Amphiphiles from Poly(3-hydroxyalkanoates)

Authored by: Baki Hazer

The Handbook of Polyhydroxyalkanoates

Print publication date:  October  2020
Online publication date:  October  2020

Print ISBN: 9780367541071
eBook ISBN: 9781003087663
Adobe ISBN:

10.1201/9781003087663-4

 

Abstract

Polyhydroxyalkanoates (PHA) are biodegradable and biocompatible microbially produced natural polyesters. However, their hydrophobic character is a disadvantage for the direct use of these polyesters. The key to biocompatibility of biomedical implantable materials is to alter their surface in a way that minimizes hydrophobic interaction with the surrounding tissue. Therefore, hydrophilic groups have been introduced into PHA in order to obtain amphiphilic polymers. This chapter has focused on the chemically modified PHA with enhanced hydrophilic character as biomaterials for medical applications.

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Amphiphiles from Poly(3-hydroxyalkanoates)

3.1  Poly(3-hydroxyalkanoates) (PHA)

Very widely produced and used commercial packaging plastic materials cause severe problems in the natural environment. Plastic pollution has been seen in the ground, in oceans, and in lakes because of its non-degradability. Mainly fuel fossil-based monomers have also been used in the production of these polymers. This is another disadvantage of the commercial non-degradable polymers, because fossil fuel reserves are limited and decreasing all the time. One of the solutions for plastic pollution is to use biodegradable polymers obtained from renewable resources. Microbial polyesters are very good candidates of these type of polymers; they can be obtained from natural products such as sugar, fatty acids, alkanoic acids, etc. via green chemistry [1,2]. PHA exhibit valuable characteristics, such as biodegradability, biocompatibility, and thermoplasticity, and therefore can be used for medical, agricultural, and marine applications. PHA accumulate to high levels in bacteria (95% of the cellular dry mass), and their structures can be manipulated by genetic or physiological strategies [3–10]. Large number of bacteria, including Ralstonia eutropha (today: Cupriavidus necator) and Pseudomonas putida, produce PHA during metabolic stresses, such as a limitation of nitrogen, oxygen, or other essential nutrients in the presence of an excess of carbon source [11–16].

The general formula of the PHA is shown in Figure 3.1. The pendent alkyl group is decisive for the physical properties and the classification of the PHA. PHA are divided into three groups according to the pendant R group; short-chain-length PHA (scl-PHA) comprising 1–2 carbon atoms, medium-chain-length PHA (mcl-PHA) comprising 3–11 carbon atoms, and long-chain-length PHA (lcl-PHA) comprising more than 11 carbon atoms. The types of PHA are listed in Table 3.1. These PHA show different thermal and physical properties. Generally, mcl-PHA show lower T m and T g and more flexibility compared with scl-PHA. These mcl-PHA which can be produced using renewable resources are biocompatible, biodegradable, and thermoprocessable. They have low crystallinity, low glass transition temperature, low tensile strength, and high elongation at break, making them elastomeric polymers. Mcl-PHA and their copolymers are suitable for a range of biomedical applications where flexible biomaterials are required, such as heart valves and other cardiovascular applications, as well as matrices for controlled drug delivery. Mcl-PHA are more structurally diverse than short-chain-length PHA and hence can be more readily tailored for specific applications.

General chemical formula of PHA.

Table 3.1   Types of PHA

Poly(3-hydroxyalkanoate), PHA

Type of PHA accumulated

Bacterium (production strain)

Side chain (R)

Name of PHA*

T g [°C]

T m [°C]

Elongation at break [%]

scl-PHA

R. eutropha (today: C. necator)

methyl

PHB

0–15

137–170

5–30

ethyl

PHV

mcl-PHA

P. oleovorans (today: P. putida)

propyl

PHHx

butyl

PHHp

−40–−20

45–90

600–800

valeryl (pentyl)

PHO

hexyl

PHN

heptyl

PHD

lcl-PHA

P. oleovorans (today: P. putida)

higher than octyl

−50

40

soft, sticky

*HB: 3-hydroxybutyrate, HV: 3-hydroxyvalerate, HHx: 3-hydroxyhexanoate, HHp: 3-hydroxyheptanoate, HO: 3-hydroxyoctanoate, HN: 3-hydroxynonanoate, HD: 3-hydroxydecanoate. T g and T m are glass transition and melting temperatures, respectively.

