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TUTORIAL REVIEW

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Water soluble polyhydroxyalkanoates: future materials for therapeutic applications Zibiao Li*a and Xian Jun Loh*abc Polyhydroxyalkanoates (PHAs) are excellent candidate biomaterials due to their exceptional biodegradability and biocompatibility. However, PHAs need to have tunable hydrophilicity, chemical functionalities, and appropriate hydrolytic stability to expand their therapeutic applications towards more advanced areas. In this Tutorial Review, we present the most recent progress in the synthetic strategies of PHA-based water soluble polymers, including the functionalisation of PHAs with polar functional groups and the block/graft copolymerization of PHAs with hydrophilic components in various polymeric architectures. These chemically modified water soluble PHAs have significant impact on materials engineering and show great value in the

Received 31st January 2015

fulfilment of smart biomaterials in emerging areas. The applications of water soluble PHAs in controlled drug

DOI: 10.1039/c5cs00089k

release, cancer therapy, DNA/siRNA delivery and tissue engineering in new aspects are discussed. In addition,

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water soluble PHA monomer production will be briefly introduced, with emphasis on its bio-significance in medical physiology and the therapeutic effect in the treatment of diseases.

Key learning points (1) (2) (3) (4) (5)

Types of PHAs and their uniqueness as biomaterials. Design strategies and synthetic methods of PHA-based water soluble polymers. Novel properties of water soluble PHA materials and their bio-significance in the emerging areas. Biodegradation of water soluble PHA polymeric materials. PHA monomer production and the therapeutic effect in medical physiology.

1. Introduction Polyhydroxyalkanoates (PHAs) are a family of natural polyesters synthesized through biological means. They are produced by microorganisms as intracellular carbon and energy storage compounds under unbalanced growth conditions.1 The general formula of PHA is shown in Fig. 1. Depending on the different number of carbons in their repeating units, PHAs can be classified into short-chain-length PHAs (sCL-PHA) with 4 to 5 carbons in one repeating hydroxyalkanoate unit, and mediumchain-length PHAs (mCL-PHA) with 6 or more carbons in monomeric constituents. To date, over 150 different types of biosynthetic PHAs have been classified, making them the largest group of natural polyesters. The representative examples of PHAs are presented in Table 1. With combination of the

various types of monomers in different proportions, PHA copolymers can be produced through metabolic engineering. PHAs have received a lot of attention in the biomedical field because of their biodegradability and their ability to promote sustainable development. With excellent biocompatibility in mammalian systems, PHA-based biomaterials show great potential as conventional medical implantation devices and tissue engineering, including sutures, repair patches, slings, orthopaedic pins, scaffold, stents and adhesion barriers.2 However, the intrinsic hydrophobicity of PHAs hinders its extensive usage in medical applications. PHAs are also lacking in chemical

a

Institute of Materials Research and Engineering, A*STAR (Agency for Science, Technology and Research), 3 Research Link, Singapore 117602, Singapore. E-mail: [email protected], [email protected]; Tel: +65-65131612 b Department of Materials Science and Engineering, National University of Singapore, Singapore 117574, Singapore c Singapore Eye Research Institute, 20 College Road, Singapore 169856, Singapore

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Fig. 1

General formula of PHA.

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Table 1

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Representative examples of PHA polymers

Chemical name

Abbreviation

Side groups

Structure

Poly(3-hydroxybutyrate) Poly(4-hydroxybutyrate) Poly(3-hydroxyvalerate) Poly(3-hydroxyhexanoate) Poly(3-hydroxyheptanoate) Poly(3-hydroxyoctanoate) Poly(3-hydroxynonanoate) Poly(3-hydroxydecanoate) Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) Poly(3-hydroxybutyrate-co-4-hydroxybutyrate) Poly(3-hydroxyoctanoate-co-hydroxyhexanoate) Poly(3-hydroxybutyrate-co-3-hydroxyoctanoate) Poly(3-hydroxybutyrate-co-3-hydroxydecanoate)

P3HB P4HB P3HV P3HHx P3HH P3HO P3HN P3HD PHBV P3/4HB PHBHHx PHBO PHBD

Methyl Hydrogen Ethyl Propyl Butyl Pentyl Hexyl Heptyl Methyl/ethyl Methyl/hydrogen Methyl/propyl Methyl/heptyl Methyl/pentyl

Homopolymer Homopolymer Homopolymer Homopolymer Homopolymer Homopolymer Homopolymer Homopolymer Copolymer Copolymer Copolymer Copolymer Copolymer

functionalities and the polyesters are often incompatible when blended with drugs. While biodegradable, PHAs are incredibly stable when unmodified, restricting their therapeutic applications in other more advanced areas. Therefore, there is a need to produce PHAs with desired amphiphilicity by anchoring water-soluble functionalities to improve biological behaviour and fine tune the materials according to different tissue engineering requirements. From the material’s point of view, the development of specific water soluble PHA polymers will result in a diverse group of new functional materials, which makes it possible to functionalize the hydrophobic PHAs into versatile and intelligent colloidal systems such as stimuli-responsive particles, hydrogels and polyelectrolytes in aqueous solution. Unusual PHAs containing some functional side groups such as hydroxyl- and/or carboxyl groups, methylated-branches and other hydrophilic derivatives were produced by some organisms to extend the scope of applications of PHAs for other types of applications. However, the complicated nature of genetic engineering resulting in bacterial modification, limited resources in large scale production, and the batch-to-batch variations in the polymer structure and composition during the biological synthesis or fermentation have led to very limited success of the use of these modified PHAs in new applications.3 On the other hand, through chemical modification of natural PHAs by the inclusion of other polymers,

chemical or surface modifications, PHAs with tunable hydrophilicities can be derived. This has significant impact in materials engineering and expands the use of PHAs in a variety of biomedical applications, while retaining the original advantageous properties of PHAs such as biodegradability and biocompatibility. Table 2 shows an overview of the design strategy and synthetic method of water soluble PHA-based polymeric materials. Such chemically modified water soluble PHAs, together with the water soluble PHA monomers, could have significant value in the fulfilment of smart biomaterials with different functionalities. The monomers, themselves, could also have therapeutic effect in diseases, such as hypoglycemia, hyperglycemia and neurodegenerative diseases. In this review, various water soluble PHA materials and their preparation techniques will be summarized, the further development of functionalized PHAs and their corresponding biomedical significance will also be discussed.

2. Water soluble PHA-based polymeric materials 2.1

PHAs with polar functional groups

The inherent hydrophobic nature of PHAs can be greatly altered by the introduction of polar groups such as hydroxyl and carboxylic groups into the side chains (Fig. 2). The presence

Zibiao Li obtained his BSc (2005) and MEng (2008) from Shantou University, China. In 2013, he completed his PhD in Department of Biomedical Engineering, National University of Singapore. Currently, he is working as a research scientist at the Institute of Materials Research and Engineering, A*STAR, Singapore. His research interests are focused on biodegradable/functional polymeric materials design, synthesis, physicochemical characterizations, and their fabrication for biomedical and personal care applications. Xian Jun Loh is the Head of Research Planning at the Institute of Materials Research and Engineering, A*STAR, Singapore, and assistant professor at the National University of Singapore (NUS). He obtained his PhD in 2009 from NUS. In 2011, he was elected into the Fellowship at Fitzwilliam College, Cambridge. He is the co-editor of an RSC-published Xian Jun Loh (left) and Zibiao Li (right) book entitled ‘‘Polymeric and Self-Assembled Hydrogels: From Fundamental Understanding to Applications’’. His main interests are in the design of supramolecular and stimuli-responsive polymers and hydrogels for biomedical applications. Currently, he has 63 journal papers and 10 patents, publishing mainly in the area of biomaterials.

