International Journal of Pharmaceutics 472 (2014) 262–275
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International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
Thermoresponsive polymers: Insights into decisive hydrogel characteristics, mechanisms of gelation, and promising biomedical applications Maja Radivojša Matanovi c, Julijana Kristl, Pegi Ahlin Grabnar * University of Ljubljana, Faculty of Pharmacy, Ašker9 ceva 7, Ljubljana 1000, Slovenia
A R T I C L E I N F O
A B S T R A C T
Article history: Received 14 March 2014 Received in revised form 9 June 2014 Accepted 16 June 2014 Available online 17 June 2014
Thermally induced gelling systems have gained enormous attention over the last decade. They consist of hydrophilic homopolymers or block copolymers in water that present a sol at room temperature and form a gel after administration into the body. This article reviews the main types of thermoresponsive polymers, with special focus on decisive hydrogel characteristics, mechanisms of gelation, and biocompatibility. Promising biomedical applications are described with a focus on injectable formulations, which include solubilization of small hydrophobic drugs, controlled release, delivery of labile biopharmaceutics, such as proteins and genes, cell encapsulation, and tissue regeneration. Furthermore, combinations of thermoresponsive hydrogels and various nanocarriers as promising systems for sustained drug delivery are discussed through selected examples from the literature. Finally, there is a brief overview of current progress in nano-sized systems incorporating thermoresponsive properties. ã 2014 Elsevier B.V. All rights reserved.
Keywords: Thermosensitive hydrogels In situ forming systems Controlled release Protein delivery Nanocarriers Tissue regeneration
Contents 1. 2.
3.
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6. 7.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Thermoresponsive polymers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Decisive characteristics of thermoresponsive hydrogels . . . Mechanisms of gelation . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Biocompatibility and biodegradability of thermoresponsive 2.3. 2.4. Drug-release mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . Thermoresponsive polymers based on polysaccharides . . . . . . . . . Cellulose derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Chitosan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Xyloglucan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Thermoresponsive polymers based on polypeptides . . . . . . . . . . . Gelatin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. PELG–PEG–PELG triblock copolymers . . . . . . . . . . . . . . . . . 4.2. Synthetic thermoresponsive polymers . . . . . . . . . . . . . . . . . . . . . . 5.1. N-isopropylacrylamide copolymers . . . . . . . . . . . . . . . . . . . PEO/PPO/PEO triblock copolymers and derivatives . . . . . . . 5.2. 5.3. PEO/PLGA/PEO triblock copolymers . . . . . . . . . . . . . . . . . . . PCL–PEO–PCL triblock copolymers . . . . . . . . . . . . . . . . . . . 5.4. Thermoresponsive systems and nanocarriers . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
* Corresponding author. Tel.: +386 147 69621; fax: +386 142 58031. E-mail address: [email protected] (P.A. Grabnar). http://dx.doi.org/10.1016/j.ijpharm.2014.06.029 0378-5173/ ã 2014 Elsevier B.V. All rights reserved.
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1. Introduction Treating a disease with multiple dosing strategies and using conventional drug formulations has many drawbacks. These include fluctuation in drug plasma concentration, various side effects due to the high plasma level of a drug, and ineffective treatment because of the low drug concentration. Another important limitation of multiple dosing regimens is poor patient compliance, especially in diseases that require daily injections for years. Therefore, for effective treatment, it is desirable to maintain drug plasma level within the therapeutic concentration range for as long as the treatment requires. Today researchers are interested in novel drug delivery systems used to direct drugs to the specific site of action and to achieve a controlled release of drug with preferable release kinetics (He et al., 2008). Considering this, there has been growing interest in developing in situ forming or gelling systems as appropriate matrices suitable for local and prolonged drug and gene delivery, but also for cell encapsulation and tissue engineering (Jankovi c et al., 2013). These formulations include low-viscous fluids which can be injected into the