Materials Science and Engineering C 45 (2014) 620–634

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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec

Towards the development of polycaprolactone based amphiphilic block copolymers: molecular design, self-assembly and biomedical applications Zibiao Li, Beng Hoon Tan ⁎ Institute of Materials Research and Engineering, A*STAR (Agency for Science, Technology and Research), 3 Research Link, Singapore 117602, Singapore

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Article history: Received 14 January 2014 Received in revised form 12 May 2014 Accepted 9 June 2014 Available online 18 June 2014 Keywords: Polycaprolactone Amphiphilic block copolymers Self-assembly Drug delivery Tissue engineering

a b s t r a c t Polycaprolactone (PCL) and its copolymers are a type of hydrophobic aliphatic polyester based on hydroxyalkanoic acids. They possess exceptional qualities: biocompatibility; FDA approval for clinical use; biodegradability by enzyme and hydrolysis under physiological conditions and low immunogenicity. These critical properties have facilitated their value as sutures, drug delivery vehicles and tissue engineering scaffolds in pharmaceutical and biomedical applications. However, the hydrophobicity of PCL and its copolymers remains a concern for further biological and biomedical applications. One promising approach is to design and synthesize well-controlled PCL-based amphiphilic block copolymers. This review summarizes recent advances in the synthesis and self-assembly of PCL-containing amphiphilic block copolymers and their bio-related applications including drug delivery and tissue engineering. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Polycaprolactone (PCL), an aliphatic polyester, has been extensively investigated for biomedical applications owing to its good biocompatibility and low immunogenicity. The degradation product of PCL is 6-hydroxyhexanoic acid, which is a naturally occurring metabolite in the human body [1]. Sutures having PCL as a main component have been approved by the Food and Drug Administration (FDA) for use in surgeries, attesting to its safe application in humans. In other applications, PCL is used in Capronor, a commercially available 1-year implantable contraceptive device. The toxicology of PCL has been thoroughly studied in the safety evaluation of Capronor and the material has been generally regarded as safe [2]. For drug release applications, the advantages of PCL include its high permeability to drugs, and less acidic degradation products as compared to other types of aliphatic polyesters, such as polylactide (PLA) and polyglycolide (PGA) [3]. However, pure PCL is typically hydrophobic solids lacking in functionality and semi-crystalline such that strategic tailoring of their structure and functionality could expand their application base considerably. A strategy is to fabricate PCL into block copolymers, which has been found as promising method to manipulate their amphiphilic behavior, mechanical and physical properties by adjusting the ratio of the constituting block or adding new blocks of the desired properties. ⁎ Corresponding author. Tel.: +65 68741985. E-mail address: [email protected] (B.H. Tan).

http://dx.doi.org/10.1016/j.msec.2014.06.003 0928-4931/© 2014 Elsevier B.V. All rights reserved.

Such structure and functionality tailoring of PCL produces desired polymers with superior properties for a wide variety of applications including targeted drug delivery, sustained gene delivery, injectables and three dimensional (3D) cell encapsulation in tissue engineering [1–10]. In this review, the most intensively studied PCL based amphiphilic block copolymers are discussed in terms of their different architectures and various self-assemblies with special focus on the biomedical applications in the delivery system and tissue engineering. 2. PCL based amphiphilic block copolymers and their self-assemblies PCL based amphiphilic block copolymers are prepared by combing a hydrophilic segment with PCL blocks in various architectures which can then self-assemble into intriguing aggregates of various shapes and sizes in selected solvents such as micelles, vesicles, and hydrogels [11–17]. Hydrophilic components such as poly(ethylene glycol) (PEG), poly(acrylic acid), (PAA), poly(2-ethyl-2-oxazoline) (PEtOz), poly(Nisopropylacrylamide) (PNIPAAm) and poly(N,N-dimethylamino-2ethyl methacrylate) (PDMAEMA) have been utilized to construct amphiphilic block copolymers with PCL as the hydrophobic segment. 2.1. Diblock copolymers and their self-assemblies PEG-PCL diblock copolymers were previously synthesized by ring opening polymerization (ROP) of ε-caprolactone using monomethyoxy poly(ethylene glycol) MPEG as the macroinitiator (Fig. 1). Calcium

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Fig. 1. MPEG-PCL diblock copolymers prepared by ring opening polymerization.

ammoniate, HCl/Et2O, rare earth catalyst yttrium tris(2,6-ditertbutyl-4methylphenolate) [Y(DBMP)3], Lewis acid and Tin compounds are effective catalysts for the ROP of ε-caprolactone when using the terminal alcohol of PEG as initiators [18–22]. The morphologies of the self-assemblies in PEG-PCL diblock copolymers aqueous solution were highly dependent on the length of the building blocks. For example, Xu and his coworkers reported that the micellar morphology of PEG-PCL block copolymers in water can be regulated by crystallization temperatures [23]. In the case of PCLn-PEG44 block copolymers with a shorter soluble block, the perfection of PCL crystals dominated the micellar morphology. Lamellar micelles are formed at a higher crystallization temperature, while spherical micelles or cylindrical micelles tend to be formed at a lower crystallization temperature. On the other hand, when PEG length increased in PCLn-PEG113 block copolymers, the tethering density determines the micellar morphology (Fig. 2). Under this circumstance, spherical micelles or cylindrical micelles with a larger length/diameter ratio are formed at a higher crystallization temperature because the chain-folding number of the crystalline PCL block becomes smaller and the grafting density increases. However, the lower crystallization temperature could trigger lamellar micelles and cylindrical micelles formation with a smaller length/diameter ratio in PCLn-PEG113 block copolymers water solutions [23]. In another example, PEG114-PCL diblock copolymers with varied PCL chain length (1900–18300 g/mol), self-assembled in aqueous solution to form regular spherical micelles in the size range of 30–80 nm [20]. The critical micellization concentration (CMC) was determined to be 0.9–6.9 mg/L, which increased with decreasing PCL length. CMC values of these PEG-PCL diblock copolymers are generally low, indicating the potential application as drug delivery carriers [20]. Through further adjustment of compositions of the building blocks, PEG-PCL diblock copolymers were able to self-assemble into well controlled vesicles, which can provide sustained drug release for various durations [17]. In addition to the micelle and vesicle formation, PEG-PCL diblock copolymers which exhibited a sol-gel-sol transition as a function of