3.2  Amphiphilic Polymers, General Introduction

Amphiphilic copolymers contain both hydrophilic and hydrophobic blocks. Amphiphilic polymers can be synthesized by introducing hydrophilic groups such as hydroxyl, carboxyl, amine, glycol, and hydrophilic polymers such as PEG, poly(vinyl alcohol), poly(acryl amide), poly(acrylic acids), hydroxyethylmethacrylate, poly(vinyl pyridine), and poly(vinyl pyrrolidone) onto a hydrophobic moiety. Such amphiphilic copolymers find numerous applications as emulsifiers, dispersants, foamers, thickeners, rinse aids, and compatibilizers [17–24].

3.3  Amphiphilic PHA via Chemical Modification Reactions

Amphiphilic PHA are hydrophobic, which is a disadvantage for some medical applications. Therefore, the hydrophobic natural polyesters, PHA, need to have a hydrophilic character, particularly for tissue engineering and drug delivery systems [25].

PHA are grafted with some hydrophilic groups in order to enhance hydrophilicity. These hydrophilic moieties are listed in Table 3.2.

Table 3.2   The Hydrophilic Moieties Used in the Preparation of the Amphiphilic PHA

Hydrophilic moieties

-COOH

-OH

-NH2

Amino acid

Poly (ethylene glycol)

Acrylic acid

Methacrylic acid

N-isopropyl acryl amide

Dimethyl amino ethyl methacrylate

3.4  Amphiphilic PHA

Microbial polyesters are excellent biodegradable hydrophobic biopolymers. Because of their limited mechanical, thermal, and non-hydrophilic properties, they are needed to diversify in reasonable chemical reactions to be able to be competitive against the convenient petroleum-based biomaterials [26].

There are three types of PHA: short-chain-length (e.g., PHB, PHBV), medium-chain-length (e.g., P(3HHx), P(3HHp), P(3HO), P(3HN), PHD, and P(3HU)), and long-chain-length (PHA derived from fatty acids) [13,14]. Despite the many PHA that are produced by many different type of bacteria and identified in detail, only a few types of PHA have undergone the hydrophilic modification reactions: poly(3-hydroxyoctanoate-co-3-hydroxyundecenoate) (PHOU), PHO, PHN, poly(3-hydroxybutyrate) (PHB), poly(3-hydroxybutyrate-co-3-hydroxyvalerate), PHBV, and unsaturated mcl-PHA obtained from polyunsaturated plant oily acids. Here, we study the chemical reactions of the specific PHA one by one to gain hydrophilicity.

3.4.1  Amphiphilic PHOU Derivatives

Double bond functionality is readily open for the modification reactions. Therefore, the first step was the production of unsaturated mcl-PHA by Lenz’s group. In an early experiment, Pseudomonas oleovorans was grown separately on 3-hydroxy-6-octenoic acid and 3-hydroxy-7-octenoic acid as the only carbon source and under ammonium nutrient-limiting conditions to produce storage polyesters. The polyesters produced contained mainly unsaturated C8 units [27].

