEMA initiated by the AIBN-COOH terminated PHBHHx (PHBHHx-AI-9) at 70oC, in DMF under argon
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The polymer yield improved by approximately 0.02 g/min as the polymerization reaction time increased from 10 to 50 min. Beyond that, yields stabilized culminating at 1.43 g in 65 min. The obtained block copolymers were characterized by 1 H NMR (Fig. 2). The PHBHHx contents of the block copolymers were calculated by using the integral ratio of the signal at 5.2 ppm (O-CH- of PHBHHx) and the integral ratio of the signal at 4.0 ppm (-COO-CH2- of DMAEMA units) (Table 1).

Fig. 2
figure 2

1 H NMR spectra of the PHDM block copolymers: (a) PHDM-AI-0, (b) PHDM-AI-1, (c) PHDM-AI-2, (d) PHDM-AI-3, (e) PHDM-AI-4, (f) PHBHHx-OH-9, (g) PHBHHx-AI.

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The FTIR spectra of the PHBHHx-PDMAEMA series also confirmed their chemical structures. Figure 3 shows the FTIR results from the synthesized copolymers with the characteristic signals at 3430 (-OH and quarternery ammonium), 2936 (-C-H), 1721 (-C = O) and 748 cm-1 (tertiary nitrogen) [70, 74].

Fig. 3
figure 3

FTIR spectra of the PHBHHx-PDMAEMA (PHDM) block copolymers: (a) PHBHHx-H9, (b) PHBHHx-OH-9, (c) PHDM-AI-9, (d) PHDM-AI-0, (e) PHDM-AI-1, (f) PHDM-AI-2, (g) PHDM-AI-3, (h) PHDM-AI-4.

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Free Radical Polymerization Kinetics of DMAEMA Initiated by the PHBHHx-macroazoinitiator

Conventional radical polymerization is usually a first order (or pseudo first order) reaction [36, 75, 76]. In this case, the rate of the polymerization reaction of DMAEMA using the PHBHHx-AI macroazo initiator at 70oC obeyed the first order kinetics and the reaction rate constant (k) was calculated according to Eq. (1).

$$Ln\left[ {{M_o}} \right]/\left[ M \right]{\rm{ }} = {\rm{ }}{k_{app}}{\rm{ }} \times {\rm{ }}t$$
(1)

Where [Mo] and [M] are the total concentrations of the monomer in the beginning and after a polymerization time (t), respectively. Figure 4 shows the plot of Ln[Mo]/[M] against the polymerization time t (s). The calculated overall rate constant was found to be k = 2.33 × 10− 4 Lmol-1s-1.

Fig. 4
figure 4

The plot of Ln[Mo]/[M] against the polymerization time t (s) in free radical polymerization kinetics of DMAEMA initiated by the PHBHHx-macro azoinitiator

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Molecular Weights of the PHBHHx-PDMA Block Copolymers and PHBHHx Precursors

The molecular weights of the obtained block copolymers and PHBHHx precursors were determined by size exclusion chromatography in CHCl3 (Table 2).

Table 2 The molecular weights of the block copolymers, the precursors, and the micelle diameters of the amphiphilic block copolymers
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Figure 5 shows the unimodal SEC chromatograms which were used to determine the molecular weights of the copolymers. Because of degradability issues associated with the PHAs upon hydroxylation with methyl amino ethanol, there was a dramatic decrease on the molecular weights of the resultant copolymers. The Mn of the pristine PHBHHx-9 was 70300 g/mol but, after capping with 4, 4’-azobis cyanopentanoic acid there was a marked decrease in the Mn value for the obtained macroazo initiator, PHBHHx-AI-9 however, once this initiator was reacted with the DMAEMA to form the block copolymer series (PHDM-AI-0 through PHDM-AI-4), the molecular weights of the block copolymers increased by 19% as polymerization time increased from 10 to 65 min.