temperature have also been reported. In these copolymers, PEG at molecular weight of 750 g/mol was fixed while the PCL chain length varied from 1,400–3,000 g/mol [19]. The prepared diblock copolymers showed good solubility in water in the concentration range of 0–20 wt%. At room temperature, the polymer solutions formed a sol while the solutions underwent two stepwise sol-gel-sol phase transitions when the temperature is increased above room temperature. The maximum viscosity appeared at both heating and cooling cycles and the values increased with increasing hydrophobic content as well as the polymer concentration. Further evidence from XRD and DSC showed that the aggregation of hydrophobic PCL segments upon heating corresponded to the sol-gel transition. This thermoresponsive PEG-PCL polymers, which have a sol-gel transition at around body temperature, could be used as potential injectables [19]. On the other hand, amphiphilic diblock copolymers containing hydrophilic PEtOz and hydrophobic PCL were synthesized by living cationic ROP of 2-ethyl-2-oxazoline followed by ROP of ε-caprolactone in the presence of Lewis acid catalyst [22]. The CMC values were in the range of 1.0–8.1 mg/L with a micelle mean diameter of 108–192 nm. The aqueous characteristics including the CMC, micelle size and partition equilibrium constants were examined to be highly dependent on the hydrophobic PCL length in the block copolymers. With larger PCL block, the micelle size and partition equilibrium increases while the CMC values reduces, indicating the stronger driving force for the self-assembly tendency [22]. PCL-poly(acrylic acid) (PCL-PAA) diblock copolymers was synthesized by the combination of ROP of ε-caprolactone and atom transfer radical polymerization (ATRP) of tert-butyl acrylate, followed by selective hydrolysis of tert-butyl ester groups to acrylic acid groups [24]. The resulting amphiphilic block copolymer showed a CMC value of 1 mg/mL. However, the PAA corona could be crosslinked below the CMC via amidation chemistry. This would lead to a shell crosslinked micelle system and the drug release from thereof showed a controlled rate compared with the non-crosslinked micelles [24]. Similar to PCLPAA, well-defined PCL-poly(N,N-dimethylamino-2-ethyl methacrylate (PDMAEMA) diblock copolymers were synthesized, and their selfassembly was investigated in aqueous solutions [25]. The DLS results show that the size of the micelles increases with the copolymer concentration and the relative amount of PCL. The amine groups in PDMAEMA block made the PCL-PDMAEMA block copolyme micelles sensitive to the pH. When the DMAEMA segment is protonated and hydrated in an acidic medium (pH = 5–6), the diameter of the micelles became larger due to the electrostatic repulsions between the ammonium groups. Under more basic conditions (pHN 7), the deprotonation of the PDMAEMA segment induced coagulation of the spherical micelles into cylindrical micelles, demonstrating the effect of pH as a critical parameter to control the size and morphology of micelles [25].

2.2. Triblock copolymers and their self-assemblies

Fig. 2. Micellar morphology of PCLn-PEG113 block copolymers at different crystallization temperatures. Adapted from ref [23] with permission.

Triblock copolymers comprising of three different segments can be built into A-B-C triblock architecture. For example, a series of welldefined amphiphilic triblock copolymer MPEG-PCL-PDMAEMA were prepared by a three step reactions in the combination of ROP, transesterification and ATRP (Fig. 3) [26].

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Fig. 3. Synthetic scheme of MPEG-PCL-PDMAEMA (A-B-C) triblock copolymer. Adapted from ref [26] with permission.

The design consideration of MPEG-PCL-PDMAEMA triblock copolymer is that: firstly, the hydrophobic PCL block has good biodegradability and the hydrophobic interaction between PCL segments could be expected to induce the formation of micellar core and provide a reservoir for drug loading [27]. Then, the hydrophilic PEG chains have the potential to protect encapsulated cargo, prolong systemic circulation, and stabilize the nanoparticles [16]. Meanwhile, the PDMAEMA block is biocompatible and pH responsive, which can serve as a binding site for gene or negatively charged drugs for different applications [27]. More interestingly, the self-assembly studies showed that these amphiphilic MPEG-PCL-PDMAEMA triblock copolymers possess distinct pHdependent critical aggregation concentrations and can self-assemble into different morphologies in PBS buffer solution, including micelles or vesicles, depending on the length of PDMAEMA in the copolymer (Fig. 4) [16]. Further exploration showed that the micelles from this A-B-C triblock copolymer could functionalize as multi-arm physical crosslinker, and facilitate a stronger supramolecular hydrogel formation with α-cyclodextrin (α-CD) for sustained gene delivery [26]. Similarly, PEG-PCL-PAA (A-B-C) triblock copolymer were also reported to self-assemble into vesicles or large compound micelles

[28]. Herein, all the three components have been approved by the United States Food and Drug Administration (FDA) for medical uses. Specifically, PAA is a bio-adhesive, which has good and instantaneous mucoadhesive properties. It provides numerous advantages as a coating material for stabilization and surface modification. In the vesicle formation, the PCL block formed the wall and the bio-amphiphilic nature of PEG and PAA flanking the central PCL segment in PEG-PCL-PAA triblock copolymer can yield different surfaces inside and outside of the vesicles [28]. These aggregates are biocompatible and biodegradable. They could combine an essential robustness needed for a long-term encapsulation or absorption of guest molecules with the ability to release them after a certain lifetime in circulation. In the case of amphiphilic triblock copolymers with A and B segments, two segmental arrangements are possible, i.e. A-B-A or B-A-B, where A and B represent hydrophobic and hydrophilic segments, respectively. New A-B-A triblock copolymers, PCL-PEG-PCL, have been successfully synthesized by ROP of ε-caprolactone in the presence of PEG as the macroinitiator [29]. The synthesis approach is similar to the technique used in the PEG-PCL diblock copolymer preparation. A series of PCLPEG-PCL triblock copolymers with PEG chain length 1000–5000 g/mol

Fig. 4. Schematic illustration of polymer vesicle and micelle obtained from the self-assembly of MPEG-PCL-PDMA (A-B-C) triblock copolymers and their TEM images. Adapted from ref [16] with permission.

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and PCL block length 2600–27000 g/mol were prepared through this method. Aqueous micelles prepared from the triblock copolymers showed that the CMC are in the range of 0.4–1.2 mg/L, depending on the length of PCL blocks [29]. In another report, PCL-PEG-PCL nanoparticles were prepared in moderate condition by solvent diffusion method without using any surfactants. The prepared blank PCL-PEG-PCL nanoparticles are mono-dispersed and smaller than 200 nm, making them suitable for hormone delivery to overcome its poor solubility [30]. Besides micelles and nanoparticles, much more attention has been given to the mechanism and structure-property relationship of the sol-gel transition. This new type PCL-PEG-PCL triblock copolymers solutions underwent the sol-gel-sol transitions as the temperature increased, which is a flowing sol at ambient temperature and turns into a non-flowing gel at around physiological body temperature [15,31, 32]. However, the sol-gel-sol transition behavior of PCL-PEG-PCL hydrogel depends on many factors, such as the hydrophilic/hydrophobic balance (PEG/PCL ratio) of the molecular structure, the total molecular weight of the copolymer and the topological variation of the triblock copolymers [31]. The temperature-dependent micellar aggregation mechanism is schematically illustrated in Fig. 5. In aqueous solution, the hydrophobic PCL blocks in the PCL-PEG-PCL triblock copolymer tend to constitute the micelle's core due to hydrophobic interaction, and the hydrated PEG blocks are located in the outer hydrophilic shell. The PCL-PEG-PCL triblock copolymer formed loops in a micelle and intermicellar bridges between different micelles. At a temperature much lower than the sol–gel transition temperature, small micelles flow freely in the aqueous solution (Fig. 5A). The micelle size increases slightly as the temperature increases, but the solution remains in the sol state (Fig. 5B). Then, with further increase in temperature to around the sol–gel transition temperature, the micelle size increases rapidly, resulting in sol–gel transition (Fig. 5C). With increasing temperature, the aggregation and packing between micelles increases, forming a denser gel (Fig. 5D) [31]. Further report on A-B-A type triblock copolymers includes PCLPNIPAAm-PCL with different molecular weights. The novel copolymers were synthesized by the combination of ROP and reversible addition-fragmentation chain transfer (RAFT) polymerization [33]. The three consecutive steps include the synthesis of 2-(2carboxyethylsulfanylthiocarbonylsulfanyl) propionic acid (CPA) at

Fig. 5. A schematic diagram of the micellar aggregation mechanism for PCL-PEG-PCL (A-B-A) triblock copolymer aqueous solution as temperature increase. See the text for more explanation. Adapted from ref [31] with permission.