However, the poly (3-hydroxyoctenoate) was rarely used in the derivatization reactions. The first attempt of the hydrophilic modification of the PHA started with the production of Poly(3-hydroxyoctanoate-co-3-hydroxyundecenoate), (PHOU) [28]. For this purpose, Pseudomonas oleovorans was grown on 10-undecenoic acid and on a mixture of sodium octanoate and 10-undecenoic acid. In the case of 10-undecenoic acid as the sole nutrient, the microorganism produced a polymer containing only repeating units with unsaturated side chains. Both the melting temperature (T m ) and glass transition temperature (T g ) were observed to decrease with increasing conversion of olefinic bonds. PHOU is a soft elastomer that is easy to handle while PHU is sticky and waxy. Interestingly, the fermentation of a mixture of nonanoic acid and 10-undecenoic acid gave a homopolymer mixture of PHN and PHU [29].

PHOU containing pendant hydroxyl and carboxylic acid groups can be obtained by varying oxidation conditions. Pendant double bonds can be oxidized by strong oxidized agents such as KMnO4 at 20°C in order to obtain hydroxylated PHOU. The polymers which were 40–60% hydroxylated were completely soluble in polar solvents including an 80/20 acetone/water mixture, methanol, and DMSO, indicating a considerably enhanced hydrophilicity of the modified PHA [30,31].

The increase in percentage of hydroxyl groups of PHU and its improved water solubility are important for preparing drug delivery systems, artificial organs, and tissue engineering applications. Nearly 100% hydroxylation of double bonds of PHU was achieved by a hydroboration–oxidation reaction using 9-borobicyclononane. The hydroxylated PHU was fully soluble in methanol and almost soluble in water [32].

The pendant double bonds can be converted to pendant carboxylic acids using a biphasic CH2C12/CH3COOH medium in the presence of KMnO4 and 18-crown-6-ether [33].

PHA containing pendant carboxyl groups was prepared by the chemical modification of unsaturated PHA using KMnO4 at 55°C, although there is a severe loss in molecular weight of PHA during the reaction. The degree of carboxylation increases to approximately 50% after 2 h of reaction time, but there is no further increase with prolonged reaction times. The polymers with a degree of carboxylation of 40–50% are completely soluble in water/Na2CO3, indicating a considerably enhanced hydrophilicity of the modified PHA [34].

With the use of osmium tetroxide and oxone (a triple salt mixture of 2KHSO5, K2SO4, and KHSO4), double bonds of PHOU are converted to carboxylic groups, and the oxidation proceeds to completion with little backbone degradation [35]. It is worth saying that these modification reactions cause a severe decrease in molar masses of the PHA.

PHO and PCL films are not degraded at 37°C at pH 7.3 over 275 days. In the case of PHO75COOH25 films and copolymers P((HOCOOH)-b-CL), the presence of the carboxyl group promotes water penetration into the polymer and participates in ester group hydrolysis through better water penetration and catalysis. Results showed that the adhesion of cells was better on PHO75COOH25 films than in PHO films. This result can be explained by the presence of carboxyl groups at the films’ surface which promoted cell adhesion [36].

The presence of carboxyl groups at the films’ surface promotes cell adhesion. The biodegradation of PHO and PCL films are not degraded in aqueous solution at 37°C at pH 7.3 for nine months while PHOU-COOH is degradable because water penetration into polymer increases. Figure 3.2 shows the comparison between biodegradability of PHOU-COOH with some polymers.

Plots of the molar masses versus time in aqueous solution. Hydrolytic degradation of the different polymers at pH 7.3 at 37°C [

Figure 3.2   Plots of the molar masses versus time in aqueous solution. Hydrolytic degradation of the different polymers at pH 7.3 at 37°C [36].

3.5  Epoxidation

Epoxidation of the double bonds is very useful tool to obtain amphiphilic PHA. PHU epoxidation with m-chloroperbenzoic acid yields to quantitative conversions of the unsaturated groups into epoxy groups. There is no side reaction on the macromolecular chain by molecular weight measurements. It has also been possible to produce new functional bacterial polyesters containing terminal epoxy groups in the side chains, in variable proportions up to 37% by growing P. oleovorans on a 10-epoxyundecanoic acid and sodium octanoate culture feed mixture. But the chemical epoxidation reaction was widely used and shown that these compounds were totally epoxidized by using meta-chloroperbenzoic acid (MCPBA) [37–39].