body prior to gelling or solidifying. In addition, they represent “user-friendly” therapeutics and diagnostics. A prepared polymer solution can gel after photopolymerization (Burkoth and Anseth, 2000), chemical crosslinking (Ossipov and Hilborn, 2006), or ionic crosslinking (Kuo and Ma, 2001). In situ gelling systems that do not require aggressive and complex preparing procedures have gained growing attention. They are called “smart materials” because they respond to change in temperature, pH, or ionic strength (Peppas et al., 2000). Thermally responsive hydrogels have been extensively investigated because of their simple application and low adverse effects on tissues compared to other stimuli (Ruel-Gariépy and Leroux, 2004). Above a certain concentration, they represent a sol at ambient temperature, whereas show gelation in a body (Lai et al., 2014). Their thermoresponsive and biodegradable properties make them potential candidates for drug delivery, cell therapy, as well for tissue engineering. Thermoresponsive systems for drug delivery are generally classified into hydrogels, interpenetrating networks, micelles, and polymerosomes. Among the most studied are hydrogels, which are insoluble matrices of hydrophilic homopolymers or block copolymer networks that swell in water or physiological fluids (Klouda and Mikos, 2008). Network architecture could be enabled by physical interactions or by chemical crosslinking. Interpenetrating
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networks are constructed of two chemically linked polymeric conjugates that interact by physical entanglements, providing unique properties to the hydrogel. Micelles are constructed from amphiphilic block copolymers that self-assemble in a solution (Rezaei et al., 2012). Polymerosomes are also formed by selfassembling of block copolymers, but they have a hydrophilic core and a hydrophobic corona. Therefore, they have been used for the delivery of hydrophilic drugs (Ward and Georgiou, 2011). This article reviews thermoresponsive polymers that form in situ gelling systems in aqueous solutions in response to temperature change. The main topics addressed are types and characteristics of polymers, their gelation mechanisms, and promising biomedical uses. Furthermore, the combination of thermoresponsive hydrogels and various nanocarriers, as an advanced system for sustained drug delivery, is discussed through selected examples from the literature. Finally, there is a brief overview of current progress in nanocarrier systems incorporated with thermoresponsive properties. 2. Thermoresponsive polymers As shown in Table 1, many polymers exhibit a thermoresponsive phase transition property. They can be divided into two major classes based on their origin: naturally occurring polymers and synthetic materials. Cellulose, chitosan, xyloglucan, and gelatin, along with their derivatives, are some examples of natural polymers. Synthetic materials include poly-N-isopropylacrylamide (pNiPAAm), poly(ethylene oxide)-b-poly(propylene oxide)-b-poly(ethylene oxide) (PEO/PPO/PEO) block copolymers, poly(ethylene oxide)-b-poly(D,L-lactic acid-co-glycolic acid)-bpoly(ethylene oxide) (PEO/PLGA/PEO) triblock copolymers, and amphiphilic triblock copolymers, composed of PEO and polye-caprolactone (PCL) (PEO/PCL/PEO). The structures of selected thermoresponsive polymers are presented in Fig. 1. Interest in thermoresponsive polymers has rapidly grown, although the set of polymer structures capable of responding at an applicationspecific temperature has not grown at the same rate. However, new thermoresponsive polymer compositions are continually being developed with a focus on better biocompatibility and biodegradability. A greater role in the next generation of smart thermosensitive materials is expected (Hennink and van Nostrum, 2012; Roy et al., 2013). Polymers that are able to respond to changes close to body temperature are further discussed in Sections 3 and 4.
Table 1 Thermoresponsive polymers with the decisive hydrogel characteristics. Thermoresponsive polymers
Naturally occurring polymers
Poly(N-isopropylacryl amide) (PNiPAAm)
Gelatine MC HPMC Chitosan/ polyol salt Xyloglucan PNiPAAm-coAA PNiPAAm-coPEO
Gelation mechanism
Gelation concent. (wt%)
Gelation Storage modulus temp. at LCST ( C) (kPa)
References
Coil to triple helix transition Hydrophobic interactions
3 1–10
16
30–35
0.1–0.3
a b
Aqueous solution of gelatin gells in response to decrease in temperature (possesses UCST). The viscosity (h) of MC-based gel.
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Fig. 1. Chemical formulas of selected thermoresponsive polymers.
