Stabilization and characterization of carboxylated medium-chain-length poly(3-hydroxyalkanoate) nanosuspensions

Stabilization and characterization of carboxylated medium-chain-length poly(3-hydroxyalkanoate) nanosuspensions

G Model ARTICLE IN PRESS BIOMAC-8476; No. of Pages 7 International Journal of Biological Macromolecules xxx (2017) xxx–xxx Contents lists availabl...

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ARTICLE IN PRESS

BIOMAC-8476; No. of Pages 7

International Journal of Biological Macromolecules xxx (2017) xxx–xxx

Contents lists available at ScienceDirect

International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac

Stabilization and characterization of carboxylated medium-chain-length poly(3-hydroxyalkanoate) nanosuspensions Benjamin Antwi Peprah 1 , Juliana A. Ramsay ∗ , Bruce A. Ramsay Chemical Engineering, Queen’s University, Kingston, ON, K7L 3N6, Canada

a r t i c l e

i n f o

Article history: Received 18 July 2017 Received in revised form 20 October 2017 Accepted 31 October 2017 Available online xxx Keywords: Carboxylated Medium-chain-length poly-3-hydroxyalkanoate Nanoparticles Stability

a b s t r a c t The effects of carboxylation (via mercaptoundecanoic acid) on colloidal properties of medium-chainlength poly(3-hydroxyalkanoate) (mcl-PHA) latexes were studied. Non-ionic surfactants tested at 0.4% solids of 11 mol% carboxylated mcl-PHA produced similar particle sizes and particle size distribution (PdI) with Triton X-100 giving the smallest size. When Triton X-100 was combined with an ionic surfactant, smaller nanoparticles (97.1 ± 1.1 to 121.7 ± 5.7 nm) with narrower PdIs (0.21 ± 0.001 to 0.25 ± 0.003) were obtained. The combination of SDS and Triton X-100 gave the smallest particle size (97.1 ± 1.1 nm) and narrowest PdI (0.21 ± 0.001). At higher solids content (10%), a mixture of 5 mM SDS and 20 mM Triton X-100 produced stable (zeta potential = −39.6± 0.9) 170.3 ± 4.6 nm nanoparticles. As carboxylation increased, particle size and hydrophobicity decreased while stability increased. When comparing nanoparticles of similar size and stability, carboxylated mcl-PHA needed ∼50% less surfactant to make stable nanoparticles compared to aliphatic mcl-PHAs, with the amount of surfactant required decreasing as carboxylation increased. This is the first study to show that stable nanoparticle suspensions of a range of carboxylated mcl-PHAs above 0.4% solids can be made using a mixture of ionic and nonionic surfactants. © 2017 Elsevier B.V. All rights reserved.

1. Introduction Medium-chain-length poly(3-hydroxyalkanoates) (mcl-PHAs), typically produced from renewable resources, are compostable, biocompatible, thermoelastomeric polyesters. They have a low melting temperature, tensile strength, and low or no crystallinity. They have potential applications as adhesives [1], coatings [1,2], in xerographic toners [3], paper-based laminates [4] and as biocarriers in the slow release of fertilizers, insecticides and herbicides [5] as well as drugs and hormones (as reviewed by Singh et al. [6]), and other biomedical applications such as tissue engineering [5,6]. Mcl-PHAs contain relatively long pendant non-polar side chains of 3–11 carbons, making the material substantially hydrophobic with water contact angles of 92–98◦ [8,9]. There are a variety of methods to incorporate functional groups at the terminal position of the pendant mcl-PHA side chains, including olefinic [10,11], aromatic [12,13], hydroxyl [14], epoxide [15,16], or carboxyl [17,18] groups. The functional groups can influence properties such as mechanical strength, surface features,

∗ Corresponding author. E-mail address: [email protected] (J.A. Ramsay). 1 Present address: Chemical and Materials Engineering, University of Alberta, Edmonton, AB T6G 1H9, Canada.