3.6  The Polycationic PHA

Epoxide groups can be opened by diethanol amine in order to obtain a water-soluble hydroxyl derivative of the PHOU, resulting in the polymer poly(3-hydroxy-octanoate)-co-(3-hydroxy-11-(bis(2-hydroxyethyl)-amino)-10-hydroxyundecanoate) (PHON) [40]. PHON binds and condenses the DNA into positively charged particles smaller than 200 nm (Figure 3.3). In this manner, PHON protects plasmid DNA from nuclease degradation for up to 30 min. In addition, treatment of mammalian cells in vitro with PHON/DNA complexes results in a luciferase expression as the result of the delivery of the encoded gene [41].

Formation of PHON/DNA complex [41].

Epoxy groups are chemically modified via the attachment of a peptide sequence such as Arg-Gly-Asp (RGD), to obtain biomimetic scaffolds. For tissue engineering, immobilizing the RGD sequence with aliphatic polyesters, amine groups of RGD were grafted onto the epoxidized PHOU through epoxy-amine chemistry. The biological response of these new functional PHA scaffolds helps have more effect on cellular response [42].

3.7  PHOU with Pendant PEG Units

PHA-PEG amphiphilic polymers find a wide variety of medical applications. For a medical application, adsorption of blood proteins and platelets on the modified PHA, PEG-g-PHU networks, are studied by Chung et al. [43]. Poly(ethylene glycol)-grafted poly(3-hydroxyundecenoate) (PEG-g-PHU) networks are prepared by irradiating the solution of PHU and the monoacrylate of poly(ethylene glycol) with UV light. The obtained cross-linked PHU and PEG-g-PHU result in lower adhesion of blood proteins and platelets. Blood compatibility increases as the PEG block increases.

Unsaturated mcl-PHA are also obtained by growing Pseudomonas oleovorans from the unsaturated fatty acids of the polyunsaturated plant oils [44]. Figure 3.4 shows the synthesis of the two types of unsaturated copolymers.

Synthesis of two types of unsaturated PHA from Pseudomonas oleovorans (I) grown on soybean oil (PHA-Sy) and (II) 10-undecenoic acid and octanoic acid (PHOU) [46].

The side chain olefinic groups of the PHA can be converted to cross-linked PHA-g-PEG amphiphilic graft copolymers. For this purpose, a macroazo initiator derived from PEG and azo-bis-cyano pentanoyl chloride, was reacted with this unsaturated PHA [45]. This free radical addition of the PEG blocks on the unsaturated PHA results in the cross-linked PHA-g-PEG conjugates amphiphilic graft copolymers.

Synthesis of a PHOU-PEG comb-type graft copolymer can be carried out by the polyesterification reaction between carboxylic acid derivative of PHOU and PEG-OH using the catalyst system dicyclohexyl carbodiimide and dimethyl amino pyridine. The graft copolymer is much more resistant to hydrolysis, at physiological pH, than PHOU-COOH. Water contact angles of PHOU-COOH and PHOU-PEG are very close to water contact angle of PHO [46].

3.8  Click Reactions

3.8.1  Thiol-Ene Click Reactions

Thiol-ene click reactions to double bonds are very attractive for producing carboxyl and hydroxyl derivatives of the unsaturated PHA without the polyester degradation. To do this, a methylene chloride solution of mercapto propionic acid or 3-thioglycerol in the presence of a photocatalyst in a Pyrex tube is irradiated by a mercury lamp. The same click reaction is also carried out by using the same thiols with the mcl-PHA obtained from soybean oil [47]. The PHOU-PEG amphiphilic copolymer leads to the formation of multi-compartment micelles [48]. Figure 3.5 shows the reaction design of the carboxylic acid (I) and hydroxyl (II) derivatives of PHOU and their 1H NMR spectra.