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the first step, followed by the synthesis of PCL-CPA-PCL by ROP using CPA as a transfer agent, and the last step is the synthesis of PCLPNIPAAm-PCL triblock copolymers through RAFT polymerization (Fig. 6) [33]. The CMC values of the resulted triblock copolymers in aqueous solution were determined in the range of 33.8-41.7 mg/L. The micelles self-assembled from the triblock copolymers exhibited well dispersed spherical morphology with diameter of around 100 nm. More importantly, the lower critical solution temperature (LCST) at physiological range could facilitate the drug loaded micelles to exhibit thermo-sensitive release behavior, indicating the great potential as a drug carrier for temperature-triggered controlled release [33]. Alternatively, B-A-B type triblock copolymers composed of two identical hydrophilic segments (B: PEG, PNIPAAm, PDMAEMA, etc.) and one hydrophobic segment (A: PCL) were also extensively explored. In an example, PEG-PCL-PEG triblock copolymers were synthesized by coupling MPEG-PCL-OH and MPEG-COOH by using dicyclohexylcar bodiimide (DCC) and 4-dimethylamino pyridine (DMAP) in a mild condition [34]. The particle size of the prepared micelles was around 40–92 nm. With the presence of hydrophobic PCL segments, the triblock copolymer could encapsulate drugs into the micelle cores and the in vitro release profiles exhibited a pH dependence and faster release at pH 5.4 than pH 7.4 [34]. On the other hand, Jeong and his coworkers reported PEG-PCL-PEG B-A-B type triblock copolymer as a poloxamer (PEG-PPG-PEG) analogue and studied the sol-gel transition behavior of PEG-PCL-PEG copolymer aqueous solutions [35]. Owning to the great thermo-sensitivity and biodegradability of these copolymers, an organic solvent-free injectable system could be desired as an in situ gel-forming system for advanced biomedical applications. Through the light scattering and 13C NMR studies, the driving force for sol to gel transition is micellar aggregation, while the increase in PCL molecular motion induces the gel to turbid sol transition. It is worth to note that PEG-PCL-PEG triblock copolymer are in powder form which could make it easy to handle and allow fast dissolution in water as compared with the previous thermogelling biodegradable polymers with sticky state [35]. PEG-PCL-PEG triblock copolymer water solution as thermoresponsive hydrogels were also examined by Qian's group [11]. Aqueous solutions of PEG-PCL-PEG triblock copolymers underwent thermo-sensitive sol-gel-sol transition as temperature increases when the concentration was above corresponding critical gel concentration (CGC). The sol-gel-sol transition temperature range could be varied through the adjustment of hydrophilic/hydrophobic balance in macromolecular structure, as well as topology of triblock copolymers and solution composition of the hydrogel (Fig. 7) [11]. Based on the reviewed content above, we understand that PEG-PCL-PEG A-B-A type triblock copolymers with different molecular weight and PEG/PCL ratio could be administrated from micelles or thermo-sensitive hydrogels respectively. Therefore, a composite system composed of PEG-PCL-PEG micelles and hydrogels could be interesting as a delivery carrier [36]. In the study, micelles were prepared by self-assembly of PEG-PCL-PEG (5000-5000-5000) copolymer triggered by its amphiphilic characteristic. Meanwhile, thermo-sensitive PEG-PCL-PEG (550-2400-550) hydrogel with a lower sol-gel transition temperature at around physiological range was also prepared successfully. The obtained PEG-PCL-PEG micelle/hydrogel complex demonstrated a significant improvement in the release property of the encapsulated cargo. A much slower and sustained release profile was observed as compared with the rapid release of free drugs. Combination of PEG-PCL-PEG micelle/hydrogel might provide a novel dosage form for clinical application [36]. In addition, thermo-responsive PNIPAAm-PCL-PNIPAAm B-A-B type triblock copolymers were synthesized by ATRP. These copolymers with PCL segment at high molecular weight (42000 g/mol) were insoluble in water. A thermo-responsive porous film could be fabricated from these copolymers. Moreover, the pore size and porosity in the membranes are controlled by the PNIPAAm content and temperature during the film casting [37]. On the contrary, PNIPAAm-PCL-PNIPAAm triblock

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Fig. 6. Synthesis of PCL-PNIPAAm-PCL (A-B-A) triblock copolymers by consecutive ROP and RAFT.

Fig. 7. Sol-gel-sol transition phase diagram of PEG-PCL-PEG triblock copolymers hydrogel: (A) effect of PCL block length, (B) effect of total molecular weight and (C) effect of topology. Adapted from ref [11] with permission.

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copolymer with a short central PCL segment with molecular weight of 2000 g/mol and various hydrophilic PNIPAAm lengths on both sides could make the copolymers water soluble [38]. The PNIPAAm-PCLPNIPAAm triblock copolymer formed micelles with a hydrophobic PCL core and a hydrophilic PNIPAAm corona as inferred from NMR spectra derived in two different environments. The mean diameters of the micelles were between 90-120 nm while the CMC values were in the range of 4–16 mg/L. The very low CMCs indicate that the micelles possess great stability under high dilution condition, which is a good characteristic for drug delivery application [38]. Likewise, PDMAEMAPCL-PDMAEMA B-A-B type triblock copolymers were achieved by ATRP of DMAEMA using di-functional PCL as macroinitiator [39]. Herein, the utilization of PDMAEMA for gene delivery is due to its relatively low toxicity and high buffer capacity. The combination of PCL biodegradation and low molecular weight PDMAEMA would be expected to further reduce their toxicity. PDMAEMA-PCL-PDMAEMA triblock copolymers, which formed cationic micelles with positive surface charges ranging from + 29.3 to + 35.5 mV, were recently discovered to exhibit high efficiency in drug and gene delivery into cancer cells [40]. 2.3. Multiblock copolymers and their self-assemblies PCL based multiblock copolymers can be built up by linking the functional groups located at the chain end of each constructing component either in a random or in an alternate manner, or by sequential polymerization of lactones/anhydride by ROP technique [41,42]. One approach is to link each segment by a coupling reagent, including diisocyanates, dicarbonyl dichloride or diols. The reactions occurrences include urethane, esterification, chloroformylation reaction etc. These approaches are also feasible in the modification of hydrophobic/hydrophobic balance of amphiphilic multiblock copolymers, which can further broaden its biomedical application with improved property. For example, multiblock copolymers composed of PCL and PEG were synthesized through one-pot copolymerization with hexamethylene diisocyanate (HDI) as a coupling agent [43]. The HDI/Diols was used to control the molecular weight. With optimal molecular weight and compositions, PCL/PEG polyurethane multiblock copolymers displayed a thermoreversible sol-gel-sol transition in water. When PEG/PCL block ratio decreased, the CGC decreased with an elevated sol-gel transition temperature on account of the enhanced hydrophobicity. The mechanism from phase separation induced gelation was proposed, in which the hydrophobic PCL blocks aggregated and formed domains when the hydrophilic PEG blocks were hydrated in water. The sol-gel transition resulted from the formation of 3D physical networks because multiPCL blocks could diffuse into different domains. On the other hand, the gel-sol transitions resulted from the melting of these domains and the collapse of physical crosslinks [43]. Interestingly, PCL/PEG polyurethane multiblock copolymers have a varied sol-gel transition range near to the body temperature, showing a high potential as new release controlled system. Another route to synthesize PCL/PEG multiblock copolymers is through coupling of the hydroxyl end groups in PCL-PEG-PCL triblock copolymers using terephthaloyl chloride [8]. The gelation investigation showed that the gel window of the PEG/PCL multiblock copolymer, the range of temperature where a gel phase exists, was narrower than that of the PCL-PEG-PCL triblock copolymer. However, similar to PCL-PEGPCL triblock copolymer, PEG/PCL multiblock copolymer keeps the powder morphology, and their aqueous solution (20 wt %) shows maximal modulus at around body temperature (35–42 °C). The powder morphology of both polymers is clearly distinguished from previous thermogelling polymers in that it is easy to weigh, transfer, and dissolve the polymer in water. The PEG/PCL multiblock copolymer aqueous solution was stable as a transparent solution at room temperature and thus gives some practical convenience during the drug formulation [8]. Pluronics or the PEG-PPG-PEG triblock copolymer is well known FDA approved polymer and has been applied in drug delivery systems as a thermogelling system for many years. Multiblock copolymers