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Design strategy and synthetic method of water soluble PHAs based polymeric material

Design strategy

PHA

PHA polarization PHOU PHOU PHOU

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Block PHB copolymerization P3HV4HB PHB PHB PHB

Synthetic method

Product

Ref.

Hydroxylation of the pendent double bonds Conversion of PHOU double bonds into epoxy groups and react with diethanol amine Carboxylation of the pendent double bonds

PHOU–(OH)x PHOU–(N(C2H4OH)2)x

4 and 5 7

PHOU–(COOH)x

8 and 35

Ester formation by Sn(Oct)2 catalyzed transesterification reaction between bacterial PHB and mPEG Ester formation by Sn(Oct)2 catalyzed transesterification reaction between bacterial P3HV4HB and mPEG Ester formation by coupling MPEG-COOH and PHB-diol oligomers Ester formation by ROP of b-butyrolactone monomers using PEG as macroinitiator Urethane formation of PHB and PEG containing hydroxyl ends

PHB–PEG

9 and 10

P3HV4HB–PEG

11

PEG–PHB–PEG PHB–PEG–PHB

12 16

PHB/PEG poly(ether ester urethane) P3/4HB Urethane formation of P3/4HB and PEG containing hydroxyl ends P3/4HB/PEG poly(ether ester urethane) PHB Urethane formation of PHB, PEG and PPG containing hydroxyl PHB/PEG/PPG poly(ether ends ester urethane) PHB ATRP of NIPAAm using PHB–diBr as macroinitiator PNIPAAm–PHB– PNIPAAm PHB ATRP of DMAEMA using PHB–diBr as macroinitiator PDMAEMA–PHB– PDMAEMA PHBV, PHBHHx, Click reaction between alkyne functionalized PHAs oligomers PHA–PEG and PHOHHx and azide-PEG PHB Click reaction between alkyne functionalized PHB and sPEG–PHB azide-terminated star PEG PHB Production through molecular biology techniques PHB–protein Graft PHB, PHBV, copolymerization and PHO P3/4HB PHOU PHOU

17–19 19 and 20 22 and 38 24, 40 and 41 25 and 39 26 and 27 28 29

Condensation with chitosan

PHA-g-chitosan

31 and 32

Michael addition between acrylated-P3/4HB and branch PEI Esterification of carboxylated PHOU with PEG Thiol–ene reaction of PHOU with thiol-terminated Jeffamine

PHA-g-PEI PHOU-g-PEG PHOU-g-Jeffamine

33 35 34

Sn(Oct)2: bis 2-ethylhexanoate tin; ROP: ring opening polymerization; ATRP: atom transfer radical polymerization; Jeffamine: a-amino-o-methoxy poly(oxyethylene-co-oxypropylene).

of the polar groups converted from the pendent unsaturated groups promotes water penetration into the polymer matrix and in some cases could make PHAs water soluble or at least water swellable. Two methods, by direct hydroxylation of unsaturated pendant groups, have been investigated to increase PHA’s hydrophilicity.

Fig. 2

In the first approach, Lee and his co-workers showed that the double bonds at the end of some lateral chains in poly(3hydroxyoctanoate-co-3-hydroxyundec-10-enoate) (PHOU) can be modified to give di-hydroxyl function by oxidation with basic potassium permanganate (KMnO4) (Fig. 2a).4 The maximum conversion rate through this approach is about 60%, regardless

Water soluble PHAs by functionalisation of the unsaturated side chains to (a) di-hydroxyl, (b) hydroxyl, and (c) carboxylic acid groups.

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of the unsaturated unit content in PHOUs, reaction time or the KMnO4/unsaturated unit molar ratios in the reaction system. The hydroxylated PHAs have a significant enhancement of hydrophilicity without significant reduction in molecular weight. Typically, PHOU with 40–60% degree of hydroxylation were completely soluble in polar solvents, such as acetone–water mixture, methanol and dimethyl sulfoxide (DMSO).4 In the other approach, hydroboration–oxidation reactions using the borane– tetrahydrofuran (BH3–THF) complex were also used to covert PHOU with hydroxyl groups.5 This reaction can proceed to complete conversion of the unsaturated double bonds in PHOU (Fig. 2b). With the increase in percentage of the pendant hydroxyl groups, the modified PHOU showed good solubility in polar solvents such as ethanol and methanol, and almost full solubility in water was also achieved.5 These PHAs containing reactive pendent hydroxyl groups are very useful in producing novel graft copolymers with desired properties.6 Enhanced hydrophilicity of PHOU has also recently been achieved by the conversion of epoxidized PHOU to the hydroxylated form in the presence of diethanol amine.7 The successful side chain conversion was further accompanied by the change in solubility. With the presence of the functionalized polar groups in the side chains, the polymer carried positive charge and became soluble in water. On the other hand, the presence of carboxylic groups are highly important in biomaterial modification, including biomolecule conjugation, the incorporation of hydrophilic or anti-fouling compounds and targeting enzyme conjugation among others. The incorporation of the carboxylic groups is also used as an important approach to enhance PHA’s hydrophilicity. The unsaturated groups in PHAs can be converted to carboxyl groups via oxidation. For example, in the presence of crown ether as a phase transfer agent and KMnO4 as a dissociating agent, carboxylation was observed at high conversion degree with no significant reduction of PHA molecular weights (Fig. 2c).8 A copolymer of poly(3-hydroxyoctanoate) (P3HO) that contains 25% of carboxyl groups in the lateral chains had enhanced hydrophilicity and the modified product was perfectly soluble in polar solvents, such as methanol and acetone–water mixture.8 The resulting products showed pH dependent solubility in water and organic solvent mixtures. Previous studies also showed that the presence of the carboxylic groups in the side chain could be utilized to adjust the time needed for complete PHA degradation. Such carboxylic acid functionalised PHAs have also been proposed to prepare drug conjugates for site-specific delivery.6,8 2.2

Water soluble PHA block copolymers

Another strategy to modulate PHAs’ properties is to prepare block copolymers with hydrophilic components. Some ester groups of the PHAs can be exchanged with an alcohol in the transesterification process to obtain a carboxylic end group and diol, quantitatively.6 Although short chain diols rarely render a hydrophilic character to the hydrophobic PHAs, hydrophilic components such as poly(ethylene glycol) (PEG), poly(propylene glycol) (PPG), poly(N-isopropylacylamide) (PNIPAAM) and poly(2dimethylaminoethyl methacrylate) (PDMAEMA) can be introduced