Reaction design of the carboxylic acid (I) and hydroxyl (II) derivatives of PHOU and their 1H NMR spectra [47].

Glycopolymers are emerging as a novel class of neoglycoconjugates useful for biological studies. Thiosugar, a maltose-containing thiol group, is a potent tool in glycobiology. In order to obtain the anti-Markovnikov adduct, the thiol end of the thiol sugar is added to the double bond of the PHOU in the presence of diethylamine [49].

Poly(3-hydroxyoctanoate-co-3-hydroxyundecenoate) (PHOU) is methanolyzed and its unsaturated side chains are quantitatively oxidized to carboxylic acid. Alkyne-containing “clickable” PHA is obtained by the esterification with propargyl alcohol [50]. A click reaction of propargyl terminated PHOU with the azide terminated PEG leads to a PHOU-PEG graft copolymer. Figure 3.6 renders the synthesis of a PHOU-g-PEG graft copolymer using an azide-acetylene click reaction.

Synthesis of PHOU-g-PEG graft copolymer using azide-acetylene click reaction [50].

An unsaturated mcl-PHA, poly(3-hydroxyoctanoate-co-3-hydroxynonenoate), PHONe, was sulfonated by a radical thiol-ene or thiol-yne reaction in the presence of sodium mercapto ethane sulfonate. Figure 3.7 shows the reaction steps to prepare a sulfonate derivative of the unsaturated PHA [51].

Synthesis of sulfonate derivative of PHOU using thiol-ene reaction with 3-mercapto-1-ethane sulfonate (redrawn from Ref. [51]).

Amphiphilic graft copolymers composed of PHA and thiol-appended PEG were synthesized by a thiol-ene addition [52]. Nanoscale polymersomes with a diameter of 63 nm and a membrane thickness of 8–9 nm were fabricated. Such a nanoparticulate system was assessed in encapsulation properties and biodegradability for potential use as delivery carriers.

A series of diblock copolymers based on a fixed poly(ethylene glycol) (PEG) block (5 000 g mol–1) and a varying poly(3-hydroxyoctanoate-co-3-hydroxyhexanoate) (PHOHHx) segment (1 500–7 700 g/mol were synthesized using “click” chemistry. These copolymers self-assembled to form micelles in aqueous media. With increasing PHOHHx length, narrowly distributed spherical micelles with diameters ranging from 44 to 90 nm were obtained, with extremely low critical micelle concentration (CMC) of up to 0.85 mg/L [53].

A glycosaminoglycan-like marine exopolysaccharide, EPS HE800, was grafted to PHA in order to enhance cell adhesion. Novel graft copolymer HE800-g-PHA was prepared to improve the compatibility between hydrophobic PHA and hydrophilic HE800. The carboxylic end groups of PHA oligomers were first activated with acyl chloride functions, allowing coupling to hydroxyl groups of HE800. Fibrous scaffolds were prepared by a modified electrospinning system which simultaneously combined PHA electrospinning and HE800-g-PHA copolymer electrospraying. Adhesion and growth of human mesenchymal stem cells on the HE800-g-PHA scaffolds showed a notable improvement over those on PHA matrices [54]. The interactions between the polymer and one of the main biomembrane components, 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) was studied using the Langmuir monolayer technique and Brewster angle microscopy. The addition of lipid to a polymer film does not change the monolayer phase behavior; however, the interactions between these two materials are repulsive and fall in two composition-dependent regimes [55]. The representative design of the synthesis of some modification reactions of the unsaturated mcl-PHA is summarized in Figure 3.8.

Representative design of the synthesis of some unsaturated mcl-PHA conjugates.

3.9  Saturated PHA with Hydrophilic Groups

Saturated PHA are mainly PHB, PHBV, and PHO without any functional groups, such as double bonds, which can be easily modified. Therefore, the modification reactions reported of the saturated PHA are very limited. Among them is transesterification with poly(ethylene glycol), adding functional groups and copolymerization with hydrophilic monomers.