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composed of PCL, PEG and PPG blocks have also been well investigated and their copolymer solutions in water show some interesting selfassembly behavior. In one report, PCL-PEG-PPG-PEG-PCL multiblock copolymers were synthesized by ROP of ε-caprolactone in the presence of PEG-PPG-PEG copolymers having hydroxyl groups at two ends of the chains as macroinitiator [44]. The nanospheres with core-shell structure showed a mean diameter of 116–196 nm, which was dependent on the type of copolymers. Due to the thermo-sensitivity of PPG moiety, the nanospheres exhibited a change of size responding to the temperature, As temperature increased, their size gradually decreased. This could be induced by the strong chain-chain aggregation occurred from the inter-molecular and intra-molecular interaction related to the solubility and hydrophobicity change of PPG block. Moreover, this change was fully reversible according to the repetitive thermal cycles. Release behavior from PCL-PEG-PPG-PEG-PCL multiblock copolymer nanospheres also showed temperature dependence and sustained release pattern [44]. In the same PCL-PEG-PPG-PEG-PCL multiblock copolymer synthesis approach, Liu et al. reported the gel-sol transition with temperature increased from 4 to 70 °C [45]. At above CGC, the results showed that the gel-sol phase diagram was dependent not only on the hydrophilic/ hydrophobic balance in macromolecular structure, but also on heating history of the copolymers aqueous solution. This is a good characteristic that can be used to broaden the temperature range of the phase transition for further application in biomedical field [45]. Instead of using PEG-PPG-PEG as macroinitator to prepare PCLPluronic-PCL multiblock copolymers with regular architecture, a random multiblock copolymers comprising the same components were synthesized through the polyurethane reaction (Fig. 8) [46]. PEG/PPG/ PCL polyurethane multiblock copolymers with various compositions were explored for biomaterials application in different forms. In one aspect, the new copolymer casted films performed thermo-responsive property, in which the films formed highly swollen hydrogel-like materials when soaked in cold water and shrank when soaked in warm water [46]. The ductile film property with reversible cyclical thermo-responsive behavior could make it a highly attractive candidate for use in biomedical devices that require regulation of behavior by temperature control. In another aspect, PEG/PPG/PCL polyurethane multiblock copolymers had been reported to have the capability to form themogels at optimal copolymer compositions [47]. The sol-gel transition diagram, showing the various sol and gel regions as a function of temperature and concentration of solution, was generated. The CGC of this new copolymer was found to be approximately 3 wt%. Drug release studies showed that sustained drug release of more than 2 weeks can be achieved within this system, and the anti-cancer drug loaded gels showed high efficiency in the control of the Hela cell growth [47]. In addition to the abovementioned copolymers, another new series of alternative PCL based amphiphilic multiblock copolymers were also reported and some interesting findings were present. Among these, novel amphiphilic alternating multiblock copolymers PCL-alt-PEtOz were synthesized by the condensation reaction of HO-PEtOZ-OH with HOOC-PCL-COOH by varying block compositions (Fig. 9). Hydrogels fabricated from PCL-alt-PEtOz multiblock copolymers exhibited phase transitions depending on the temperature increase. Upon heating, the optical transmittance of the hydrogel increased but further heating induced the decrease in the transmittance. The melting of the crystalline PCL domain and the hydrophobic interaction within the hydrogel could be correlated with the increase and decrease in optical transparency, respectively. The hydrogels exhibited reversible thermo-negative swelling-shrinking behavior. The swelling capacity of PCL-alt-PEtOZ multiblock copolymers prepared hydrogels could be manipulated by adjusting the PCL/PEtOz block ratios. In the mechanical tests, the dried gels and hydrogels of PCL-alt-PEtOZ multiblock copolymers prepared from longer PCL exhibited maximum strengths in the range of 10.6–12.5 and 3.2–7.3 MPa, and ultimate elongation up to 880–930 and 320–1000%, respectively [48]. In another paper,

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Fig. 8. Synthesis of PEG/PPG/PCL polyurethane multiblock copolymers. Adapted from ref [46] with permission.

PCL-alt-PEG polyurethane multiblock copolymers with regular and controlled block arrangement in an alternating fashion was prepared via selectively coupling reaction between PCL-diol and diisocyanate end-capped PEG [49]. PCL-alt-PEG polyurethane multiblock copolymers possess well controlled and determined chemical structure as well as regular microstructure. The regular structures endow materials with more special and intriguing properties, such as better biocompatibility, mechanical, and shape forming properties, giving us capacity for more sophisticated applications [49]. In addition, the biological interface test showed that PCL-alt-PEG polyurethane multiblock copolymers films could be optimized to give not only suitable mechanical properties and hemacompatibility, but also good cell compatibility, attachment, growth, and proliferation of rat glial cells thus rendering the copolymer films suitable candidate in nerve regeneration (Fig. 10) [49]. 2.4. Star-shape block copolymers and their self-assemblies Compared to the liner copolymers, star-shaped block copolymers have many characteristic properties due to the unique structure. Firstly, star-shaped copolymers have a smaller hydrodynamic radius and lower solution viscosity in comparison with linear polymers that have the same molecular weight and composition. In addition, the unimolecular micelles prepared from star-shaped block copolymer have great advantages in the micelle stability over that of its linear counterpart [53]. There are two methods to prepare the star-shaped block copolymers, “arm-first” and “core-first”. The “arm-first” route involves construction

of polymer arms on a macroinitiator that contains a precise number of reactive sites while the “core-first” approach utilizes multifunctional low molecular weight initiators allowing the synthesis of block copolymer chains [50]. With the fast evolution of polymerization techniques, several synthetic strategies have been reported for the effective synthesis of PCL based star-shaped amphiphilic block copolymers. Firstly, 3-arm star-shaped PCL/PEG block copolymers were synthesized. During the synthesis, star-shaped PCL was synthesized by OP of ε-caprolactone using tri-functional glycerol as initiator. Then, the 3-arm star-shaped PCL was further activated with succinic anhydride, and MPEG2000 was subsequently conjugated to this activated star-shaped PCL with DMAP as catalyst under a nitrogen atmosphere. The obtained 3-arm star-shaped PCL/PEG block copolymers can form micelles by self-assembly without contact with any organic solvent. The micelle properties as investigated by size distribution, zeta potential, and TEM visualization of the micelles indicated that the micelles are homogeneous and stable. The core-shell structure of the micelles can amorphously enclosed the honokiol and released it slower than the free one, demonstrating its potential as a new dosage form for drug delivery [51]. In another paper, 3-arm star-shaped [PCL2/PEG] block copolymers was made by a combination of Michael-addition type reaction and ROP polymerization [50]. A Michael-addition reaction yielded a PEG end-capped having two hydroxyl groups macrointiator which was used for sequential building of PCL blocks. This operational simplicity, high yield and purify of the products have been demonstrated to be the advantages of the applied synthetic strategy to the

Fig. 9. Synthesis route of PCL-alt-PEtOZ multiblock copolymers. Adapted from ref [48] with permission.