and hydrophilicity to suit the requirements of a specific application. Carboxyl and hydroxyl groups enhance hydrophilicity. Water or polar solvents can penetrate into the polymer to enhance its solubility or swellability [19]. As the degree of carboxylation increases, solubility in polar solvents such as acetone, methanol and acetonewater [17] and aqueous hydrolysis [19] increase. Non-carboxylated mcl-PHAs are insoluble in water, and are very stable to aqueous hydrolysis [19]. Since aqueous hydrolysis can be controlled by the carboxylic acid content [7], carboxylated mcl-PHA would be a superior slow release agent in agricultural and biomedical applications. Furthermore, carboxyl groups are useful in material modification such as making block or graft co-polymers and for attaching bioactive compounds [20]. Many potential applications for carboxylated mcl-PHAs require a dense nanoparticle formulation. Although carboxylated mclPHAs have been suggested for use in xerographic toners [21], hot melt adhesives and protective coatings [1] and as microbeads in personal care products such as cosmetics and toothpaste [22], there is no publication on making dense stable nanoparticles of carboxylated mcl-PHAs. Beauregard et al. [23] made a stable latex of only 0.4% (w/v) mcl-PHA with 5.5 mol% carboxylation without a surfactant. The latex was likely stable due to the “ouzo effect” which only occurs at low polymer concentrations [24,25]. Publications from the group of Renard and Langlois (e.g. [14,17,19]) describe making a mixture of micro- and nano-particles using 0.17% (w/v) carboxy-

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lated mcl-PHA by the precipitation-solvent evaporation method using a non-ionic surfactant (1% (w/v) Pluronic F-68). The nanoparticles were separated by centrifugation then stabilized in 1% (w/v) Pluronic F-68. Kurth et al. [17] found that 10 and 25 mol% carboxylated mcl-PHA aggregated without a surfactant but only the 25 mol% carboxylated mcl-PHA was stable with the non-ionic surfactant. Most industrial applications require suspensions well above 10% solids with particle sizes below 300 nm [23], but to date no data have been reported on the fabrication of stable carboxylated mcl-PHA latexes above 0.4% solids or with mcl-PHA with carboxylation of less than 25 mol%. Ionic surfactants have been used in combination with non-ionic surfactants to make stable polymer dispersions at high solids content [26] and has been applied to aliphatic [27] but not to carboxylated mcl-PHA. Carboxylated PHA latexes have different surface properties [17,19]; they have ionizable groups, are significantly more hydrophilic (more soluble in polar solvents) and have lower particle-water interfacial tension than non-carboxylated mcl-PHA. This is the first study to investigate the formulation of dense (10% (w/v)) carboxylated mcl-PHA latexes using a combination of ionic and non-ionic surfactants in the emulsification-solvent evaporation method.

2.3. NMR spectroscopy

2. Materials and methods

2.4. Particle size characterization

2.1. Biosynthesis of mcl-PHAs

The mean particle diameter (Z-average), the width of the particle size distribution (polydispersity index (PdI)) and the zeta potential were measured by dynamic light scattering (Malvern Zetasizer Nano ZS, Worcestershire, UK) at 25 ◦ C. Samples were diluted with distilled, deionized water to 1 g/L and measured at day 1. All plots of the particle size distribution by intensity showed a single peak with a binomial distribution. Data are the average of three measurements ± the standard deviation.

Four mcl-PHAs were produced by fed-batch fermentation of Pseudomonas putida KT 2440 [28] for further modification or study. They were: (1) poly(3-hydroxynonanoate) (PHN) (70 mol% 3-hydroxynonanoate and 30 mol% 3-hydroxyheptanoate), (2) poly(3-hydroxydecanoate) (PHD) (60 mol% 3-hydroxydecanoate, 20 mol% 3-hydroxyoctanoate, and 20 mol% 3-hydroxyhexanoate), two PHN with some unsaturation in the side-chains (vinyl groups) (PHNUs): (3) 11 mol% unsaturation composed of 61 mol% 3hydroxynonanoate, 28 mol% 3-hydroxyheptanoate, 6 mol% C9:1 3-hydroxynonenoate, 4 mol% C11:1 3-hydroxyundecenoate, and 1 mol% C7:1 3-hydroxyheptenoate (PHNU-11), and (4) 18 mol% unsaturation composed of 72 mol% 3-hydroxynonanoate, 10 mol% 3-hydroxyheptanoate, 7 mol% C9:1 3-hydroxynonenoate, 9 mol% C11:1 3-hydroxyundecenoate, and 2 mol% C7:1 3hydroxyheptenoate (PHNU-18). All PHAs were purified with at least one precipitation step to remove biomass impurities and were above 96% purity as determined from a known mass of sample and PHA analysis using benzoic acid as the internal standard [28].

1 H NMR spectroscopy was performed on mcl-PHA samples (∼20 mg) dissolved in 0.8 mL of deuterated chloroform (SigmaAldrich 99.8 atoms%D) using a Bruker AVANCE 300 spectrometer at 300 o K. The intensity of individual peaks in the 1 H NMR spectrum was used to calculate the percent conversion of double bonds (C C) into carboxyl groups based on equation 1 [29].