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into telechelic PHA macromonomers by copolymerization to form amphiphilic block copolymers with various architectures and functionalities. This strategy has been utilised to manipulate PHAs hydrophilicity, as well as other desired properties including mechanical strength and degradation profiles. In addition, more advanced biomedical applications in the considered living system could be fulfilled by using water soluble PHAs. In this section, the synthetic strategies for water soluble PHA macromonomer based block copolymer preparation with the most recent advancements will be discussed. (a) Block copolymerization in ester formation. Ester formation in the PHA block copolymer reaction is popularly designed due to the mild reaction conditions. PEG, as a hydrophilic and biocompatible polyether is extensively used as a biomaterial in a variety of drug delivery vehicles and is also being tested as a surface coating for biomedical implants. When dissolved in water, PEG has a low interfacial free energy and exhibits rapid chain motion, its large excluded volume could lead to steric repulsion of approaching molecules. These properties make PEG an excellent candidate for the construction of PHA amphiphilic block copolymers. With precise control of the ratio between constituting PHA/PEG blocks, water soluble characteristics can be endowed on the long hydrophobic PHA chains. Previously, Ravenelle et al. reported the synthesis of PHB–PEG diblock copolymers through a tin(II) 2-ethylhexanoate catalyzed transesterification reaction. Bacterial PHB of high molecular weight underwent a concomitant depolymerisation to lower molecular weights to an approximate range of 2300 to 7300 Da.9 However, because PHB is hydrophobic, crystalline and had no appreciable mobility in water, the diblock copolymer itself did not generate water soluble self-assembled structures spontaneously. Once formed from oil-in-water emulsion, the PHB–PEG diblock copolymer could self-assemble into nanoparticles in the selected oil/water suspension, which could be potentially be used as drug carriers, binders and other specialty applications. Such drug carriers may show a longer lifetime in the bloodstream for they were robust against dilution, owing to the hydrophobic character of PHB.10 In a similar approach, an amorphous amphiphilic block copolymer P(3HV-co-4HB)– mPEG was synthesized from the coupling of non-crystallizing poly(3-hydroxyvalerate-co-4-hydroxybutyrate) and PEG via a transesterification reaction.11 Core–shell nanoparticles in water medium were prepared from this copolymer by an emulsification– solvent evaporation method. The amorphous core domain of the nanoparticles could be used as a reservoir for chemotherapeutic drugs and provided sustained release of the embedded cargo.11 In another study, amphiphilic PEG–PHB–PEG triblock copolymers were synthesized by coupling two chains of methoxyPEG-monocarboxylic acid (MPEG-COOH) with a low molecular weight PHB-diol chain through esterification formation which was catalyzed by DCC and DMAP (Fig. 3).12 The PHB-diol was obtained by the transesterification reaction of high molecular weight natural PHB with ethylene glycol. A group of PEG–PHB– PEG triblock copolymers with PEG chain lengths of 1820 and 4740 Da and PHB chain lengths in the range of 470 to 5490 Da

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Fig. 3 PEG–PHB–PEG triblock copolymers preparation by esterification reaction between carboxylated PEG and hydroxylated PHB as building blocks.

were used. Crystallinity studies showed that the condensed state behaviour of the PHB and PEG blocks in the copolymers were greatly affected by each other, and the phase separation was not observed with very low molecular weight PHB. PEG–PHB–PEG triblock copolymers with high PEG content were water soluble and could self-assemble into spherical micelles. These micelles contained the insoluble PHB blocks as the dense cores, which were surrounded by soluble PEG blocks as the outer shells. Interestingly, water soluble PEG–PHB–PEG triblock copolymer solutions at certain concentrations (e.g. 13.3%) could be transformed from free flowing solutions into semi-solid hydrogels by mixing with a-cyclodextrins (a-CD). These gels could be used to provide controlled release of the encapsulated molecules within the gel matrix.13,14 In a similar reaction fashion, PEG-alt-PHB multiblock copolymers were also synthesized to increase hydrophilicity of PHB.15 The esterification between the building blocks in PEG-alt-PHB multiblock copolymers were realized by using telechelic carboxylated PEG (PEG-diacid). Interesting phase separation patterns were also observed on the PHB-alt-PEG block copolymer coated surface.15 In contrast to the above mentioned copolymers prepared from the esterification reaction with microbial PHB, an alternative synthetic approach to obtain PHB/PEG block copolymers is via ring opening polymerization (ROP) of the b-butyrolactone monomer using PEG as a macroinitiator (Fig. 4).16 An important feature of this strategy is the ability to produce atactic PHB. Different from microbial isotactic PHB, atactic PHB is completely amorphous and does not retain any crystallinity in its microstructure. At PEG molecular weight of 3.32 kDa flanking with two PHB segments at around 0.86 kDa, an amphiphilic PHB–PEG–PHB triblock copolymer was easily dispersible in water and formed flower-like micelles with a critical micellization concentration (CMC) of 0.216 mg mL1. The micelle formation strongly points to the highly associative behavior of the PHB segments in water while the middle PEG chain forms the hydrophilic coronas.

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Fig. 4 Synthesis of PHB–PEG–PHB triblock copolymers through ring opening polymerization of b-butyrolactone monomer using PEG as a macroinitiator.

(b) Block copolymerization in urethane formation. Urethane formation is another synthetic method that has been well studied to develop PHA based amphiphilic block copolymers. Owing to their good biocompatibility and easily modified properties to suit specific applications, polyurethanes are a very appealing class of polymers for use in the biomedical fields. Telechelic PHA-diol obtained by esterification can be used in the preparation of poly(ether ester urethane)s via a diisocyanate chain extension reaction with hydroxyl-capped polyethers such as PEG and PPG. Amphiphilic PHAs which are water-swellable or completely water soluble were produced through this approach. Among the PHA based amphiphilic poly(ether ester urethane)s block copolymers, PHB and PEG segments as the building blocks have been widely investigated. Li et al. reported the synthesis of a series of PHB/PEG poly(ether ester urethane)s block copolymers coupled by hexamethylene diisocyanate (HMDI).17 The structure–property relationship of these copolymers were studied using PHB and PEG polymer blocks with different chain lengths as building blocks. The hydrophilicities of the PHB/PEG poly(ether ester urethane)s, as ascertained by water contact angle measurements, were greatly improved through incorporation of PEG segments. Results showed that at an identical PHB length of 1740 Da, the water contact angle of PHB/PEG poly(ether ester urethane)s decreases with increasing PEG segment content. In addition, the bulk hydrophilicity and swelling property of PHB/PEG poly(ether ester urethane)s also showed significant improvement compared with the unmodified PHB. At the same PHB length, the equilibrium water uptake of PHB/PEG poly(ether ester urethane)s increased from 55% to 575% with increasing PEG segment length from

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2000 to 8000 Da. However, the equilibrium water uptake of the natural source PHB is only 5%, due to its hydrophobic nature. These changes greatly affect the degradation profile of PHB and increase its bioavailability in tissue engineering.18 In another aspect, the mutual interference between PHB and PEG segments made the block copolymers ductile. The ductility was enhanced in hydrated states with one particular example showing increment in strain at break from 1090 to 1962%.18 In addition to PHB, a similar type of PHA/PEG based poly(ether ester urethane)s have also been studied by including PHBV, P3/4HB, PHBHHx as building blocks.19–21 Many new features and various biomedical applications were achieved by modulating the surface hydrophilicity and bulk hydration of the polymers. However, to ensure good water solubility, the molecular weight and composition of the poly(ether ester urethane)s should be well controlled. In general, PHA block lengths have to be short enough to reduce the crystallinity and hydrophobic nature, and the incorporated PEG content should be above a certain threshold to provide sufficient hydrophilicity. In one particular example, P3/4HB/PEG poly(ether ester urethane)s block copolymers were easily dispersed in water only when amorphous P3/4HB (4HB in mole = 36.3%) and PEG (450 wt%) were used as building blocks. The copolymer aqueous solution prepared from oil-in-water colloidal suspensions could assemble to form different shapes ranging from a leaf-like structure to irregular particles, dominated by the solubilizing effect of PEG.19 On the other hand, the structure–property relationship provided specific considerations in the PHA/PEG poly(ether ester urethane)s block copolymer development. For example, Pan and his coworkers reported the synthesis of amphiphilic