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Fig. 10. Synthesis of 3-arm star-shaped [PCL2/PEG] block copolymers. Adapted from ref [50] with permission.

previously described ones. The 3-arm star-shaped [PCL2/PEG] block copolymers can form spherical polymeric micelles with a smaller mean diameter, and the CMC values were lower than those of their linear counterparts [50]. The degradation study showed that 3-arm starshaped PCL/PEG block copolymers with shorter PCL block length degraded faster. The core-shell micelle sizes fluctuated during the initial degradation period and then increased slightly before finally dropping off. The degradation, which is significantly influence by the CL/EG ratios within the copolymers, occurred firstly at CL-CL linkages followed by the EG-CL linkages [52]. On the other hand, 4-arm star-shaped PCL/PEG block copolymers consisting of PEG and PCL were synthesized by ROP of the εcaprolactone monomer with hydroxyl-terminated 4-arm PEG as initiator [53]. These amphiphilic star block copolymers showed micellization and sol–gel transition behaviors in aqueous solution with varying concentration and temperature. In the dilute aqueous solutions of 4-arm star-shaped PCL/PEG block copolymers, micellization behavior occurred over specific concentrations. These new block copolymers with larger hydrophobic block formed micelles at a lower concentration. The obtained micelle sizes were in the range of 20–40 nm as measured by DLS. With increasing hydrophobic PCL block lengths, the micelle sizes increased. The temperature-sensitive sol–gel transitions were also observed on this 4-arm star-shaped PCL/PEG block copolymers at high concentrations (above 10 wt%). Morphology study of the two selfassemblies from the copolymers showed a core-corona spherical structure within the micelle and a mountain-chain-like morphology picture within the gel [53]. In addition, a 4-arm star-shaped PCL/PEG diblock copolymers were also explored. The 4-arm star-shaped PCL macromers with two or four hydroxyl end groups were prepared by ROP of ε-caprolactone and condensed with α-methoxy-ω-carboxyl PEG [54]. The copolymers were semi-crystalline and could selfassemble in aqueous media, giving perfect core-shell supramolecular structures. Polymeric micelles with a size less than 50 nm and a slightly negative surface could be prepared. The CMC values were in the range 0.010–0.023 mg/mL. The biological properties envisaged their use for potential drug targeting after intravenous injection [54]. Some interesting works have also been performed to compare the architecture of block copolymers and micellar properties and gelation between the 3-arm and 4-arm star-shaped PCL/PEG block copolymers, as well as the relationship with diblock copolymers [13,55]. Results showed that the CMC values decreased in the order of di-, 3-arm star

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shaped and 4-arm star shaped block copolymer. The size of the micelles increased in the same order of CMC. Theory predicted that the formation of micelles was easier for 4-arm star shaped block copolymers than that for di-, and 3-arm star shaped PCL/PEG block copolymers [55]. The aggregation behavior for sol-gel transitions of four different types of PCL/PEG block copolymers were also studied [13]. They are PEG-PCL di-, tri-, 3-arm and 4-arm star shaped block copolymers with PEG fraction between 66 and 86%. It is found that, for these block copolymers with the same PEG block lengths and molecular architecture, their aggregate diameters decreased as increasing PEG weight fraction. These results can be understood by considering that shorter hydrophobic PCL blocks led to smaller aggregates at the same hydrophilic PEG block length. The sizes of the aggregate diameters were below 50 nm. At 10 w/v% copolymer concentration in water, the copolymers with high PEG contents, such as PCL-PEG-PCL3, MPEG-PCL3, 3-arm PEGPCL3 and 4-arm PEG-PCL3 are transparent at 4 °C as compared to the copolymers with low PEG contents, such as PCL-PEG-PCL1, MPEG-PCL1, 3-arm PEG-PCL1 and 4-arm PEG-PCL1, which are semitransparent, turbid gels or turbid suspensions at the same temperature. This results are related to the water solubility of the copolymers architectures [13]. Amphiphilic PCL/PEG star shaped block copolymers with more arm numbers have also been investigated. In one case, the core of the starshaped block copolymers is polyamidoamine (PAMAM) dendrimer, the inner block in the arm is PCL and the outer block in the arm is PEG [56]. This new copolymer was synthesized first by ROP of ε-caprolactone with a PAMAM-OH dendrimer as initiator. The PEG was then attached to the PCL terminus by an ester-forming reaction. The unimolecular micelles from the 16-arm star shaped PCL/PEG block copolymers formed a relatively loose outer PEG shell, which was not sufficient to hinder the intermolecular association of the inner hydrophobic PCL blocks. It is expected that more stable unimolecular micelles may be obtained when the number of arms in a star polymer increased to a higher number. It has been demonstrated that when the number of arms reached 64, star-shaped PEG/PCL block copolymers exhibited characteristics of a hard sphere, and the repulsion between the star molecules became much stronger [56]. Recently, multi-arm star-shaped PCL/PEG block copolymers were prepared and used to deliver doxorubicin (Dox) [57]. During the synthesis, an acrylate MPEG-PCL diblock copolymer was synthesized, which can self-assemble into micelles with a core shell structure in water. Then the double bonds at the end of PCL blocks were conjugated together by radical polymerization, forming starshaped PCL/PEG micelles. The micelles were monodispersed with mean diameter of 25 nm. Moreover, the CMC of the star-shaped micelles were five times lower than that formed from MPEG-PCL diblock copolymers, indicating better stability against dilution, showing promising application in drug delivery [57]. 2.5. Hyperbranched block copolymers and their self-assemblies Amphiphilic hyperbranched core-shell polymers containing PCL and PEG with folate moieties as the targeting groups were synthesized and characterized [3]. In this copolymer design, the core of the amphiphilic polymers was hyperbranched aliphatic polyester Boltorn H40. The inner part and the outer shell of the amphiphilic polymers were composed of hydrophobic PCL segments and hydrophilic PEG segments, respectively. To achieve tumor cell targeting property, folic acid was further incorporated to the surface of the amphiphilic polymers via a coupling reaction between the hydroxyl group of the PEG segment and the carboxyl group of folic acid. The schematic illustration of the PEG/PCL hyperbranched block copolymers is presented in Fig. 11. In this new block copolymer, all the building components were selected with special reasons. H40 is commercial biodegradable hyperbranched aliphatic polyester, which has shown potential in biomedical applications. PCL is an important member of the aliphatic polyester family and of interest for biomedical applications owing to its good biocompatibility and low immunogenicity. For drug release applications, the

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Fig. 11. Schematic illustrations of amphiphilic PCL/PEG hyperbranched block copolymers. Adapted from ref [3] with permission.