% Conversion =

100[(% C = C)initial − (%C = C)unreacted ] (%C = C)unreacted

[1]

where (%C = C)unreacted =

100 × Peak 8 Peak 3

Peak 3 (Fig. 1b), the integration of the peak centered at 5.18 ppm, is the initial mol% concentration of the methine backbone protons ( HC O). Peak 8, the integration of the peak centered at 5.75 ppm, is the initial mol% concentration of the methine group ( HC ) of the terminal olefin. The intensity of Peak 3 was set to 1 in each spectrum.

2.5. Molecular weight determination The molecular weight of the polymers was determined by gel permeation chromatography (GPC) using a Waters 2690 gel permeation chromatograph equipped with a Waters 410 differential refractometer and polystyrene standards. Each sample was prepared by dissolving 40 mg of polymer in 10 mL of tetrahydrofuran (THF) (Fisher Scientific, Canada) overnight to ensure complete dissolution, then filtered through a 0.2 ␮m nylon filter. THF was used as the eluent at a flow rate of 0.3 mL/min through four Waters Styragel columns (4.6 × 300 mm) of pore sizes 104 , 103 , 102 and 500 ␮m maintained at 40 ◦ C with an injection volume of 30 L.

2.2. Carboxylation of PHNUs 2.6. Contact angle Synthesis of PHNCs (carboxylated products of PHNU) was performed as described by Hany et al. [18] (reaction scheme shown in Fig. 1a). PHNU was dissolved in a mixture of toluene (1:10 wt PHNU per vol, Anachemia Canada Inc., Montreal QC, Canada), 11-mercaptoundecanoic acid (3.5 equiv. per unsaturated units in PHNU, unless otherwise stated, W & J PharmaChem Inc., MD, USA), and 2,2 -azobis(2-methylpropionitrile) (AIBN, 0.2 equiv. per unsaturated units in PHNU, Sigma-Aldrich, Oakville ON, Canada) under nitrogen. The reaction solution was heated to 75 ◦ C for 17 h, cooled to room temperature, then slowly added drop wise into ice cold methanol (1:10 v/v, ACP, Montreal QC, Canada), and left in the fume hood overnight. After the solvent was removed, the product was dissolved in a minimal amount of methylene chloride (ACP, Montreal QC, Canada), which was slowly added to ice cold methanol (1:10 v/v) for further purification. The precipitated product was left in the fume hood overnight, then dried under vacuum in a desiccator. Two carboxylated PHAs were prepared: PHNC-11 with 11% carboxylation and PHNC-18 with 18% carboxylation.

Films were formed by spin coating 0.1 g of the polymer dissolved in 20 mL of tetrahydrofuran using a Laurell WS-650-MZ spin coater, and dried in a desiccator. Drops (2 ␮L) of Milli-Q water were automatically dispensed on the surface of the polymer film at room temperature and allowed to equilibrate (10–15 s) before the static image of the drops was captured with a precision camera and the contact angles calculated by the computer with an accuracy of ± 0.5◦ using a VCA Optima goniometer (AST Products Inc, Billerica, MA, USA). 2.7. Preparation of mcl-PHA suspensions Mcl-PHA dissolved in methylene chloride was added to an aqueous surfactant solution, and homogenized for 3 min using a rotor-stator system (SDT Tissumizer, Tekmar Company, USA, equipped with an 18 mm diameter stator at 10% of the power output of the Tekmar power controller (model TR-10)), to form a

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Fig. 1. a) Reaction scheme for conversion of alkenes to carboxylic acids via a radical addition reaction with 11-mercaptoundecanoic acid [18], b) 1 H NMR spectrum of PHNU-11 in deuterated chloroform.

premix emulsion in an ice bath. The premix emulsion was ultrasonicated (20 KHz Ultrasonic Homogenizer 4710 Series, Cole Parmer Instrument Co., IL, USA, equipped with a microtip probe of 3.2 mm diameter) in an ice bath to produce a suspension in water. For example, 10% (w/v) suspensions were prepared by dissolving 1 g of the desired PHA in 10 mL of methylene chloride before adding to 9 mL of aqueous surfactant solution. The homogenized emulsion was then subjected to ultrasonication. Methylene chloride was removed by rotary evaporation, and the suspension transferred to a graduated centrifuge tube. Distilled, deionized water was added to obtain 10% (w/v) solids content.