Some ester group(s) of the PHA is exchanged with an alcohol in the transesterification process. Transesterification is carried out in melt or in solution. Hydroxylation of the PHBs via chemical modification is usually achieved by the transesterification reactions to obtain diol ended PHB. Transesterification reactions in the melt between poly(ethylene glycol), mPEG, and PHB yield a diblock amphiphilic copolymer with a dramatic decrease in molecular weight. Catalyzed transesterification in the melt is used to produce diblock copolymers of poly([R]-3-hydroxybutyric acid), PHB, and monomethoxy poly(ethylene glycol), mPEG, in the presence of a catalyst, in a one-step process. The formation of diblocks is accomplished by the nucleophilic attack from the hydroxyl end group of the mPEG catalyzed by bis(2-ethylhexanoate) tin [56,57].

PHB-b-mPEG diblock copolymers can self-assemble into nanoparticles which could find use as drug carriers, binders, and other specialty applications. Such drug carriers may show a longer lifetime in the bloodstream for they are robust versus dilution, that is, they have no critical micelle concentration because their core is hydrophobic and crystalline, and hence will not dissociate because of low concentration [58].

The refluxing of PHB and poly(ethylene glycol) bis (2-aminopropyl ether) with M w 1 000 and 2 000 gave the block copolymers in a one-step transesterification reaction. According to the equivalency, AB and ABA type of block copolymers could be obtained.

PHB and PHU were microbially synthesized from Alcaligenes eutrophus (today: C. necator) fed with oleic acid and from Pseudomonas oleovorans fed with 10-undecenoic acid, respectively, according to the procedure cited in the literature. 1,2-Dichlorobenzene (DCB), 1,4-butanediol, poly(ethylene glycol) bis (2-aminopropyl ether) of average M w 1 000 g/mol (PEG1KNH2) and M w 2 000 g/mol (PEG2KNH2) were gifts from the Huntsman Co. (Switzerland) [59]. Transamidation reactions of PHB with primary amine-terminated poly(ethylene glycol) yield linear block copolymer of PHB with amine ends. PHO reacts with this amine-terminated PHB to give PHB-b–PEG-b–PHO block copolymers [60].

PEG is a polyether that is known for its exceptional blood and tissue compatibility. It is used extensively as biomaterial in a variety of drug delivery vehicles and is also under investigation as a surface coating for biomedical implants [25]. PHB-PEG amphiphilic polymers find wide variety of applications such as drug delivery systems [61] and adsorbents for metal cations [62].

3.10  Polyesterification of PHB-Diol and PEG-Diacid

Condensation polymerization of PHB-diol and PEG-diacid at equal molar amounts renders PHB-alt-PEG block copolymers. This polymerization is carried out in the presence of DMAP and DCC in dried methylene chloride [63]. PHB-alt-PEG alternating block copolymer can be seen in Figure 3.9.

Designed formulation of PHB-alt-PEG alternating block copolymer (redrawn from Ref. [63]).

Hydrogels can be prepared from cross-linked PEG-diacrylate and covered with PHB which is called a semi-interpenetrating network (IPN) hydrogel. IPN hydrogels are prepared by UV irradiation of the mixture of PEG-diacrylate and PHB. Net-PEG-based hydrogels all show higher equilibrium water contents (EWCs), while a remarkably decreased EWC is observed for the hydrogel containing 75% PHB. However, the crystallinity of PEG segments is noticeably decreased by cross-linking and would further drop with increasing amounts of PHB. Incorporation of a semi-IPN structure with PHB could significantly improve the mechanical properties of hydrogels when compared with those of pure net-PEG [64].