advantages of PCL include its high permeability to drugs, and less acidic degradation products as compared to polylactide and polyglycolide. Biocompatible PEG was used as flexible spacers to connect with folate moieties. Folic acid (FA) is a targeting group which is extensively used to deliver therapeutic and imaging agents to folate receptor (FR) overexpressed cancer cells. The obtained amphiphilic PCL/PEG hyperbranched block copolymers can self-assemble to form nanoparticles with mean diameter less than 100 nm, and the size of nanoparticles increases with increasing molecular weight of the copolymer. Through incorporation of FA, the amphiphilic hyperbranched polymers are endowed with tumor cell targeting property [3]. Another new approach through the molecular recognition was recently reported to develop the amphiphilic PCL/PEG hyperbranched block copolymers [58]. In this approach, adenine-terminated H40-starPCL-adenine (H40-star-PCL-A) and uracil-terminated PEG (PEG-U) were successfully prepared. Due to the molecular recognition between A and U moieties, PCL/PEG hyperbranched block copolymers were obtained by simply mixing the hydrophobic H40-star-PCL-A core and hydrophilic PEG-U shell. They not only have similar properties to conventional covalent-linked multi-arm hyperbranched copolymers, but also possessed a dynamic and tunable nature. These new copolymers were found to self-assemble into pH-responsive micelles with low CMC because of non-covalent connection and hyperbranched architecture. The micelle sizes were easily tailored by simply adjusting the ratio of hydrophilic/hydrophobic segments. Furthermore, these micelles were found to release DOX inside the cells rapidly in an acidic environment to yield significantly enhanced drug efficacy. They are expected to be attractive new carriers for drug delivery or other medical applications [58]. Additionally, a novel bulk click polymerization approach was recently determined to prepare amphiphilic PCL/PEG multiblock and hyperbranched block copolymers [59]. During the synthesis, PCL and PEG were first modified into azide and alkyne terminated macromonomers by esterification, respectively. Subsequent azide– alkyne click polymerization of the two macromonomers in bulk or through the A2 + B3 strategy afforded the targeted block copolymers with higher degrees of polymerization than those obtained via solution polymerization in the control experiments, showing high efficiency of the click copolymerization. The self-assembly behavior of the amphiphilic multiblock copoylmers was investigated, and interesting structures such as globules, fibers, and worm were formed implying its usefulness as versatile self-assembly precursors to construct many supramolecular structures [59]. A2 + B3 strategy using urethane bond as the linkage was also widely adopted to prepare amphiphilic PCL hyperbranched block copolymers. Due to the strong hydrogen bonding effect between the urethaneurethane and urethane-ester linkage, interesting hydrogel assemblies were formed from this type of block copolymers aqueous solution with enhanced rheological properties. Recently, a new kind of thermogelling

polymers consisting of PPG, PEG and PCL in hyperbranched macromolecular architecture (HBPEC) were investigated [60]. In this report, trifunctional hydroxyl groups terminated PCL-triol was used as branching unit to lead the targeted polymer into hyperbranched structure. A comparison study with linear counterpart (LPEC) was carried out, in terms of their self-assembly and aggregation behaviors and thermoresponsive properties. Results revealed that the effect of hyperbranch architecture was more prominent in the gelation of the copolymers. The aqueous solutions of HBPEC copolymers exhibited thermogelling behaviors at critical gelation concentrations (CGCs) ranging from 4.3 to 7.4 wt %. These values are much lower than those reported on other PCL-contained linear thermogelling copolymers and Pluronic F127 copolymer. In addition, the CGC of HBPEC copolymers is much lower than the control LPEC copolymer. The hyperbranched structure would be expected to give an enhanced hydrogen bonding effect from increased urethane linkages and intensified hydrophobic interactions among polymer branches (Fig. 12) [60]. In a similar approach, biodegradable amphiphilic PCL polyurethane hyperbranched block copolymers were synthesized by copolymerizing PCL and PEG together with glycerol. Hydrogels were formed from these copolymers by swelling of water at low polymer concentrations. The morphology of the solid copolymers was semicrystalline, while the hydrogels were totally amorphous without crystallinity, showing the potential of providing a mild aqueous environment for living cells. The use of the branching agent glycerol is expected to further enhance the mechanical strength of the otherwise weak physical PCL/PEG hydrogels. The results found that the hyperbranched block copolymer hydrogels demonstrated superior elastic, thixotropic property, high water swelling capability, and copolymer composition-controlled hydrolytic degradation profile [1]. 3. Biomedical applications of amphiphilic PCL block copolymers The development of biodegradable PCL amphiphilic block copolymers has broadened the application into more advanced area. An overview for biomedical application, in particular delivery carrier and tissue engineering is provided. 3.1. Delivery carrier Current drug delivery systems are facing a great challenge in maintaining the optimum efficacy of the drug at a therapeutic concentration in the blood plasma. The drug efficacy decreases significantly when the concentration is below the critical value while the toxicity becomes a big concern when the concentration is too high after repeat dosage [61]. Controlled drug delivery technology, on the other hand, offers numerous advantages including improved efficacy, reduced toxicity, and improved patient compliance and convenience when compared to conventional dosage forms [62]. Biodegradable PCL based amphiphilic block copolymers could possibly provide the encapsulated drugs, a release profile in a controlled manner by means of the various selfassembly morphologies. In general, amphiphilic block copolymers self-assemble into a micelle-like structure with a hydrophobic inner core and a hydrophilic outer shell in selective solvent. In amphiphilic PCL-based block copolymeric micelles, hydrophobic core formed by PCL is stabilized and protected by hydrophilic components in an aqueous medium. Therefore, the drugs with a hydrophobic character can be easily incorporated into the core of nanoparticles by covalent or non-covalent bonding through hydrophobic interactions in aqueous media [63]. For example, PCL-PEG-PCL triblock copolymer nanoparticles were successfully prepared for honokiol delivery in vitro [33]. Blank or honokiol loaded PCL-PEG-PCL nanoparticles were prepared in moderate condition by solvent diffusion method without using any surfactants. The encapsulation of honokiol in the hydrophobic core of the monodispersed nanoparticles (~200 nm) retained the potent anticancer activity and did not induce hemolysis in vitro. Honokiol could be

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Fig. 12. Graphic showing the intramolecular interation model for the stable network-like packing of HBPEC copolymer chains (A). Sol–gel phase diagrams of HBPEC copolymers in comparison with LPEC and Pluronic F127 copolymers (B). Adapted from ref [60] with permission.

efficiently loaded into PCL-PEG-PCL triblock copolymer nanoparticles and released from thereof in an extended period in vitro. The released honokiol can effectively inhibit both cisplatin-sensitive (A2780s) and -resistant (A2780cp) human ovarian cancer cells proliferation in a dose-dependent manner in vitro [30]. In other reports, PCL-PEG-PCL triblock copolymer micelles was loaded with Dox to demonstrated the pharmacological application as drug carriers [34]. The in vitro release profiles of drugs from the micelles exhibited a pH dependence. Furthermore, in comparison with free DOX, DOX-loaded micelles showed a similar cytotoxicity to MCF-7/adr cells. Additionally, the micelles displayed higher cellular internalization and cytoplasm residence in drug-resistant cells [34]. In the case of using PCL-PEG-PCL triblock copolymers fabricated micelles/hydrogel composite system for drug delivery; the system was a free-flowing sol at ambient temperature and became a nonflowing gel at body temperature. The cytotoxicity results showed a safe carrier and the encapsulated honokiol retained its potent antitumor effect. In addition, the in vitro release profile demonstrated a significant difference between rapid release of free honokiol and much slower and sustained release of honokiol micelles/hydrogel. The results suggested that the PCL-PEG-PCL triblock copolymer based micelles/ hydrogel complex might have great potential applications in cancer chemotherapy [36]. Smart micelles self-assembled from thermosensitive PCL-PNIPAAm-PCL triblock copolymers exhibited well dispersed spherical morphology with diameter of around 100 nm [33]. The prednisone acetate loaded micelles of triblock copolymers exhibited thermosensitive drug release behavior, indicating the triblock copolymers have great potential as a drug carrier for temperature-trigged controlled release (Fig. 13). Similarly, release behavior of indomethacin from PCL/ Pluronic multiblock copolymer micelles also showed temperature dependence and a sustained release pattern [44]. On the other hand, ligand-directed delivery of hydrophobic drugs with polymeric micelles has been reported. For example, a cyclic pentapeptide c(Arg-Gly-Asp-d-Phe-Lys) (cRGD) as an αvβ3 ligand to the surface of dox-loaded PCL-PEG micelles. cRGD was selected as the targeting ligand since it can selectively bind to the αvβ3 integrin that are overexpressed on the angiogenic endothelial cells of the tumor vasculature or on tumor cells. Development of RGD-decorated PCL-PEG block copolymeric micelles that can increase the delivery of anticancer drugs to angiogenic tumor endothelial cells and metastatic tumor cells with high affinity (Fig. 14) [64]. Folic acid (FA) is a targeting group which is extensively used to deliver therapeutic agents to folate receptor overexpressed cancer cells. Through the incorporation of FA, amphipihlic PCL based hyperbranched block copolymers are also endowed with tumor cell targeting property by using 5-Fluorouracil (5-Fu) and Paclitaxel as model in the A549 cell culture [3]. Amphiphilic PCL based block copolymers containing cations in the hydrophilic components have also been extensively explored as gene