2.8. Selection of surfactants at 0.4% solids of 11% carboxylated mcl-PHA (PHNC-11) 2.8.1. Without surfactant Colloidal suspensions of 0.4% (w/v) mcl-PHA with 11% carboxylation (PHNC-11) were made without surfactant. Suspensions were

fabricated with and without the premix step, then subjected to ultrasonication for 10 min at 20% amplitude. 2.8.2. Selection of a non-ionic surfactant The particle size was evaluated with four non-ionic surfactants (5 mM) of 0.4% (w/v) PHNC-11 at a sonication amplitude of 20% for 10 min. The non-ionic surfactants were polyoxyethylene ® octyl phenyl ether (Triton X-100, OmniPur ), sorbitan monooleate (Span 80), polyoxyethylene (20) cetyl ether (Brij 58), and polyoxyethylene (20) sorbitan monooleate (Tween 80), purchased from Sigma-Aldrich Canada (Oakville ON, Canada). 2.8.3. Selection of an ionic surfactant with triton X-100 Suspensions containing 0.4% (w/v) PHNC-11 were prepared as described above at a sonication amplitude of 20% for 10 min in 5 mM Triton X-100 plus 2 mM of an ionic surfactant, dodecyltrimethylammonium bromide (DTAB), cetyltrimethylammonium bromide ® (CTAB), alkyldiphenyloxide disulfonate (Dowfax 2A1) or sodium

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Table 1 Mcl-PHA molecular weights measured by GPC. Mn is the number average molecular weight, Mw is the weight average molecular weight and Mw /Mn is the polydispersity index. Mcl-PHA

Carboxylation (mol%)

Mn (g mol−1 )

Mw (g mol−1 )

Mw /Mn

PHNU-11 PHNC-11 PHNU-18 PHNC-18

0 11 0 18

23377 30641 52408 55684

37871 48413 94334 101345

1.6 1.6 1.8 1.8

NMR spectroscopy. Without 11-mercaptoundecanoic acid, no double bonds were lost nor carboxyl groups added. As the molar ratio of 11-mercaptoundecanoic acid to unsaturated units was increased from 0 to 4, the −COOH content increased and a minimum of 3.5 equiv. of 11-mercaptoundecanoic acid was needed for the complete conversion of the olefin groups. The increase in molecular weight (Table 1) was due to the grafting of an additional 11 carbons (from 11-mercaptoundecanoic acid) via the thioester linkage to the C9 and C7 monomers of PHNU (Fig. 1a). 3.2. Preliminary experiments at 0.4% (w/v) solids

dodecyl sulfate (SDS). The first two are cationic and the latter two are anionic surfactants. All surfactants were obtained from SigmaAldrich Canada (Oakville ON, Canada). The HLB of a surfactant mixture was calculated as HLB = a1 HLB1 + a2 HLB2 where a1 and a2 are the weight fractions of the two surfactants which have HLB1 and HLB2 values respectively [30]. 2.9. Evaluations at 10% solids 2.9.1. Effect of SDS concentration Suspensions of 10% (w/v) PHNC-11 were made as described above with an increasing SDS concentration from 1 to 8 mM, using an ultrasonication time and amplitude of 10 min and 20% respectively. 2.9.2. Combining SDS with triton X-100 Suspensions of 10% (w/v) PHNC-11 were fabricated as described above in 5 mM SDS with 5–25 mM Triton X-100, at a sonication amplitude of 20% for 10 min. 2.9.3. Effect of carboxyl group Colloidal suspensions were made with carboxylated (PHNC-11 with 11% carboxylation and PHNC-18 with 18% of carboxylation) and non-carboxylated (PHN and PHD) mcl-PHAs in 5 mM SDS and 20 mM Triton X-100 at 10% solids, at an ultrasonication time and amplitude of 10 min and 20% respectively. 2.9.4. Statistics One-way ANOVA analyses were performed to determine the statistical significance between different surfactants. The hypothesis that the average particle sizes being compared were equal was rejected if the p-value was less than 0.05 (i.e. 95% confidence). Data compared were the mean ± standard deviation for n = 3 values. 3. Results and discussion 3.1. Conversion of vinyl to carboxyl groups The oxidizing agent, 11-mercaptoundecanoic acid, was selected since the degree of carboxylation is more easily controlled with little to no loss of molecular weight [18] compared to methods such as using KMnO4 [17]. PHNU was carboxylated as described by Hany et al. [18] and the composition of the product was determined by 1 H