3.11  Stimuli Responsive PHA Graft Copolymers

PHOU-g-PNIPAM comb-type thermoresponsive graft copolymer films show surface hydrophilicity improvement, thermoresponsive properties at different temperatures, and good biocompatibility for cell growth as well as thermoresponsive cell detachment ability [65,66]. Chain transfer agent terminated PNIPAM is obtained by the RAFT polymerization of NIPAM and then transformed to thiol-terminated PNIPAM, PNIPAM-SH, via aminolysis by the reaction with n-butyl amine. Then the unsaturated PHA, PHOU, is grafted with the PNIPAM-SH via a thiol-ene click reaction.

PNIPAAm-PHB-PNIPAAm triblock copolymers are non-toxic and soluble in water. Diol-terminated PHB is transformed to the dibromo-terminated PHB macroinitiator which is used in the atom transfer polymerization of NIPAM in order to obtain a thermoresponsive triblock copolymer. Dibromo-terminated PHB is prepared from PHB-diol by the reaction of the terminal hydroxyl end groups of PHB-diol with 2-bromoisobutyryl bromide [67]. Paclitaxel (PTX) is a common drug for cancer therapy. But it suffers from drug resistance. An amphiphilic cationic polyester PHB-PDMAEMA was designed to show better cell biocompatibility and comparable gene transfection efficiency. The bromo-terminated PHB can also be used in the atom transfer polymerization of DMAEMA in order to obtain PHB-b-PDMAEMA cationic polyester to encapsulate chemotherapeutic paclitaxel in a hydrophobic PHB domain [68–70]. Figure 3.10 shows the schematic illustration of amphiphilic cationic PHB-PDMAEMA copolymer with nanoparticulated polyplex formation ability to encapsulate chemotherapeutic paclitaxel (PTX) in its hydrophobic core.

Schematic illustration of amphiphilic cationic PHB-PDMAEMA copolymer [69].

PHB-PDMAEMA diblock copolymer with the ability to co-deliver PTX and Nur77 could effectively inhibit the growth of the drug-resistant cancer cells [71]. Mono brominated PHB is used in the ATRP of the DMAEMA to obtain amphiphilic cationic AB-type block copolymer. The reaction scheme is rendered in Figure 3.11.

Chemical reaction route of PHB-b-PDMAEMA cationic copolymer: (i) transesterification of natural PHB with hexanol, (ii) bromo esterification, and (iii) ATRP reaction of DMAEMA monomer, in the presence of HMTETA and CuBr [71].

3.12  Enhanced Hydrophilicity via Radical Formation onto Saturated PHA

Free radicals can be formed on the PHA films leading to the free radical polymerization of the hydrophilic monomers (e.g., acryl amide, hydroxyethylmethacrylate). For example, PHO films are treated with plasma of different discharge powers (10–50 W) and then treated with acrylamide solutions in order to prepare films with hydrophilic surfaces. The acrylamide-grafted PHO can be used as cell-compatible biomedical applications [72]. In this manner, the number of Chinese hamster ovary cells is investigated and it is observed that they adhere to and grow on the film surfaces depending on the hydrophilicity degree.

Radical formation on PHBV can be carried out by thermally heating with benzoyl peroxide in the presence of acrylamide. Graft polymerization of acrylamide onto PHBV leads to an amphiphilic PHBV-g-PAAm graft copolymer [73,74]. The Langlois group grafted a hydrophilic monomer, HEMA, onto PHBV film at 80°C in the presence of benzoyl peroxide as a thermally radical producing source. Wettability has been obviously improved by grafting a hydrophilic monomer such as HEMA for a high graft yield (>130%) [75].

3.13  PEG Grafting onto Saturated mcl-PHA

Radical terminated PEG was grafted onto medium-chain-length PHA with a double bond in order to obtain Poly(3-hydroxyalkanoate)-g-poly(ethylene glycol) cross-linked graft copolymers. PHA with low concentration of the double bonds lead to a branched graft copolymer instead of the cross-linked one [76].