2.1. Decisive characteristics of thermoresponsive hydrogels The decisive characteristics of thermoresponsive hydrogels are presented in Table 1. The ideal system is a solution that represents a free-flowing liquid at room temperature, while gel after administration into a body (Ruel-Gariépy and Leroux, 2004). The structure of thermoresponsive polymers reflects a fine balance between the hydrophobic and hydrophilic groups. Due to this balance, a small change in the temperature in the aqueous polymer solution can create a new adjustment of the hydrophobic and hydrophilic interactions among the polymer blocks and water molecules
(Bajpai et al., 2008). The common feature of these polymers is the critical solution temperature (CST), which is defined as the temperature at which the polymer solution undergoes phase separation. Below the lower critical solution temperature (LCST), the polymers are soluble, and above the LCST they become hydrophobic and insoluble, causing the gel formation. This is actually a crucial process for the application of thermoresponsive gels in drug delivery (Bromberg and Ron, 1998). In contrast, a polymer solutions that gel upon cooling have an upper critical solution temperature (UCST) (Peppas et al., 2000). Generally speaking, the decisive characteristics of gels being
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thermoreversible are LCST, gelation polymer concentration, and viscosity parameter after gelation. It is important to point out that both gelation temperature and viscosity of thermoreversible gels are highly influenced by polymer concentration. As presented in Fig. 2a, with an increase in polymer concentration, the sol–gel transition temperature decreases as hydrophobic polymer interactions occur faster (Jeong et al., 1999). In addition, the effect of the polymer concentration on the LMWH and storage modulus (G0 ) value is shown in Fig. 2b. As obvious, at the beginning, G0 is low but increases drastically with temperature increase as a result of the gelation. Once the gel is formed, G0 becomes independent of the temperature. Further, the highest G0 is obtained with the most concentrated polymer solution. Once more, the transition temperature decreases with increasing concentration, and the curve is displaced to the left (Ricci et al., 2002). The thermoresponsiveness of hydrogels can be experimentally verified by various techniques which include spectrometry, differential scanning calorimetry, and rheology. 2.2. Mechanisms of gelation Various processes are responsible for thermally induced gelation of aqueous polymer solutions. The thermogelation mechanisms include coil-to-helix transition, hydrophobic interactions, micelle packing, and entanglements, as well as micellar growth combined with hydrophobic interactions (Klouda and Mikos, 2008). The common characteristic of thermosensitive hydrophilic homopolymers is the presence of hydrophobic groups such as methyl, ethyl, and propyl groups (Qiu and Park, 2001). The interactions that take place in an aqueous polymer solution are: polymer–polymer, polymer–water, and water–water (Klouda and Mikos, 2008). As mentioned above, below the LCST the polymers are soluble, and above the LCST they become increasingly hydrophobic, forming the hydrogel. At LCST, the scattering of light in the solution is increased due to collapse and aggregation of polymer chains. Subsequently, at cloud point, the two phases appear: collapsed gel and the water expelled from the gel. From the thermodynamic perspective, this abrupt change in solubility in response to the change in temperature is governed by the free energy of mixing (Schild, 1992). A change in environmental temperature results in negative free energy of the system, which makes polymer–water association unfavorable, allowing interactions between polymer chains and between water molecules. This negative free energy (DG) of association is attributed to the higher entropy term (DS) with respect to the increase in the enthalpy term (DH) in the thermodynamic relation: DG = DH TDS. The
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governing water–water interactions increase the entropy term (DS). Subsequently, as DG is negative polymer chains associate. These hydrogen (water–water) and hydrophobic (polymer–polymer) molecular interactions depend on temperature, and thus, determine the sol–gel transition (Klouda and Mikos, 2008; RuelGariépy and Leroux, 2004). At the sol–gel transition point, the solvated polymers quickly dehydrate and change to a more hydrophobic structure. On the other hand, some amphiphilic block copolymers, which undergo self-assembly in solution, exhibit micelle packing and thermogelation ability due to the polymer–polymer interactions (Mortensen and Pedersen, 1993). Thermoresponsive amphiphilic copolymers can form micelles or hydrogels in an aqueous environment at different temperatures, depending on their concentration. Several amphiphilic biodegradable copolymers, which contain hydrophobic segments such as poly(e-caprolactone) (PCL), poly(D,L-lactide), poly(glycolide) (PGA), and their copolymers, poly(lactide-co-glycolide) (PLGA), and hydrophilic segments such as polyethylene glycol (PEG), in di- or tri-block copolymers, have been used (Lai et al., 2014). The thermosensitivity of these micelles can be modulated by altering the concentration in aqueous solution or the composition of the copolymers. In addition, those block copolymers constructed of a hydrophobic core and hydrophilic corona have been mostly used for the delivery of poorly water-soluble or lipophilic drugs. As typical micelle size is between 20 and 100 nm, the renal clearance and rapid uptake by the reticuloendothelial system are avoided, and thus, the sustained drug delivery is provided. Finally, it is important to mention that all these mechanisms include reversible physical linkage of the polymers, so that gel–sol transition is enabled after the gelling stimulus is removed. 2.3. Biocompatibility and biodegradability of thermoresponsive polymers For most applications of biomaterials, their contact with cells and tissues via surfaces is inevitable (Drotleff et al., 2004). According to that, hydrogels intended for biomedical applications are required to have acceptable biocompatibility and biodegradability. Indeed, their hydrophilic surface has low interfacial free energy in contact with body fluids, which results in a low tendency for proteins and cells to adhere to these surfaces. (Hennink and van Nostrum, 2012). The characteristic of hydrogels with various chemical compositions and structures rely not only on the preparation methods but also on the monomers used in their synthesis. Numerous crosslinking methods routinely used for hydrogel preparation, both the
Fig. 2. (a) The phase diagram of PEG–PLGA–PEG triblock copolymer aqueous solution, gelation temperature vs. polymer concentration. Sol (flow) to gel (no flow) transition temperature was measured by the test tube inverting method increasing 2 C/step. Ref. (Jeong et al., 1999); (b) curves of the sol–gel transition temperature for Poloxamer 407 and lidocaine 2% solution shown as changes in elastic modulus (G' (Pa), at a frequency of 1 rad s1) at different polymer concentrations: (&) 20, () 25, and (*) 30%. Ref. (Ricci et al., 2002).