Fig. 5
figure 5

 s chromatograms of the block copolymers and the precursors: (a) PHBHHx-9, (b) PHBHHx-OH-9, (c) PHBHHx-AI-9, (d) PHDM-AI-0, (e) PHDM-AI-1, (f) PHDM-AI-2, (g) PHDM-AI-3, (h) PHDM-AI-4.

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Because of the high hydrophilic effect of PDMAEMA, micelle formation of the obtained block copolymers was measured in aqueous medium. The micelle sizes of the block copolymers varied from 602 to 1351 nm with polydispersity indices varying from 0.317 to 0.437. From this, we can infer that micelle size increases with the increase in DMAEMA content in the block copolymer. Size distribution can be seen in Fig. 6.

Fig. 6
figure 6

Micelle size distribution of the PHBHHx-PDMAEMA amphiphilic block copolymers: (a) PHDM-0, (b) PHDM-1, (c) PHDM-2, (d) PHDM-3, (e) PHDM-4.

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Thermal Properties

The thermal properties of the PHBHHx-PDMAEMA block copolymers were determined using DSC and TGA techniques. The thermal analysis results are shown in Table 3 While the actual curves are shown in Fig. 7. Glass transition (Tg) and melting transition temperatures (Tm) were determined by DSC. The typical Tm values of the PHBHHx block copolymers were observed at around 125 ± 3oC. Additionally, multiple Tgs and Tms were observed in some of the copolymers. Presumably, during the polymerization, small amounts of degraded polymer segments can cause these multiple Tgs and Tms. Homo PDMAEMA is an amorphous polymer with a Tg of around 10 °C [77].

Table 3 Results of thermal analysis of the PHBHHx-PDMAEMA block copolymers. aDetermined by DSC analysis. bDetermined by TGA analysis
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Fig. 7
figure 7

DSC analysis of the PHBHHx-b-PDMAEMA block copolymers: (a) PHDM-AI-0, (b) PHDM-AI-1, (c) PHDM-AI-2, (d) PHDM-AI-3, (e) PHDM-AI-4, (f) PHBHHx-9 (pristine), (g) PHBHHx-dea-OH, (h) PHBHHx-mae-OH, (i) PHBHHx-AI-9.

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The carboxyl ends of PHBHHx were converted into hydroxyl groups using methyl amino ethanol amine. Thermal properties of the hydroxylated PHBHHx were also compared with their melting transitions in Fig. 7(g and h). The hydroxylated PHBHHx obtained using dea showed a bimodal Tm whose peak values were both approximately 15% higher than those corresponding to the mae derived copolymers. The effect of the PDMAEMA blocks lowered the Tm of pure PHBHHx (ca. 145oC).

Figure 8 shows the TGA (I) and DTG (II) curves. PHBHHx intermediates showed a single decomposition at around 250oC (Fig. 8a-c) while the synthesized block copolymers showed multiple decomposition temperatures (Fig. 8d-h) whose initial Td (Td1) values were approximately 4% on average lower than those from the PHBHHx intermediates. The very low peak of Td4 decomposition temperature presumably occurs during the side reactions of the PHBHHx and PDMAEMA carbonaceous residues.

Fig. 8
figure 8

TGA (I) and DTG (II) curves: (a) PHBHHx-9, (b) PHBHHx-mae-OH, (c) PHBHHx-AI-9, (d) PHDM-AI-0, (e) PHDM-AI-1, (f) PHDM-AI-2, (g) PHDM-AI-3, (h) PHDM-AI-4, (i) PDMAEMA.

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Conclusion

Microbial polyesters require modification to improve the hydrophilicity, hydrophobicity, thermal resistance, and the mechanical properties. To modify the PHAs, block copolymers were prepared using functional intermediates. PHBHHx macroazo initiators were prepared and used in the synthesis of DMAEMA amphiphilic block copolymers with a reasonable polydispersity. This type of macroazo initiator can be used to prepare block copolymers with a wide range of vinyl monomers to obtain very versatile PHA derivatives. Because of the mild polymerization conditions, PHBHHx macroazo initiators can also be promising macrointermediates to prepare bioconjugates for medical applications.