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alternating block polyurethane block copolymers based on P3/4HB and PEG by a coupling reaction between P3/4HB-diol and PEG-diisocyanate, with different 3HB, 4HB, PEG compositions and segment lengths.20 The synthetic methodology for the alternating polyurethane block copolymers provided a way to control the exact structure of the products and tailor the properties to specific requirements. Comparing with the random P3/4HB/PEG poly(ether ester urethane)s, alternating P3/4HB/PEG poly(ether ester urethane)s was more hydrophilic with a higher surface energy, rendering a better hemocompatibility and more favorable glial cell attachment on the copolymer surface. In addition, functionalization of PHAs through the incorporation of PEG together with PPG to form poly(ether ester urethane)s have also been reported to introduce hydrophilic characteristics to PHAs and the newly developed polymers have achieved significant alternation to the behaviour of PHAs from hydrophobic solids to hydrogels. For example, multiblock amphiphilic and thermosensitive poly(ether ester urethane)s consisting of PHB, PEG, and PPG blocks were synthesized (Fig. 5).22 This represents the first time that PHB has been incorporated in a thermogelling copolymer. It was found that the incorporation of PHB below 11.4 wt% rendered the copolymers soluble in water, which exhibited interesting self-assembly properties in the aqueous solutions. The water soluble PHB/ PEG/PPG poly(ether ester urethane)s had very low CMC ranging from 5.16  104 to 9.79  104 g mL1. On the basis of the NMR experiments, the micelles were concluded to have a hydrophobic core made up of PHB and PPG segments and an outer hydrophilic corona of PEG segments. Aqueous solutions of the PHB/PEG/PPG poly(ether ester urethane)s underwent a

Fig. 5 (A) Synthesis of PHB/PEG/PPG poly(ether ester urethane) block copolymers, and (B) sol–gel–sol transition of the copolymer aqueous solution with increasing temperature. Adapted with permission from ref. 22. Copyright (2007) American Chemical Society.

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sol–gel–sol transition as the temperature increased from 4 to 80 1C, and showed a very low critical gelation concentration (CGC) ranging from 2 to 5 wt%.22 The sol–gel transition of the copolymer solution at increasing temperatures was due to the increased association of the micelles was brought about by the multiple segments that link the micelles together in a networklike structure. The incorporation of PHB segments into the copolymer significantly reduced the CGC and enhanced the cell adhering ability on the hydrogel surface when compared to the commercialized Pluronic (PEG–PPG–PEG) gel.23 (c) Block copolymerization in other formation. With the recent advances in polymerization techniques, water soluble PHA block copolymers have been synthesized by various means, such as atom transfer radical polymerization (ATRP) and ‘‘click’’ reaction. One example reported the synthesis of thermoresponsive amphiphilic triblock copolymers with two hydrophilic PNIPAAm blocks flanking a central hydrophobic PHB block (PNIPAAm–PHB–PNIPAAm) by ATRP.24 In the designed strategy, the starting Br–PHB–Br, as a macroinitiator, was prepared from the reaction of terminal hydroxyl end groups of PHB-diol with 2-bromoisobutyryl bromide. The PNIPAAm–PHB–PNIPAAm triblock copolymer was synthesized via ATRP of NIPAAm from the Br–PHB–Br macroinitiator units in dioxane at 45 1C (Fig. 6A). By changing the monomer feed, a series of PNIPAAm–PHB–PNIPAAm triblock copolymers with different PNIPAAm block lengths were obtained. The water soluble copolymers formed micelle aggregates in water with a hydrophobic PHB core and hydrophilic PNIPAAm corona at 25 1C. The CMCs of the triblock copolymers were in the range of 1.5 to 41.1 mg L1. These values are very low and indicate that PNIPAAm–PHB–PNIPAAm copolymer micellar solutions have great stability under high dilution conditions. More interestingly, when the temperature of the solution was increased, the hydrophobicity of PNIPAAm increased and PNIPAAm chains in the micelle corona collapsed. The increased hydrophobicity of the micelles leads to micelle aggregation, which leads to the

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formation of larger particles. The micelles are temperature responsive and the incorporated drugs can be made to release on-demand by a temperature trigger.24 Using a similar synthesis method, pH sensitive triblock copolymers with two hydrophilic PDMAEMA blocks flanking a central hydrophobic PHB block were also prepared (Fig. 6B).25 Core–shell micelle formation was formed from the PDMAEMA–PHB–PDMAEMA triblock copolymers aqueous solution, with a hydrophobic PHB core surrounded by a hydrophilic DMAEMA shell. The CMC of the triblock copolymers was also very low, indicating the great stability of the micellar solution, which can be used as controlled drug release carriers. pH and temperature triggered release of encapsulated cargo can be achieved by this system. Another approach relies on the synthesis of well-defined PHA amphiphilic block copolymers by ‘‘click’’ coupling using functionalized PHAs and hydrophilic PEG as building blocks.26 In one example, a series of PHBV, PHBHHx, and poly(3-hydroxyoctanoate-cohydroxyhexanoate) (PHOHHx) oligomers prepared by thermal degradation were functionalized with alkyne function. The ‘‘click’’ coupling reaction of alkyne-terminated PHAs and azideterminated PEG could be carried out highly efficiently to afford well-defined PHA–PEG diblock copolymer structures. Among these copolymers, PHOHHx–PEG diblock copolymers can selfassemble into aqueous micelles (CMC = 0.85 mg L1) and micelles with diameters ranging from 44 to 90 nm were obtained.27 Another example lies in water soluble PHA starblock copolymers (sPEG–PHB) synthesized through the combination of ROP and alkyne–azide coupling.28 In aqueous medium, the sPEG–PHB block copolymers self-assembled into nanogel-like large compound micelles, and transformed into vesicular nanostructures under b-CD derived host–guest interaction. Recently, water soluble PHA–protein block copolymer based micelles with a targeting capability were also produced through molecular biology techniques.29 The protein engineering approach provided another potential method for tailoring the amphiphilic properties of PHAs.

Fig. 6 Synthesis of water soluble PNIPAAm–PHB–PNIPAAm (A) and PDMAEMA–PHB–PDMAEMA (B) triblock copolymers using PHB–diBr as a macroinitiator in ATRP.