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Fig. 3. Drug release from a thermosensitive polymer micelle upon temperature increase (Nakayama et al., 2006).
chemical and physical approach, can be found in the literature (Hennink and van Nostrum, 2012; Roy et al., 2013). However, the use of physically crosslinked gels is more favorable due to the fact that crosslinking agents are not required for their preparation (Hennink and van Nostrum, 2012). Today much newer synthesized thermoresponsive polymers are available because they have undergone extensive biocompatibility examinations (Roy et al., 2013). As biodegradability is a desirable hydrogel property, weak bonds are, therefore, frequently introduced in their structure. The labile bonds can be broken under physiological conditions usually by hydrolysis, which means that the compounds formed can either be metabolized into harmless products or can be excreted by the renal filtration process. Biodegradable properties are also considered preferable for micelle-forming block copolymers because polymers obtained after degradation of the hydrophobic block are expected to be quickly excreted by the kidneys if the molecular weight of the thermoresponsive block is below the critical value (approx. 40,000) (Nakayama et al., 2006). Regarding the description above, it is clear that the attention should be paid to a variety of material design principles and a number of material classes used for thermoresponsive polymer synthesis. Interdisciplinary research between polymer chemistry and biomedical science has yielded several promising thermoresponsive systems for different application routes. Biomaterials are materials that show a marked compatibility with the biological environment or are able to replace or restore biological function or deliver therapeutic substances. Mimicking the biological environment could also be a matter of shaping a material at the micrometer and nanometer scale (Drotleff et al., 2004). However, certain drawbacks prevent their broader application, such as low cell adhesion and local acidity. An acidic environment due to the formation of acids by polymer degradation is deleterious to encapsulated bioactive proteins or cells and may induce in vivo inflammation in the surrounding tissue (Lai et al., 2014). Therefore, precise knowledge of the degradation and metabolism of polymers and excretion of metabolites is necessary, even before the testing is started. To monitor thermoresponsive polymers in a laboratory environment or an in vivo environment, there are a number of standardized methods to follow today, such as in vitro degradation and pH value change of hydrogel, in vitro biocompatibility, in vitro and in vivo drug release, gelling time, degree of swelling, degree of crosslinking, viscosity along with the heating process, sol–gel transition, morphology, and others.