Initial screening experiments to select surfactants were done at 0.4% (w/v) of carboxylated mcl-PHA with particle size as the main criterion. Later experiments performed at 10% (w/v) solids focused on particle size and stability. Nanoparticles were made without surfactant by adding a solution of 11 mol% carboxylated mcl-PHA dissolved in methylene chloride to water alone to form an oil-inwater emulsion. Once the solvent was evaporated, nanoparticles were left suspended in water. Without a surfactant, the average particle size in suspension was 320.1 ± 7.5 nm (Table 2), but the dispersion was poor. Immediately after mixing, some agglomerates were observed floating on the surface of the mixture, and adhering to the inside surface of the tube in which the emulsion was prepared as well as to the ultrasonic probe. This indicates that van der Waals attraction between the particles was greater than the repulsive forces [30] and that addition of surfactant(s) would be required to increase repulsive forces. 3.2.1. Evaluation of non-ionic surfactants with PHNC-11 Non-ionic surfactants were evaluated to prevent nanoparticle aggregation. Surfactants with a hydrophilic-lipophilic balance (HLB) between 8 and 18 form oil-in-water (O/W) emulsions [30]. Of the non-ionic surfactants tested, Triton X-100, Tween 80 and Brij 58 will form oil-in-water emulsions while Span 80 forms water-in-oil emulsions. A concentration of 5 mM was chosen based on a previous study with aliphatic mcl-PHA [27] to ensure excess surfactant. All surfactants tested significantly reduced the particle size (Table 2), at a p value less than 0.001 (i.e.>99.9% confidence) in a one-way ANOVA analysis and there was no visible agglomeration over a period of 30 days. Furthermore, particle size distribution (PdI) was significantly narrower with a surfactant. The hydrophobic tails of the surfactant would have adsorbed onto the particles while the hydrophilic heads created a steric repulsive barrier between particles to achieve smaller particles and prevent agglomeration. The particle size distribution showed a single population of nanoparticles with a symmetrical peak, indicating good miscibility of the two phases. HLB had no effect on particle size. Triton X-100, which has the shortest hydrophobic tail, formed the smallest particles (155.6 ± 8.4). Only one research group has previously published data stabilizing carboxylated mcl-PHA nanoparticle suspension using a surfactant [14,17,19]. They used Pluronic F-68, a triblock copolymer, (polyethylene oxide-polypropylene oxide-polyethylene oxide) non-ionic surfactant, in a precipitation-solvent evaporation

Table 2 Effect of non-ionic surfactants (5 mM) on particle size for 0.4% (w/v) PHNC-11 with premixing the emulsion (3 min homogenization) before ultrasonication at an amplitude of 20% for 10 min in an ice bath except at * when there was no premixing. Data are mean ± standard deviation n = 3. Non-ionic surfactant

HLB

Hydrophobic tail

Particle size (nm)

PdI

No surfactant Span 80 Triton X-100 Triton X-100* Tween 80 Brij 58

– 4.3 13.6 13.6 15 15.7

– C17 C9-10 C9-10 C17 (3 tails) C16

320.1 ± 7.5 163.4 ± 11.3 155.6 ± 8.4 194.2 ± 11.2 165.8 ± 10.1 168.2 ± 9.3

0.59 ± 0.08 0.42 ± 0.03 0.43 ± 0.03 0.49 ± 0.04 0.45 ± 0.04 0.45 ± 0.04

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Table 3 Effect of 5 mM non-ionic surfactant (Triton X-100 (HLB = 13.6), Tween 80 (HLB = 15), or Span 80 (HLB = 4.3)) and 2 mM ionic surfactant on particle size for 0.4% (w/v) mcl-PHA (11% carboxylation). Data are the mean ± standard deviation for n = 3. Surfactants

HLB of ionic surfactant

HLB of surfactant mixture

Hydrophobic tail

Particle size (nm)