delivery carriers. For instance, complexation of plasmid DNA (pDNA) with hyperbranched PEI-PCL-PEG block copolymers was investigated to achieve particles of ca. 200 nm diameter. The gene transfection efficiency of polyplexes was found to be dependent on the copolymer composition. All the DNA/copolymer complexes showed a much lower ζ-potential (i.e., neutral or negative) than the DNA/PEI25 kDa complex (21 mV), indicating lower toxicity of PEI-PCL-PEG copolymer-based complexes. Lower cytotoxicity of DNA/copolymer complexes was also demonstrated by the viability of cells in the transfection experiments. These results indicate that these ternary copolymers are promising candidates for gene delivery, featuring good biocompatibility, potential biodegradability, and relatively high gene transfection efficiency [65]. Cationic MPEG-PCL-PDMAEMA triblock copolymer, on the other hand, was reported to have the ability to effectively condense pDNA to form polyplexes [16]. Recently, a sustained gene delivery system was developed by using this copolymer/pDNA polyplexes as anchors in the cyclodextrin (CD) based supramolecular hydrogels system (Fig. 15) [26]. In the designed system, MPEG-PCL-PDMAEMA triblock copolymers were used to condense pDNA into polyplexes. The multiple MPEG chains that are located at the corona of these polyplexes can be used as cross-linking moieties to anchor the DNA nanoparticles within the α-CD/PEG supramolecular hydrogel system. Such supramolecular cross-linking phenomenon also led to formation of stronger hydrogels. Upon hydrogel dissolution, the anchored DNA polyplexes provided controlled DNA release with high activity. In view of the ease of preparation that is free of organic solvent, the hydrogel's thixotropic nature, and controlled release property, this simple DNA entrapment strategy has immense potential in injectable sustained gene delivery systems [26]. Interestingly, co-delivery of drug and gene with MPEG-PCLPDMAEMA copolymers as carrier was also investigated to achieve the synergistic/combined effect on cancer therapies [27]. In the study, MPEG-b-PCL-b-PDMAEMA polyplexes with or without paclitaxel were

Fig. 13. Schematic illustration of thermally induced drug release from micelles selfassembled from PCL-PNIPAAm-PCL triblock copolymers. Adapted from ref [33] with permission.

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Fig. 14. RGD-functionalized PCL-PEG diblock copolymer and the Dox-loaded micelles for targeting drug delivery. Adapted from ref [64] with permission.

able to complex with pDNA completely when N/P ratio is equal to or above 3, and the paclitaxel-loaded copolymer/pDNA complexes have the equivalent transfection efficiency compared to blank complexes when N/P ratio is equal to or above 15. Whether or not carrying paclitaxel, the NPs/pDNA complexes can be efficiently internalized in 293 T cells after transfection for 2 h. The drug release rate of paclitaxelloaded polyplexes in pH 5.0 release medium is higher than that in

pH 7.2 release medium, which is a potential great value for anticancer drug delivery [27]. In another example, Zhong and his coworkers reported PDMAEMA–PCL–PDMAEMA triblock copolymers that were able to apply for the co-delivery of siRNA and paclitaxel into cancer cells (Fig. 16) [40]. Three different molecular weights of PDMAEMA blocks varied from 2700, 4800 to 9100 (denoted as polymer 1, 2 and 3, respectively) were used in their study. These PDMAEMA-PCL-

Fig. 15. Schematic illustration of MPEG-PCL-PDMAEMA copolymer/pDNA polyplexes anchored supramolecular hydrogel for sustained gene delivery.

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Fig. 16. Illustration of the combinatorial delivery of hydrophobic anti-cancer drugs and siRNA into cancer cells by using biodegradable cationic micelles self-assembled from PDMAEMA– PCL–PDMAEMA triblock copolymers. Adapted from ref [40] with permission.

PDMAEMA triblock copolymers formed nano-sized micelles in water with positive surface charges ranging from +29.3 to +35.5 mV. Notably, GFP siRNA complexed with micelle 1 exhibited significantly enhanced gene silencing efficiency as compared to that formulated with 20 kDa PDMAEMA or 25 kDa branched PEI in GFP-expressed MDA-MB-435GFP cells. Moreover, micelle 1 loaded with paclitaxel displayed higher drug efficacy than free paclitaxel in PC3 cells, due to most likely

improved cellular uptake. The co-delivery of VEGF siRNA and paclitaxel has revealed a further improved gene knockdown efficiency [40]. Importantly, these micellar carriers are easy to prepare with controlled molecular characteristics, are biodegradable, and have low cytotoxicity. These biodegradable micellar carriers are promising for cancer therapy with therapeutic siRNA and chemotherapeutics. Besides drug and gene delivery, a controlled protein release from amphiphilic self-assembled

Fig. 17. 3D cell encapsulation in hydrogel prepared from hyperbranched PCL-PEG-Glycerol (CEG) block copolymers. Adapted from ref [1] with permission.