In general, drug-release mechanisms from hydrogels are classified as: (1) diffusion-controlled, (2) erosion-controlled, (3) swelling-controlled, and (4) chemically controlled (Hamidi et al., 2008). Delivery systems with diffusion-controlled drug release are known as reservoir or matrix type systems. Drug diffusion out of a hydrogel primarily depends on the mesh sizes within the gel matrix, but also on hydrodynamic radius of the drug molecule. Typical mesh sizes reported for hydrogels in their swollen state range from 5 to 100 nm, and thus, larger than most small-molecule drugs. Consequently, the diffusion of small drugs is not considerably sustained, whereas macromolecules such as oligonucleotides and protein drugs have a prolonged release (Kristl et al., 1991). In cases in which the hydrodynamic radius of a drug molecule is larger than hydrogel pores, erosion or swelling will be the driving mechanism for release. In erosion-controlled hydrogels, the release is conducted by the surface or bulk erosion. Another interesting issue that has to be addressed is controlled drug release from thermoresponsive polymeric micelles. They consist of block copolymers that in a solution form micelles with a hydrophobic core and a thermoresponsive outer block. Drug molecules are embedded into the inner core, while the outer shell has a thermoresponsive ability (Fig. 3). These polymeric micelles show dramatic thermoresponsive on/off switching behavior for drug release according to the thermally induced structural changes in the micellar shell structure (Fig. 4). At temperatures below the polymer LCST, micelles are constructed of the hydrophilic outer shell and the insoluble inner core. At temperatures above the LCST, the hydrated shell shrinks and the drug is released by diffusion. Moreover, the drug release is quit by simply cooling below the LCST and accelerated again upon another heating. Hence, in this case, the drug release is governed by the change in temperature and the rate of drug diffusion (Chung et al., 1999; Nakayama et al., 2006).
2.4. Drug-release mechanisms During the development phase of a new delivery system, it is very important to know the transport processes in the gelled matrix. If injectable fluid consisting of a thermoresponsive polymer is introduced into the body and changes its structure from sol to gel, then a prolonged release is expected.
Fig. 4. On/off switching behaviour of drug release from thermoresponsive polymeric micelles, according to the change in temperature (Chung et al., 1999).
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3. Thermoresponsive polymers based on polysaccharides 3.1. Cellulose derivatives Originally, cellulose is a natural polysaccharide, insoluble in water. However, substitution of the hydroxyl groups with some hydrophobic segments renders the cellulose water-soluble. Certain cellulose derivatives show thermogelation properties in aqueous solutions (Calejo et al., 2012). In the concentrations 1–10%, aqueous solutions of such derivatives are liquid at low temperature, whereas yield gels when the temperatures is elevated. Methylcellulose (MC; Fig. 1a) and hydroxypropyl methylcellulose (HPMC; Fig. 1b) represent these polymers. MC exhibits thermoreversible gelation in aqueous solution, gelling at temperatures between 40 and 50 C, while HPMC shows a phase transition between 75 and 90 C (Sarkar, 1979). The thermogelation mechanism of aqueous MC and HPMC solutions is attributed to hydrophobic interactions among polymer chains containing methoxy groups, and therefore, the gelation is highly affected by substitution at the hydroxyl group of cellulose. Upon heating, polymer–polymer interactions become dominant, causing the gel formation. The important aspect is that phase transition temperatures of cellulose derivatives can be modified by some chemical and physical alterations. In addition, by reducing the hydroxypropyl molar substitution of HPMC, its transition temperature can be lowered to about 40 C (Sarkar, 1979). MC has been grafted with synthetic pNiPAAm in various ratios to produce fast reversibly thermogelling hydrogels and enhance the mechanical strength of the hydrogel (Liu et al., 2004a). Furthermore, recently a gel combined of low-molecular-weight MC and poloxamer micelles has been developed for subcutaneous and prolonged delivery of docetaxel. These mixtures could form a gel at temperatures between 15 and 40 C, depending on the drug concentration. The combined system released docetaxel for more than 30 days; moreover, the docetaxel stability was maintained (Kim et al., 2012). Another attractive approach towards a thermoresponsive MC systems is functionalization of MC with protein laminin, and thus, creating a bioactive scaffold for neural tissue engineering (Stabenfeldt et al., 2006). 