PdI

Triton X-100 alone Anionic +SDS +Dowfax 2A1



13.6

C9-10

155.6 ± 8.4

0.43 ± 0.03

40 16.7

17.6 14.7

C12 C10

97.1 ± 1.1 120.5 ± 3.9

0.21 ± 0.001 0.25 ± 0.001

Cationic +DTAB +CTAB

12.9 10

13.3 11.1

C12 C16

115.6 ± 3.3 121.7 ± 5.7

0.24 ± 0.003 0.25 ± 0.003

Tween 80 alone Anionic +SDS



15

C17

165.8 ± 10.1

0.45 ± 0.04

40

17

C12

122.6 ± 4.4

0.31 ± 0.03

Cationic +CTAB

10

14.4

C16

136.2 ± 7.5

0.37 ± 0.03



4.3

C17

163.4 ± 11.3

0.42 ± 0.03

12.9

6.1

C12

128.8 ± 6.7

0.32 ± 0.05

Span 80 alone Cationic +DTAB

method and obtained a broad range of particles sizes requiring a separation step to recover the nanoparticle fraction. Pluronic F68 has an HLB >24 [31]. Such a high HLB value may not be desirable for making nanoparticles of carboxylated mcl-PHA. The surfactants in the present study had lower HLB values (<16), and with intensive mixing, made smaller particles with a much smaller PdI and eliminated the need for a post-emulsification fractionation. Since carboxylated mcl-PHAs are more hydrophilic, the interfacial tension between the “oil” and water phases is lower than for non-carboxylated mcl-PHA, and hence, less energy should be required to make smaller particles. However, although ultrasonication alone resulted in 194.2 ± 11.2 nm particles, the combination of both homogenization to “premix” and ultrasonication were necessary to make significantly smaller nanoparticles of 155.6 ± 8.4 nm with a similar PdI. “Premixing” using a homogenizer disrupts droplets to form smaller ones, producing a large surface area to be coated with surfactant. Ultrasonication further disrupts these droplets, which should not coalesce if the amount of surfactant is sufficient to cover the larger total surface area and provide steric repulsion. For the purpose of this study, all further suspensions were prepared with a homogenization step prior to ultrasonication.

3.2.2. Evaluation of non-ionic and ionic surfactant combinations with PHNC-11 Typically, stable nanoparticles at high solids content are made with a non-ionic surfactant in combination with an ionic surfactant to achieve a synergistic stabilization not obtained with the individual surfactants [32]. In this study, both cationic and anionic surfactants were evaluated in combination with a non-ionic surfactant. It was found that any combination tested resulted in significantly smaller particles (Triton X-100, p < 0.004), Tween 80, p < 0.015) or Span 80, p 0.01)) and smaller PdIs compared to what was obtained with the non-ionic surfactant alone (Table 3). While non-ionic surfactants stabilize by steric repulsion, ionic surfactant creates an electrical double layer causing electrostatic repulsion and further contributing to stability. The addition of an ionic with a non-ionic surfactant likely further reduced the interfacial tension between the PHA solution and the water phase, allowing smaller particles to be formed during emulsification and reducing the amount of energy needed to form them [33]. The narrowest distributions (∼0.24) were found with Triton X-100 in combination with any ionic surfactant, and the smallest particle size with Triton X-100 and the anionic SDS. Cationic and anionic surfactants gave similar average particle sizes. Even though Bouchemal et al. [34] found that the droplet size

of oil-in-water nanoemulsions decreased as the HLB of non-ionic surfactant mixtures increased from 10 to 21, the data in Table 3 show no statistically significant difference in particle size or particle size distribution with HLB values between 6.1 and 15. However, when the HLB of the mixture of non-ionic and ionic surfactants was ≥ 17, the particle sizes were significantly smaller. This occurred when SDS was the ionic surfactant. Since there was no relationship between HLB of the non-ionic surfactants and particle size (Table 2), this effect was likely influenced by the very hydrophilic SDS (HLB = 40). The higher water solubility of the charged surfactant head may have enhanced the electrical double layer, and hence the electrostatic repulsion resulting in smaller particles. Since the Triton X-100 and SDS combination gave the smallest particles (p < 0.001), it was selected to evaluate making stable nanoparticles at a higher solid content (10% (w/v)).

Fig. 2. Effect of SDS concentration on (a) zeta potential and (b) particle size in 10% (w/v) PHNC-11 at sonication amplitude of 20% for 10 mins. Data are the mean ± standard deviation for n = 3.

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3.3.2. Effect of SDS with triton X-100 on PHNC-11 particle size and stability At 10% (w/v) PHNC-11 and 5 mM SDS, increasing the Triton X100 concentration from 5 to 20 mM significantly decreased the particle size from 221 ± 6.8 to 170 ± 4.6 nm (Fig. 3b). Suspensions with ≤ 10 mM Triton X-100 would be considered unstable as the zeta potential was between −30 and 30 mV. At 20 mM Triton X100, stability as indicated by the zeta potential (-39.6 ± 0.92 mV) plateaued (Fig. 3a). Hence, the combination of 5 mM SDS and 20 mM Triton X-100 was selected to investigate other mcl-PHAs at the same solids content.

Fig. 3. Effect of 5 mM SDS and Triton X-100 concentrations on (a) zeta potential and (b) particle size in 10% (w/v) mcl-PHA suspensions at 20% amplitude and 10 min of sonication. Data are the mean ± standard deviation for n = 3.