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2.3

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Water soluble PHA graft copolymers

Various graft copolymers containing PHAs as main or side chains prepared from ‘‘graft onto’’, ‘‘graft from’’ and ‘‘graft through’’ were discussed in a previous review.30 Here we summarize the modification of the water solubility of PHA graft copolymers through the control of polymer chemical compositions by modifying the graft chains. The new properties obtained from these PHAs through this approach, and preparation of PHA grafted copolymers with the desired hydrophilicity to suit specific applications are also discussed in this section. The ‘‘graft onto’’ method consists of the covalent coupling of reactive sites located along pendant chains or the main chain with end groups of oligomers, polymers, or copolymer segments. In the grafting from strategy, functional sites along the polymer backbone are activated and graft copolymerization with a second monomer takes place from the active sites to give the graft copolymer architecture. The third method involves copolymerization of a low molecular weight monomer with a macromonomer. The amidation reaction between the carboxyl groups in PHAs and amine functions in chitosan has been used to synthesize PHA-g-chitosan graft copolymers. PHB-g-chitosan graft copolymers were synthesized by the treatment of chitosan in dilute acetic acid solution with various amounts of PHB oligomers in the feed ratios. Although both PHB and chitosan are water insoluble polymers, the obtained PHB-g-chitosan graft copolymers could dissolve in water to form viscous solutions, showing a significant enhancement in hydrophilicity through the graft copolymerization.31 In a similar fashion, both PHBV and PHO oligomers have also been reported to graft onto chitosan, giving the respective PHBV-g-chitosan and PHO-g-chitosan copolymers. The solubility of the PHA-g-chitosan graft copolymers were controlled by the degree of substitution of chitosan amine groups in the final products.32 Recently, a PHA grafted poly(ethylene imine) (PEI) copolymer that was synthesized by utilising the Michael addition between acrylate groups in functionalized mP3/4HB-acrylated prepolymer and branched PEI (P3/4HB-g-bPEI) (Fig. 7) was reported. P3/4HB with different chain lengths were employed in the graft copolymerization. The obtained P3/4HB-g-bPEI copolymer showed good solubility in 4-(2-hydroxyethyl)-1piperazineethanesulfonic acid (HEPES) buffered glucose (HBG) solution. When the P3/4HB-g-bPEI solution was mixed with siRNA solution, well condensed copolymer–siRNA complexes were formed. The electrophoretic retardation analysis indicated that the PHA-g-bPEI copolymer could effectively bind siRNA and protect it from degradation by nucleases, which is a prerequisite for efficient gene therapy.33 In another aspect, PEG in various derivative forms such as diazo-linked PEG, hydroxyl-terminated PEG, acrylate-PEG and PEG copolymers have been used to produce PHA-g-PEG copolymers by free radical chemistry, etherification reaction, UV irradiation and thiol–ene chemistry.34 In view of the high molecular weight of the PHA in the final product, most of the obtained PHA-g-PEG copolymers were fabricated into grafted amphiphilic films or membranes with increased tensile strength when compared with the untreated PHAs. The substrate surfaces of the PHA-g-PEG

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Fig. 7 Synthesis route of the mP3/4HB-g-bPEI graft copolymer by Michael addition.

copolymers became more hydrophilic when PEG graft density in the copolymer was increased, leading to water swelling characteristics. These surface characteristics make the graft copolymers useful in the prevention of protein adsorption and platelet adhesion. In one particular study, amphiphilic PHOU-g-PEG copolymers with improved solubility were synthesized through the grafting of PEG onto PHOU side chains. The reaction between carboxylic acid groups in PHOU side chains and the hydroxyl group in mPEG was utilised in PHOU-g-PEG copolymer synthesis (Fig. 8). In this approach, PEG grafting was achieved with a high conversion yield of 76%. This enhanced the hydrophilic character of the modified polymers to a higher degree and made PHOU-g-PEG copolymers soluble in typical polar solvents, such as alcohols and water–acetone mixtures.35 Nanoparticles were prepared using PHOU-g-PEG copolymers in different saline concentrations. Suspension stability studies showed that the hydrophilic PEG in PHOU-g-PEG copolymers could act as a protective agent to prevent particle aggregation while the unmodified PHOU led to obvious particle aggregation when NaCl concentration was increased.35 PEG grafting provides an opportunity to modify the solubility and functionality of pristine PHAs, therefore potentially improving their ability to function as biomaterials. On the other hand, enhancement of the surface hydrophilicities of PHAs was also achieved by grafting copolymerization of PHAs with hydrophilic sugars, 2-hydroxyethylmethacrylate (HEMA), N-vinylpyrrolidone (VP) and poly(acrylic acid) (PAA) through different graft techniques. As expected, all the resulted new PHAs grafted copolymers are more hydrophilic than their parent PHAs. For example, through the grafting of HEMA onto

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Fig. 8 PHOU-g-PEG copolymers prepared from PEG esterification with carboxylic acid groups in PHOU side chains.

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PHB blocks (1750 or 3140 Da) and long PEG block lengths (5000 Da) were found to be water soluble. Along with the self-assembled spherical micelle formation, in situ hydrogels were also formed from aqueous mixtures containing PEG–PHB–PEG triblock copolymers and a-CD through region-selective inclusion complexation between PEG and a-CD (Fig. 9).13 Typical hydrogel formulations contain 13.3 wt% of PEG–PHB–PEG triblock copolymers and 9.7 wt% of a-CD. The hydrophobic interactions within intramolecular PHB chains during the micellization process were important to facilitate the hydrogel network and stabilize the hydrogels. Fluorescein isothiocyanate labeled dextran (dextran-FITC) was used as a model for drug release study. It was found that the a-CD/PEG–PHB–PEG hydrogels were slowly disassociated during the release course, and eventually disappeared when all the encapsulated dextran-FITC was completely released. The in vitro release profile can be controlled from 5 days to more than 1 month by applying PEG–PHB–PEG copolymers with different chain lengths. The sustained release of dextran-FITC for more than 30 days makes the a-CD/PEG–PHB– PEG hydrogel system a potential material for well-controlled long term release applications.13,14 In addition, thermo-responsive PHB/PEG/PPG poly(ether ester urethane)s copolymers were another type of interesting

PHBV films, the wettability of obtained HEMA-g-PHBV specimen was improved dramatically because of the presence of many hydroxyl groups.36 Surface contact angle measurements showed a value of about 751 for unmodified PHBV, while these values recorded on HEMA-g-PHBV films decreased with the increasing grafted PHEMA quantity. At a graft yield of 150%, the contact angle was around 371, indicating significant enhancement of the surface hydrophilicity. The modified HEMA-g-PHBV substrates also showed potential for bioactive molecule attachment through the exposed hydroxyl groups in HEMA.36 In another study, hydrophilic VP was grafted onto PHBV using 2,2 0 azobisisobutyronitrile (AIBN) as an initiator, and a PVP content of 45% was achieved through this method.37 Compared to the ungrafted PHBV, the obtained PHBV-g-PVP graft copolymer possessed improved swelling properties due to the hydrophilic nature of PVP. More interestingly, the presence of PVP in PHBVg-PVP afforded the copolymer anti-bacterial activity. When tested against Gram-negative and Gram-positive bacteria, the antibacterial activity detected was proportional to the PVP grafting density in PHBV-g-PVP copolymers.37 2.4

Bio-significance of water soluble PHAs in emerging areas

Previous applications of natural PHAs were concentrated in the areas of packages, conventional medical devices fabrication and implanted tissue engineering.2 The functionalization of PHAs into amphiphilic or water soluble polymers allows for the modulation of PHAs with favourable mechanical properties, stimuli responsiveness and desirable degradation times under specific physiological conditions. All these new features have expanded PHAs into emerging applications. (a) Water soluble PHAs system for controlled release of chemotherapy. PEG–PHB–PEG triblock copolymers with short

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Fig. 9 (A) Schematic illustration of the PEG–PHB–PEG triblock copolymer and a-CD; (B) optical photographs showing PEO–PHB–PEO aqueous solution and (C) its self-assembled hydrogel with a-CD; (D) illustration of the inner structure a-CD–PEO–PHB–PEO hydrogel formation. Adapted with permission from ref. 13. Copyright (2006) Elsevier.