In order to obtain biomaterials for packaging, PHB is grafted onto cellulose using a simple reactive extrusion process in the presence of dicumyl peroxide. The grafting reaction reduces the crystallinity because the grafting reaction occurs in the amorphous region and also slightly in crystalline regions of both cellulose and PHB. So the smaller crystal sizes cause the decrease of brittleness of PHB [77].

3.14  Ozonization of PHB and PHBV

Chitosan is natural biocompatible cationic polysaccharide. When sticking to the bacterial cell wall, chitosan can suppress the metabolism of bacteria. When PHA films are ozonized in acidic aqueous media, the film surface is peroxidized and then oxidized to carboxylic acid (Figure 3.12). The PHA film with carboxylic acid is grafted with chitosan in order to obtain antibacterial biomaterial against Escherichia coli, Pseudomonas aeruginosa, methicillin-resistant Staphylococcus aureus, and S. aureus. Acrylic acid grafting increases the biodegradability with Alcaligens faecalis, whereas chitosan grafting reduces the biodegradability [78].

Synthesis of PHA-grafted chitosan [78].

3.15  Chlorination of PHA

Chloride derivatives of the PHA open new modification reactions. Chlorine gas is passed into the carbon tetrachloride solution of the PHA moiety such as PHB, PHO, and also PHOU in order to obtain chloride derivatives (e.g., PHB-Cl, PHO-Cl, etc.) [79]. Decrease in methyl signals in 1H NMR spectra can be attributed to the chlorination starts from the methyl groups of the PHA. Quaternization of the chlorinated PHA with triethyl amine makes them soluble in methanol and diethyl ether. For another amphiphilic derivative of the PHB-Cl, sodium thiosulfate is reacted with PHB-Cl to obtain the sulfonate derivative of bacterial polyester (PHB-SO3) [80].

3.16  PHB Graft Copolymers with Natural Hydrophilic Biopolymers

Hyaluronic acid (HA) (Figure 3.13) end capped PHA graft copolymers are successfully used to encapsulate hydrophobic drugs. In order to obtain these graft copolymers, PHA oligomers were obtained by refluxing the PHA with a mixture of glacial acetic acid and distilled water at around 105°C. The carboxylic end of the oligo-PHA was esterified with the hydroxyl group of the hyaluronan: Poly(3-hydroxybutyrate)-HA, Poly(3-hydroxyoctanoate)-HA, Poly(3-hydroxyoctanoate-co-3-hydroxydecanoate)-HA, and Poly(3-hydroxyoctanoate-co-3-hydroxydecanoate-co-3-hydroxydodecanoate)-HA. HA and oligo (3-hydroxyalkanoates) copolymers were [81].

Hyaluronan (hyaluronic acid). The hydroxyl group in the circle reacts with the carboxylic acid end of the PHA oligomer.

Carboxylic acid ends of the partially depolymerized PHB and PHO react with the amine groups of chitosan via the amidation reaction at 90°C for 5 h [82]. Chitosan-g-PHBV graft copolymers shown in Figure 3.14 exhibit different solubility behavior such as solubility, insolubility, or swelling in 2%wt. acetic acid and in water as a function of the degree of substitution of NH2, while pure chitosan does not swell in water.

PHA-g-chitosan graft copolymer.

3.17  Conclusions and Outlook

PHA are excellent biodegradable materials for medical and industrial applications. In medical applications such as tissue engineering and drug delivery systems, their high hydrophobicity makes them limited usage. PHA-enhanced hydrophilicity is a very useful candidate for these medical applications. We have mentioned the hydrophilization process of the PHA which finds very different medical application areas. However, there are gradual increases in producing new amphiphilic PHA for these application areas. There is still a great challenge to diversifying PHA in view of the enhanced hydrophilicity including amphiphilic polymers, copolymers with hydrophilic monomers, and stimuli-responsive monomers.

Acknowledgments

This work was supported by the Kapadokya University Research Fund (#KÜN.2018-BAGP-001) and Zonguldak Bülent Ecevit University, Faculty of Engineering.

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