3.3. Evaluation of surfactants at 10% (w/v) 3.3.1. Effect of SDS on PHNC-11 particle size and stability Increasing the SDS concentration from 1 to 8 mM decreased the nanoparticle size from 285.2 ± 12.3 to 198.7 ± 1.92 nm (Fig. 2b) while the particle size distribution decreased from 0.42 to a minimum of ∼ 0.22 at SDS concentrations ≥5 mM. Thus, at 10% (w/v) carboxylated mcl-PHA, ≥ 5 mM SDS will cover the nanoparticles. This is supported by the zeta potential values, which give an indication of the strength of the energy barrier created by repulsive forces to prevent particles or droplets from approaching each other. Nanoparticles prepared with ≥ 5 mM SDS are stable, as the zeta potential values (−31.3 ± 0.86 to −36.1 ± 0.92 mV) were not between +30 and −30 mV [35] (Fig. 2a). Since a minimum of 5 mM SDS was needed to maintain particle stability, this concentration was chosen for further study to determine the effect of Triton X-100.

3.3.3. Nanoparticles of different mcl-PHAs Similar particle sizes, particle size distributions and stability (zeta potential values) were obtained for both PHN and PHD (Table 4). These two materials have similar hydrophobic pendant side chains and the small difference in the length of the side chain did not affect how they behaved in aqueous suspension. As carboxylation increased, hydrophilicity increased as the ␪H2O decreased from ∼103◦ (no carboxylation) to 81.8 ± 0.6◦ and 72.4 ± 1.4◦ at 11 and 18% carboxylation respectively. This resulted in even smaller particles and further improved stability (Table 4). Higher zeta potential values indicate a stronger repulsive force as carboxylation increased. More importantly, the presence of carboxylic acid side groups reduced the amount of surfactant required to stabilize the nanoparticles. To make a similar particle size of ∼145 nm with comparable stability (i.e. zeta potential ∼ –40 mV) at 10% (w/v) solids, an aliphatic mcl-PHA, PHD, needed twice the molar concentration of the non-ionic surfactant (Triton X-100) and four times more ionic surfactant (SDS) [27] than the 18% carboxylated PHNC-18 (Table 5). At higher carboxylation, even less surfactant should be needed. In an aqueous medium, the carboxylic groups would be oriented at the polymer/water interface decreasing the interfacial tension to help achieve a smaller particle size. In addition, they may contribute to electrostatic repulsion between particles to maintain stability. Kurth et al. [17] were unable to make a stable suspension of 10% carboxylated mcl-PHA using only one non-ionic surfactant (Pluronic F68) and were only able to make particles from 25% carboxylated mcl-PHA with a very wide PdI. Our results show that stable nanoparticle suspensions of carboxylated mcl-PHA, even with low carboxylation, can be made using a mixture of a non-ionic and an ionic surfactant. Since the carboxylic acid groups are likely

Table 4 Effect of 5 mM SDS ± 20 mM Triton X-100 on zeta potential and particle size of 10% (w/v) mcl-PHA suspensions. Data are the mean ± standard deviation for n = 3. Water contact angles are the average of three independent drops on spin coated films. The PHAs were poly-3-hydroxynonanoate (PHN), poly-3-hydroxydecanoate (PHD), PHNU-11 and PHNU-18 with 11 and 18% unsaturation respectively in the pendant side chains, and PHNC-11 and PHNC-18 with 11 and 18% of carboxyl groups in the pendant side-chains. * n.d. − not determined. Mcl-PHA

Particle sizea (nm)

PdI

Zeta potential (mV)

Contact angle (◦ )

PHD PHN PHNU-11 PHNU-18 PHNC-11 PHNC-18

181 ± 3 184 ± 5 n.d.* n.d. 170 ± 5 143 ± 3

0.16 ± 0.002 0.15 ± 0.003 n.d. n.d. 0.12 ± 0.001 0.08 ± 0.002

−35.2 ± 1.3 −34.4 ± 2.6 n.d. n.d. −39.6 ± 0.9 −44.1 ± 0.5

103.5 ± 0.6 102.9 ± 1.0 106.6 ± 0.2 108.4 ± 1.0 81.8 ± 0.6 72.5 ± 1.4

Table 5 Comparison of the amount of surfactant(s) needed to make nanoparticles of a similar size and stability of mcl-PHA with and without carboxylation. All suspensions were made at 10% (w/v) mcl-PHA from which the amount of surfactant needed at 100 g PHA was extrapolated. The PHAs were poly-3-hydroxydecanoate (PHD), PHN with 11% of carboxyl groups in the pendant side-chains (PHNC-11), and PHN with 18% of carboxyl groups in the pendant side-chains (PHNC-18). PHA