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materials that exhibit spontaneous physical gelation upon heating.22 The as-formed hydrogel could maintain its integrity for a desired duration when it was exposed to physiological conditions. Pharmaceutical formulation could be easily formed by solution mixing at low temperature, and the formation of the hydrogel at higher temperatures could allow this system to serve as drug reservoirs and provide controlled release of the embedded cargo through diffusion means. For example, when the model protein bovine serum albumin (BSA) was encapsulated in PHB/PEG/PPG poly(ether ester urethane)s thermogels, the period of sustained release varied from 15–80 days. This was achieved by simply controlling of the copolymer concentration in water solution.38 As a control, the Pluronic thermogels (PEG–PPG–PEG F127 copolymer gels) with similar PEG/PPG content to that of PHB/PEG/PPG poly(ether ester urethane)s released the entire BSA amount in less than 4 h. The different release duration is mainly caused by the packing structure between the two thermogels. PHB/PEG/PPG poly(ether ester urethane)s thermogels were observed as a more tightly packed structure than F127 gels, leading to the formation of a more robust hydrogel, which extends the diffusion of the solute.38 On the other hand, water soluble PDMAEMA–PHB–PDMAEMA triblock copolymers with pH and thermo-responsiveness could selfassemble into micelles at 20 1C, and the micelle core comprised of hydrophobic PHB which could be used as a reservoir to encapsulate hydrophobic drugs such as chemotherapeutics.25 The higher PHB content in the copolymer provided a higher drug loading content in the system. Moreover, the particle size changes in response to the external environments are advantageous in drug controlled release. For example, the particle size increased at higher temperature because of the increased hydrophobicity of PDMAEMA chains and micelle aggregation. At the same time, the PDMAEMA chains became protonated upon decreasing the pH of the solution. The repulsion force within PDMAEMA chains reduces the micelle aggregation, leading to the formation of smaller particles. The difference in the size and the exposed surface area of the particles lead to a different drug release profile. The effectiveness of temperature and pH in the control of drug release was studied by using doxorubicin as a model drug. The obtained results showed that when incubating the drug loaded micelle system under different temperatures, the stimulus-triggered drug release profile can be obtained. For instance, the incubation at 20 1C could slow down the rate of drug release, and upon returning to the incubation temperature to 37 1C the release of the drug would become faster than that at 20 1C (Fig. 10A).25 This is because the collapse of PDMAEMA chains at a higher temperature would disturb the micelle structure, leading to a faster drug release rate at the lower temperature. The pH effect on the drug release was also investigated. As shown in Fig. 10B, for the same PDMAEMA–PHB–PDMAEMA copolymer system, decreasing the medium to a lower pH 5 could trigger a faster release than that at pH 7. The proposed mechanism behind this observation is that the aggregated micelles formed at pH 5 showed a greater surface area than the micelles formed at pH 7, which enhanced the release of the drug from the system.25 More interestingly, the cell culture results showed that the responsive behavior of

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PDMAEMA–PHB–PDMAEMA micelles could enhance the cell uptake by HeLa cells, and the in vitro drug delivery is more efficient than the commercially available Pluronics F127 (Fig. 10C and D).25 Recently, drug delivery systems developed from PHAs based water soluble copolymers were also explored in cancer therapy. For example, cisplatin is a typical anticancer drug showing chemotherapeutic effects against a variety of tumors. When cisplatin was loaded onto amphiphilic P(3HV-co-4HB)–mPEG copolymer self-assembled micelles, the in vitro drug release profile showed a sustained release behavior. Moreover, the cisplatin-loaded micelles could be more effectively internalized by tumour cells and accumulated to control the growth of the tumours. With the presence of P(3HV-co-4HB)–mPEG micelles, the drug accumulation became more obvious and can be sustained for a longer period of time when compared with the free drug in solution.11 Another functional micelle system with targeting capability was investigated as a drug carrier to cancer cells through molecular recognition with cancer markers. The micelles were formed from a water soluble PHA–protein block copolymer with PHA as the hydrophobic core and hydrophilic protein block as the stabilized corona. Cys–Asp–Cys–Arg–Gly–Asp–Cys–Phe–Cys (RGD4C) was conjugated to the protein during PHA–protein block copolymer synthesis and it was used as a ligand for integrin targeting in cancer cells. Hydrophobic drugs or other functional biomolecules could be easily incorporated into the PHA core during the micellization of the copolymer (Fig. 11). The incubation of the micelles with MDAMB-231 breast cancer cells indicated that the RGD4C ligated PHA– protein micelles bonded effectively to the targeted cells.29 (b) Water soluble PHAs system for DNA/siRNA delivery. Gene delivery could potentially be used for the treatment of diseases via the introduction of corrective genetic information to the malfunctioning cells. However, this area is facing great challenges in the development of a suitable delivery vector with low toxicity and high transfection efficiency. Water soluble cationic PDMAEMA–PHB–PDMAEMA triblock copolymers showed positive charge in aqueous solution. This provides the copolymer with a strong condensation ability with the negatively charged plasmid DNA (pDNA) to form copolymer/ pDNA polyplexes, which is an important process to prevent pDNA degradation by nucleases.39 When the ratios of the amino group in PDMAEMA to the phosphate group in pDNA in the polyplexes, defined as the N/P ratio, reached a critical value, the particles size of the polyplex remained fairly constant at about 100 nm, which is in the suitable range for cell uptake. The presence of PHB in the copolymer improved the biocompatibility of PDMAEMA–PHB–PDMAEMA as revealed by cytotoxicity assay in HEK293 cells. It showed significantly lower toxicity as compared with the PDMAEMA homopolymer and benchmark PEI (25 kDa). The above study illustrated that the incorporation of nontoxic PHB in the PDMAEMA–PHB–PDMAEMA copolymer could disperse the cationic groups and the subsequently decrease the overall surface charge of the self-assembled polyplex particles.39 More importantly, when compared with the homopolymer PDMAEMA, the less toxic PDMAEMA–PHB–PDMAEMA copolymers showed a great

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Fig. 10 PDMAEMA–PHB–PDMAEMA triblock copolymers are denoted as DHD, and the numbers in brackets show the indicative molecular weight of the respective block in hundred g mol1. The effect of temperature (A) and pH (B) on the drug release profile of PDMAEMA–PHB–PDMAEMA micelles. Confocal images of HeLa cells uptake by incubating with (C) Pluronic F127 micelles and (D) PDMAEMA–PHB–PDMAEMA, DHD(21-21-21) micelles. The scale bar denotes 20 mm. Adapted from ref. 25.