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vesicles were reported by using PEG-PCL-PAA triblock copolymers [28]. Immobilization of the hydrophilic Bovine Serum Albumin (BSA) protein molecules was achieved either by encapsulation or by adsorption of the protein on the corona of the vesicles. The different natures of the inner and outer interfaces of the vesicles particles permitted preferential adsorption of the protein to one of the interfaces. This provided an essential robustness needed for a long-term encapsulation of guest molecules with the ability to release them after a certain lifetime in circulation. Such PEG-PCL-PAA copolymers vesicles may meet the requirements for technical or therapeutic applications requiring both biocompatibility and biodegradability [28]. 3.2. Tissue engineering In another aspect, amphiphilic PCL based block copolymers also show great potential in tissue engineering, comprising of 3D scaffold in tissue support, advanced treatment in the prevention of post-operative intestinal adhesion, nerve repair in tissue augmentation and cartilage regeneration. Recently, engineered hydrogels from hyperbanched PCL-PEGGlycerol (CEG) were investigated for 3D cell encapsulation application. The fabricated CEG hydrogels from direct water swelling process were found to have tunable rheological properties particularly suitable for living cell encapsulation (Fig. 17) [1]. These studied hydrogels underwent hydrolytic degradation in a controllable manner. Cells were encapsulated into the hydrogels, which were found to homogeneously distribute within the hydrogels. The encapsulated cells maintained good cell viability, and the cells recovered from the hydrogels showed good cell proliferation ability. Based on our results, it is expected that the hydrogels could provide a mild and suitable 3D environment for cells just like a cell suspension. Together with its injectable property in nature, the hydrogels could be promising injectable systems for cell delivery application, which is applicable to transfer cells into specific spot for tissue support and special disease treatment [1]. On the other hand, In situ gel-forming scaffolds from MPEG–PCL diblock copolymers were previously demonstrated to be successfully utilized in bone tissue engineering [66]. In the report, MPEG-PCL diblock copolymers were found to be biocompatible, maintained adequate mechanical properties for the intended period, be sterilizable to prevent infection and easy to handle. In light of this, solutions of these copolymers containing rBMSCs and dexamethasone injected into rats formed gel scaffolds at the injection sites and subsequently afforded viable bone formation over 4 weeks. The in situ gel scaffolds developed from MPEG-PCL diblock copolymers could potentially be used as an alternative to current treatments for debilitating orthopedic conditions [66]. In another example, supramolecular hydrogels selfassembled by α-CD and MPEG-PCL-MPEG triblock copolymers were prepared and characterized using 3D cell culture matrix [67]. Because of the rapid gelation property, the hydrogels could effectively entrap biologically active additives such as drugs and cells for in situ injection. In addition to the sustained drug release property, the ECV 304 and MSC cells were encapsulated in hydrogels, and the cell morphologies could be kept during the cell culture. The in vitro cytotoxicity and the in vivo histological studies demonstrated that the hydrogels were promising as injectable scaffolds for tissue engineering applications because of their good biocompatibility [67]. Moreover, PCL and PEG containing amphiphilic PCLA-PEG-PCLA triblock copolymer solution (poly(ε-caprolactone-co-lactide)-PEGpoly(ε-caprolactone-co-lactide)) were reported to serve as a barrier material to prevent post-operative intestinal adhesion, as schematically presented in Fig. 18. The hydrophobic blocks was copolymerized by CL and D,L-lactide (LA) to avoid the crystallinity of PCL blocks in the desired sol state. Besides, the presence of CL reduces the acidic effect of degradation products compared with using LA alone. The injectable physical hydrogel could be prepared from this copolymer solution. Little cytotoxicity and hemolysis of this polymer were found, and the in vivo inflammatory response was mild. Both the in vitro and in vivo

degradation experiments illustrated that the physical hydrogel was biodegradable, while its integrity could be retained as long as several weeks. In vivo applications of this hydrogel on prevention of postoperative adhesion indicated that the hydrogel is very convenient in operation and highly effective in reducing the formation of intraperitoneal post-operative intestinal adhesion [68]. A further study on the effect of encapsulation of RGD peptides in the anti-adhesion efficiency was performed [69]. The sustained release of RGD from this physical hydrogel was successfully achieved for 1 week. More importantly, this period fits the therapeutic window of anti-adhesion quite well. In vivo experiments confirmed a better efficacy of using RGD-encapsulated hydrogels to prevent tissue adhesions in rabbits than using RGD or hydrogel alone. The convenient operation of this peptide-loaded hydrogel and spontaneous gelation at body temperature could afford a facile and efficient system of anti-adhesion biomaterials integrating both pharmaceutical treatments and barrier-based devices [69]. Interestingly, when the RGD was immobilized on the hydrophilic blocks of this amphiphilic block copolymer, it increased the cell adhesion on the corresponding hydrogel surface much more significantly than that in the hydrophobic blocks. The difference may come from the RGD in hydrophilic blocks which were exposed on the micellar surface in water for a longer duration compared to those in hydrophobic blocks. This finding would guide the molecular design of bioactive materials for controlled cell adhesion in tissue engineering [70]. On the other hand, using of PCL/PEG based amphiphilic block copolymers as the recovery of nerve in tissue engineering was also explored. For example, PEG-PCL diblock copolymer micellar containing FK506 was used to investigate the promoting functional recovery in the crushed nerve. By using well-established animal model of peripheral nerve injury (crushed sciatic nerve), the rate of functional recovery of injured nerve is significantly enhanced in rats treated with micellar FK506. These findings supports the notion that PEG-PCL is a suitable polymer material for FK506 and also suggests that tailored PCL-b-PEG with different core-forming properties could offer versatile and convenient carriers to best suit the physical and pharmacological properties of drugs to be incorporated for advanced treatment in tissue engineering [71]. In another instance, the preliminary results of biological interface test showed that alternative PEG-PCL polyurethane multiblock copolymers could provide regular and controlled structure on the prepared film surface. The platelet adhesion assay on this new copolymer revealed that it possessed excellent hemocompatibility. In the cell culture assay, rat glial cells were favorable for the attachment and proliferation on surface. This would make the materials highly potential in nerve regeneration [49]. In another aspect, cartilage defects are common, painful conditions and none of the currently available treatment options are satisfactory. Tissue engineering techniques involving PCL/PEG amphiphilic block copolymers based hydrogels and scaffolds have shown great promise for the future because PEG is known to have anti-fouling properties and has been proven to prevent thrombogenesis in cartilage [72,73]. PCL on the other hand degrades at a much slower rate than other aliphatic polyesters such as PLA, PGA, and PLGA, which makes it more attractive when long-term implants are desired [74,75]. In one report, the mixture of PCL-PEG-PCL copolymer solution with autologous bone marrow stem cells (BMSCs) and chondrocytes (CH) were injected into rabbit articular defects and gelation was induced by photocrosslinking. The results revealed that co-culturing CHs and BMSCs in PCL-PEG-PCL hydrogels provides an appropriate in vitro microenvironment for chondrogenic differentiation and cartilage matrix production. The co-culture system exhibited a hyaline chondrocyte phenotype with excellent regeneration, resembling the morphology of native cartilage. These findings suggest that the co-culture of these two cell types promotes cartilage regeneration and that the PCL-PEG-PCL hydrogel scaffold, has potential in cartilage tissue engineering [76]. In another report, biodegradable elastic hydrogel scaffolds based on hydrophilic PEG and hydrophobic PCL were fabricated and investigated as a delivery vehicle of rabbit

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Fig. 18. Demonstration of amphiphilic PCLA-PEG-PCLA triblock copolymer based themogels and their application as anti-adhesive materials. Adapted from Ref [68] with permission.

chondrocytes for the formation of neocartilage [77]. Based on the swelling properties, chondrocytes cells could be directly and uniformly seeded into the scaffold sections without any further pre-wetting treatments. The in vitro and in vivo cell culture showed that the cells and tissue had different swelling ratios in the scaffolds for effective cell growth and tissue regeneration. Considering the versatile properties, including hydrophilicity, swelling, biodegradability, and mechanical strength, it is concluded that PEG/PCL-based hydrogel scaffolds could be a good tool for systematic studies of the interactions between cells or tissues and scaffolds [77]. 4. Conclusion The hydrophobicity of PCL and its copolymers enhances the uptake of drug-loaded NPs, resulting in their short residence time in circulation, thus leading to a decrease in drug efficiency in vivo. One promising approach to address the problem is to design and synthesize amphiphilic block copolymers that enable self-assembly toward the formation of core/shell nanoparticles consisting of a hydrophobic core surrounded by hydrophilic coronas. A variety of strategies for the synthesis of novel PCL based amphiphilic block copolymers have been developed. Hydrophilic polymers such as poly(ethylene glycol) (PEG), poly(acrylic acid), (PAA), poly(2-ethyl-2-oxazoline) (PEtOz), poly(N-isopropylacrylamide) (PNIPAAm) and poly(N,N-dimethylamino-2-ethyl methacrylate) (PDMAEMA) have been utilized to construct amphiphilic block copolymers with PCL as the hydrophobic segment. These PLA-based

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