Carboxylic acid (%)

Particle size (nm)

Zeta potential (mV)

Surfactant concentration

g Surfactant per 100 g PHA

Reference

PHD PHNC-11 PHNC-18

0 11 18

148 ± 1.72 170 ± 5 143 ± 3

−40.9± 0.9 −39.6 ± 0.9 −44.1± 0.5

40 mM Triton + 20 mM SDS 20 mM Triton + 5 mM SDS 20 mM Triton + 5 mM SDS

31.6 14.4 14.4

[19] This work This work

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oriented at the polymer/water interface in aqueous suspension, carboxylated mcl-PHAs are good candidates as a biocompatible material to attach bioactive molecules for biomedical applications [14,17,36] or as a potential biomaterial for soft tissue engineering to make heart valves, skin grafts or to regenerate nerves [37] in addition to use in xerographic toners [21], coatings [1] and for slow release of agricultural products. This is the first study to show that stable carboxylated mcl-PHA latexes with less than 25 mol% carboxylation can be made above 0.4% solids using a combination of nonionic and ionic surfactants. 4. Conclusions A minimum of 3.5 equiv. of 11-mercaptoundecanoic acid per double bonds in PHNU was needed for the complete conversion of the olefin groups to carboxyl groups. At 0.4% (w/v) of 11% carboxylated mcl-PHA, a combination of SDS and Triton X-100 was found to give the smallest particle size (97.1 ± 1.1 nm) and the narrowest size distribution (PdI = 0.21 ± 0.001). When this combination was evaluated at 10% solids, a mixture of 5 mM SDS and 20 mM Triton X-100 was the best compromise to make stable nanoparticles. At this same surfactant concentration, particle size decreased and stability increased as the degree of carboxylation increased. The hydrophilicity of these materials also increased since the water contact angle decreased from ∼ 103◦ for non-carboxylated PHD and PHN to 81.8 ± 0.6◦ and 72.4 ± 1.4◦ for 11 and 18% carboxylated PHN respectively. Furthermore, carboxylated mcl-PHA needed less surfactant to make nanoparticles of a similar size and stability compared to non-carboxylated mcl-PHAs. Conflicts of interest None. Acknowledgement This research was funded by the Natural Science and Engineering Research Council of Canada (NSERC). References [1] R.S. Whitehouse, L. Zhong, S. Daughtry, Compositions comprising low molecular weight polyhydroxyalkanoates and methods employing same, US Pat. 7,094,840 B2 (2006). [2] J. Asrar, J.R. Pierre, P. D’Haene, Polyhydroxyalkanote coatings, US Pat. 6,025,028 (2000). [3] G. McAneney-Lannen, G.G. Sacripante, E.G. Zwartz, M.N.V. McDougall, Toner compositions and processes, US Pat 8,187,780 (2012). [4] C.S. Cleveland, T.S. Reighard, J.L. Marchman, Biodegradable paper-based laminate with oxygen and moisture barrier properties and method for making biodegradable paper-based laminate, US Pat 8,637,126 B2 (2014). [5] S. Philip, T. Keshavarz, I. Roy, Polyhydroxyalkanoate: biodegradable polymers with a range of applications, J. Chem. Technol. Biotechnol. 82 (2007) 233–247. [6] P.K. Singh, A.K. Sen, A.S. Vidyarth, Diversified application of polyhydroxyalkanoates, Biopharm. J. 2 (2016) 14–26. [7] Z. Li, X.J. Loh, Water soluble polyhydroxyalkanoates: future materials for therapeutic applications, Chem. Soc. Rev. 44 (2015) 2865–2879. [8] L. Luo, X. Wei, G.-Q. Chen, Physical properties and biocompatibility of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) blended with poly(3-hydroxybutyrate-co-4-hydroxybutyrate), J. Biomat. Sci. Polym. Ed. 20 (2009) 1537–1553. [9] L. Mauclaire, E. Brombacher, J.D. Bünger, M. Zinn, Factors controlling bacterial attachment and biofilm formation on medium-chain-length polyhydroxyalkanoates (mcl-PHAs), Colloids Surf. B: Biointerf. 76 (2010) 104–111. [10] H.W. Ulmer, R.A. Gross, M. Posada, P. Weisbach, R.C. Fuller, R.W. Lenz, Bacterial production of poly(␤-hydroxyalkanoates) containing unsaturated repeating units by Rhodospirillum rubrum, Macromolecules 27 (1994) 1675–1679.

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