Fig. 11 Schematic illustration showing PHA–protein block copolymers with RGD4C as a tumor targeting ligand and the drug loading process into the micelles by self-assembly. Adapted from ref. 29.

improvement in the gene transfection efficiency. For example, when tested in Cos7 and HEK293 cells by using luciferase as a marker gene, the transfection capability for PDMAEMA–PHB– PDMAEMA copolymers displayed significantly better efficacy when compared to the linear PDMAEMA and PEI counterparts under both full serum and reduced serum conditions. A typical example of the copolymer at constituting composition of DND(110-21-110) showed 6 times better than the ‘gold’ standard of polymeric transfection agents PEI. Considering the low toxicity and high transfection efficiency together, the water soluble PDMAEMA–PHB–PDMAEMA copolymers could have great potential as a gene delivery vector to transport plasmid to cells.39 On the other hand, a P3/4HB-g-bPEI copolymer based delivery carrier was developed for efficient siRNA delivery.33 Although previous studies demonstrated that PEI is a high gene transfection candidate in pDNA delivery, the efficiency of

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siRNA delivery using PEI is less effective. This is because of the different properties in terms of molecular weight, charge density, stability behaviour of pDNA and siRNA and their interaction methods with polymers. The incorporation of P3/4HB onto PEI was shown to be more advantageous in siRNA delivery than untreated PEI and other types of polycations. For example, the P3/4HB-g-bPEI copolymer could form nano-sized complexes with siRNA with particle sizes of below 200 nm, which protected it from serum degradation. The positive surface charge of the P3/4HB-g-bPEI/siRNA polyplexes was maintained between 33 and 43 mV to facilitate the necessary binding of the polyplexes with the anionic cell membranes, which is expected to further promote cellular uptake for siRNA delivery. For example, there is no detectable red fluorescence after incubation of free Cy3-siRNA with A549 cells (Fig. 12A). On the contrary, high intracellular fluorescence was observed in cells transfected with the P3/4HB-g-bPEI copolymer, especially for the sample with a moderated P3/4HB graft degree of 1.03% (Fig. 12B). The fluorescence intensity was even higher than the counterparts Lipofectamine and PEI (Fig. 12C and D), indicating a better cellular uptake and subcellular distribution.33 Furthermore, the incorporation of PHA in the copolymer could decrease the stability of the P3/4HB-g-bPEI/siRNA complexes, and the degradation of the ester bonds in P3/4HB could make it easier to release siRNA into the cytoplasm for efficient gene silencing. In A549-Luc

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Fig. 13 Temperature induced cell detachment by using PNIPAAm–PHB– PNIPAAm triblock copoylmer coated surface. Adapted with permission from ref. 41. Copyright (2009) Wiley-VCH.

Fig. 12 Confocal microscopy images showing the A549 cell uptake of (A) free siRNA and (B) P3/4HB-g-bPEI/siRNA complexes. (C) Lipofectamine and (D) bPEI were used as control. Cy3-lablled siRNA appeared red fluorescence and the cell nuclei were stained blue by using DAPI. Adapted with permission from ref. 33. Copyright (2011) Elsevier.

cells and MCF-7-Luc cells, P3/4HB-g-bPEI copolymers/siRNA complexes displayed remarkable knockdown of luciferase expression up to 70–90%, showing its potential as safe and efficient siRNA carriers for human gene therapy. (c) Water soluble PHAs system in tissue engineering. Water soluble PNIPAAm–PHB–PNIPAAm triblock copolymers could self-assemble into micelle aggregates at very low concentrations.24 The temperature responsivness of PNIPAAM chains from hydrophilic to hydrophobic transitions could allow the PNIPAAm–PHB–PNIPAAm copolymer coated surface for the nonenzymatic thermal-induced cell detachment in tissue engineering.40,41 For example, our group demonstrated that the presence of PHB in the PNIPAAm–PHB–PNIPAAm micelle core could be attached onto a polyester cell culture surface to form a stable coatings through hydrophobic–hydrophobic interactions. The coated surface had the thermo-responsive properties with hydrophobic behavior at higher temperatures such as 37 1C. When the temperature of the system was lowered below the LCST of PNIPAAM (32–32 1C), the surface became hydrophilic (Fig. 13). At the coating density of just 5.66 mg cm2, vast changes in the surface hydrophilicity can be observed as a function of temperature. The surface contact angle decreased from 60.31 at 37 1C to 16.51 when cooled to a temperature of 4 1C. Embryonic stem cells were used in the cell cultures and it showed similar cell morphology to the standard culture techniques, showing that the surface does not affect the viability of these cells.41 More importantly, the cells were detached by cooling at 4 1C without trypsin digestion and harvested in high efficiency. In the similar approach, human mesenchymal stem cells was used in the cell culture. The thermally detached cells showed strong intercellular associations and a sheet of cells adhered onto the cell culture plate was obtained during the cell sub-culture, which is different from the single cell seeding observed in a

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traditional trypsinization method.40 The maintenance of the interaction between cell–cell and cell–extracellular matrix could allow using the PNIPAAm–PHB–PNIPAAm coated surface as a new approach for cell harvesting for tissue regeneration therapies, bypassing the need for trypsin in the conventional cell harvesting process. On the other hand, the increased hydrophilicity of modified PHB via the formation of PHB/PEG poly(ether ester urethane)s copolymers was investigated for the enhancement of mineralization capability. The simple process in the polymer preparation could allow eliminating the complicated process involved in plasma or alkaline treatment for the same purpose of improving PHA hydrophilicity. Through rational control of the PEG content below 50 wt%, the water swelling property of the PHB/PEG poly(ether ester urethane)s electrospun scaffold was kept in an optimal range for mineralization, after which the scaffold would be ready to use without additional surface treatment. The mineralization in simulated body fluid (SBF) showed that the mineral content in the mineralized PHB/PEG poly(ether ester urethane)s is around 54 wt% and the mineralization could enter deep into the scaffold construct. This is in contrast with the 10 wt% of minerals content found in the PHBV scaffold, and the mineralization was largely confined to the top few layers of the scaffold.18

3. Biodegradation of water soluble PHA-based polymeric materials PHAs are susceptible to degradation through biodeterioration or depolymerisation to produce water soluble products. Two steps are involved in this form of degradation. The first step is to absorb the enzyme to the polymer surface by the binding domain of the enzyme and subsequently to hydrolyze the polymers through the active site of the enzyme. The rate of PHA hydrolysis degradation is dependent on many factors including the monomeric composition of PHAs, molecular weights, microstructure, surface morphology and concentration of enzyme. Inducing the hydrophobic PHA degradation through simple hydrolysis is not readily achieved and in many alternative approaches, researchers in the field have synthesized new amphiphilic or water soluble PHAs. The degradation of the polyester chains in these chemically modified PHAs has been altered as well. However, the precise control of PHA degradation

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is complex. Based on the different types of applications, the degree of hydrophilicity can be adjusted, by varying the content and structure of the hydrophilic component in the functionalized PHAs. For example, hydrogels prepared from PHB/PEG/PPG poly(ether ester urethane)s copolymers eroded during the hydrolytic degradation process in PBS, and the erosion time increased with decreasing PHB content in the copolymer. The molecular weight of the degraded products decreased sharply after 2 month; and as the hydrolysis proceeded for 6 months, significant amount of water soluble products degraded from PHB/PEG/PPG poly(ether ester urethane)s leached into the buffer solution.38 The degradation of the copolymer chains was expected to be a random occurrence via the cleavage of ester bonds in PHB. The degradation products were confirmed as 3-hydroxybutyric acid or its oligomers with 3–5 repeating units, and some PEG, PPG segment fragments.38 In another report, different degradation patterns were obtained by using amphiphilic P3/4HB/PEG poly(ether ester urethane)s copolymer films. The increasing hydrophilicity provided by the introduction of PEG would make the P3/4HB/PEG poly(ether ester urethane)s copolymer degrade at a faster rate than the neat P3/4HB. Compared with the hydrolytic degradation samples in surface erosion, the lipase could accelerate the chain scission of the ester bonds in P3/4HB, and followed a bulk diffusion-controlled degradation pattern. This would thus lead to a rougher and highly porous surface for the enzymatic degradation samples.20 A similar phenomenon was also observed in the degradation investigation of PHBHHx/PEG poly(ether ester urethane)s copolymers based films.21 However, it is important to note that the control of hydrophobic/hydrophilic balance in chemically modified PHAs is not enough to modulate the degradation behavior. The situations for real polymer degradation are more complex, and degradation has to be still studied with combinations of other hydrolysis factors.

4. Water soluble PHA monomers in medical physiology and therapeutic effects on diseases treatments Other than the chemically modified amphiphilic or water soluble PHAs-based polymeric materials, the degradation products of these polymers, PHA oligomers with the monomeric unit or a few repeating units, are also important water soluble materials. Similar to the classification of PHAs polymers, PHA monomers, noted as 3-hydroxyalkanoic acids (3-HA), can also be categorized into two groups depending on the side chain length; namely short chain and medium chain monomers. There are several strategies to produce PHA monomers, including chemical degradation of PHAs polymers, epoxidation and hydroxylation of allylic alcohols, reduction of keto esters, and some other biotransformation approaches.42 Although many PHA monomers have been identified, only few of these compounds were actively studied, such as 3-HB and 4-HB, showing potential in medical physiology and therapeutic effects in the treatment of some diseases. 3-HB is the simplest monomer among PHA monomers, and is found in human blood and tissue as a normal metabolite. The cellular responses to 3-HB were investigated by different

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cell types including murine fibroblast L929 cells, human umbilical vein endothelial cells, and rabbit articular cartilages. It was found that 3-HB in the concentration range of 0.005–0.10 g L1 was able to promote cell proliferation of each cell line.43 Specifically, in L929 cells, 3-HB at 0.02 g L1 could stimulate a rapid increase in cytosolic calcium (Ca2+) concentration. The 3-HB enhanced cell proliferation was more significant when the cells were cultured in high density. This is because the presence of 3-HB can prevent apoptotic and necrotic cell death. Furthermore, the effects of 3-HB on cell differentiation were evaluated by using murine osteoblast MC3T3-E1 cells.44 The results showed that the intensity of cell differentiation was proportional to 3-HB concentration up to 0.01 g L1. Moreover, the positive effect of 3HB on cell differentiation was also shown by the increased calcium deposition, and a higher expression level of osteocalcin (OCN) mRNA in MC3T3-E1 cells after 3-HB administration.44 On the other hand, oxidative stress was involved in the death of neurons and this would lead to cell apoptosis. The ability of 3-HB to protect neurons in culture was reported. For example, a previous work on PC12 cell culture showed that 3-HB inhibited the decrease of cell viability and showed neuroprotective effects through antioxidant activity. The results suggested that 3-HB and ketogenic diet would be of potential for therapy in the treatment of neurological diseases. Another study using glutamate excitotoxicity model showed that a combination of ketones 3-HB and acetoacetate could decrease neuronal death and prevent neuronal membrane properties changes. In addition to the reduced effect of 3-HB on oxidative injury and death, it was proposed that 3-HB could contribute to neuroprotective activities in mitochondria by maintaining bioenergetic processes and preserving cellular function.45 It has been well researched that glucose is the main nutritional composition that fulfils brain’s energy demands. However, ketone bodies 3-HB could also sustain neuronal function as energy substrates under some special conditions such as diabetes, starvation, and a ketogenic diet.46 Previous investigations showed that 3-HB is not only able to substitute for glucose as an energy substrate at low blood concentration but also able to preserve neuronal integrity and stability. The hyperketonemia effect on the glucose metabolism behaviour under stress conditions was previously studied by administration of 3-HB into rats in haemorrhagic hypotension. The plasma concentration of metabolites results suggested that the administrated 3-HB had a suppressive effect on glycolysis during haemorrhagic shock.47 PHA monomers also showed great potential in diagnostic and therapeutic applications in diseases treatment. For example, there is a significant need for rapid and accurate diagnosis for patients with diabetes so as to provide an appropriate level of care efficiently. Previously, diagnosing diabetic ketoacidosis (DKA) for a hyperglycemic patient required at least two tests before obtaining the relevant measurement results including blood glucose, serum bicarbonate, serum pH, and serum anion gap levels, as well as ketone body measurements in urine or serum. Recently, a commercial point-of-care device for easy 3-HB measurement has become readily accessible at low cost, allowing clinicians to diagnose hyperglycemic patients.48 In addition, the

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ketone body 3-HB was also reported as a positive marker for sudden deaths in chronic alcoholics due to hypoglycemia.49 In recent years, more therapeutic applications of PHA monomers in emerging areas have also been discovered, including treatment of epilepsy and neurodegenerative disorders etc. A previous study showed that the increasing level in the concentrations of blood 3-HB was useful to control seizures. For example, the effect of 3-HB on pilocarpine-induced seizures was evaluated in animal models. The mean latency to the onset of seizure was significantly prolonged in 3-HB treated mice. This suggests that ketone body 3-HB may represent a unique, additional therapeutic modality to treat patients with epilepsy.50

5. Conclusion PHAs are a class of biologically-derived polyesters produced under unbalanced growth conditions in bacteria. With useful properties such as biodegradability and biocompatibility, PHAs have gained great interest in the development of advanced medical devices and implanted tissue engineering. PHAs with no chemical modifications usually have high hydrophobicity and crystallinity, which have shown low drug loading and poor compatibility between polymers and drugs in drug release applications. The slow degradation rate and high hydrolytic stability in tissues make PHAs more appropriate for long-term (6 months to 2 years) applications. However, the possibilities of chemically modified PHAs into water soluble polymers afford new materials features such as novel chemical functionalities, bioactivities, as well as smart properties tailored towards specific applications. In this Tutorial Review, the latest development of novel water soluble PHA materials was addressed. We first summarized the design strategies and synthetic methods to produce water soluble PHA based polymers. This included the modification of PHAs polymers by introducing hydroxyl groups, amine groups and carboxylic groups, or inserting a wide range of hydrophilic components into PHAs to form block/graft copolymers through various polymerization techniques. Then, the biomedical significance of the functionalized water soluble PHAs polymers in advanced drug delivery, cancer therapy, DNA/siRNA delivery and tissue engineering in new aspects were discussed. Moreover, the production of water soluble PHA monomers has been shown to have a profound physiological impact. The effect of 3-HA monomers on different aspects of medical physiology were discussed, covering the promotion of cell proliferation, inhibition of apoptosis and suppression of oxidative stress. Finally, the 3-HA monomers enhanced metabolism efficiency and potential in diagnostic and therapeutic applications in some diseases treatments. The many possibilities to customise PHAs in a wide range of accessible properties have shown this type of materials to have great potential as therapeutic materials.

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Chem. Soc. Rev.

Water soluble polyhydroxyalkanoates: future materials for therapeutic applications.

Polyhydroxyalkanoates (PHAs) are excellent candidate biomaterials due to their exceptional biodegradability and biocompatibility. However, PHAs need t...
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