3.2. Chitosan Chitosan is a linear polysaccharide composed of randomly distributed b-(1,4)-linked D-glucosamine and N-acetyl-D-glucosamine (Fig. 1c), which has been used extensively in drug delivery. It is biocompatible, biodegradable, and considered non-toxic. Chitosan is a hydrophilic, cationic polymer with gelation ability (Kristl et al., 1993). Gelation appears at body temperature when the pH of chitosan solution is increased to 7.2. Chitosan-based thermoresponsive gelling systems have also been investigated. Chenite et al. developed a thermosensitive neutral liquid formulation based on chitosan/polyol salt combinations (Chenite et al., 2000). Namely, pH-responsive chitosan solutions were transformed into thermoresponsive gelling systems after addition of polyol salts such as b-glycerophosphate. At pH 7, the solution was a clear liquid at ambient temperature, whereas exhibited gelation around 37 C. The dominant gelation mechanism of chitosan/b-glycerophosphate solution is assigned to hydrophobic interactions of chitosan chains that can be improved by the structuring glycerol–water interactions upon heating. A chitosanbased thermoresponsive gelling system was proposed for local application of the hydrophobic drug paclitaxel in order to prevent tumor recurrence. In vitro release profiles displayed prolonged paclitaxel delivery for over a month (Ruel-Gariépy et al., 2004). Furthermore, Bhattarai et al. embed hydrophilic poly(ethylene glycol) (PEG) into chitosan, the water solubility of chitosan was
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enhanced, and gelation occurred at physiological pH values (Bhattarai et al., 2005a). This approach was also applied in the development of a advanced system for the controlled albumin release (Bhattarai et al., 2005b). A thermoresponsive chitosanbased hydrogel for sustained release of insulin has been thoroughly investigated in vitro. According to the obtained results, the insulin was embedded into the aqueous gel regions, showing a sustained release over a period of 2 weeks. Additionally, the amount of released insulin increased with an increasing proportion of b-glycerophosphate (Kempe et al., 2008). Generally speaking, the chitosan b-glycerophosphate thermogelling systems seem to be an attractive delivery system for peptides and proteins. When it comes to tissue engineering applications, various thermogelling chitosan systems have been studied as potential cell carriers. A hydrogel based on watersoluble chitosan and copolymer of NiPAAm was investigated for chondrogenic differentiation of human mesenchymal stem cells both in vitro and in vivo (Cho et al., 2004). Moreover, Crompton et al. concluded that polylysine modified chitosan-glycerophosphate salt hydrogel could be an excellent candidate for neural tissue engineering (Crompton et al., 2007). In addition, Yan et al. developed chitosan-based injectable hydrogels modified with glycerophosphate and hydroxyethyl cellulose whose biocompatibility for culturing mouse mesenchymal stem cells was investigated. It was concluded that this system can be used as an interesting injectable in situ forming scaffold that represents a delivery system for biologically active therapeutics (Yan et al., 2010). 3.3. Xyloglucan Xyloglucan (Fig. 1d) exhibits thermoresponsive behavior when more than 35% of its galactose residues are removed. For treated xyloglucan, a lower and upper transition temperature from sol to gel and from gel to sol, respectively, were found. When temperature is increased, hydrophobic parts aggregate to minimize the hydrophobic surface area contacting the water, and thus, causing gelation (Hoare and Kohane, 2008). Xyloglucan gel is produced from solutions with concentrations in a range from 1 to 2% (Miyazaki et al., 1998), but strong hydrogels were only obtained at 3 wt% at 37 C (Nisbet et al., 2006). Even though rheological and morphological data of xyloglucan hydrogels are scant, this cytocompatible polysaccharide has been studied as a delivery vehicle for a number of applications (Ruel-Gariépy and Leroux, 2004). More recently, Chen et al. tested properties of xyloglucan hydrogel as biomedical sustained-release carriers. According to rat experiments, the results indicated the potential application of xyloglucan hydrogel as carriers for the oral drug delivery system of paracetamol. It was non-toxic and had remarkable water-retaining capacity (Chen et al., 2012). 4. Thermoresponsive polymers based on polypeptides 4.1. Gelatin Typical natural polymers showing thermally induced phase transition are gelatin and carrageenan. When the temperature is raised above 30 C, they adopt a random coil conformation at sol state. Upon lowering the temperature below 25 C, gelatin solution solidifies, and a continuous network is formed by partial helix formation (Fig. 5; Joly-Duhamel et al., 2002). As biomedical applications require the opposite behavior, gelatin has been grafted with other polymers that exhibit thermogelation upon heating. The main advantage of gelatin is its biodegradability and biocompatibility. Moreover, it is available for easy modifications at the amino acid level. A proper mixture of gelatin and
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Fig. 5. Thermoreversible gelation of gelatine and carrageenan caused by coil-triple helix transitions (Bromberg and Ron, 1998).
monomethoxy poly(ethylene glycol)-poly(D,L-lactide) produced a thermoresponsive hydrogel that underwent fast gelation upon cooling at 37 C (Yang and Kao, 2006). Gelatin grafted with poly-Nisopropylacrylamide (pNiPAAm) when the pNiPAAm:gelatin weight ratio was above 5.8 resulted in a thermoresponsive matrix analogue. Namely, it was shown that a high pNiPAAm:gelatin ratio improved the cell proliferation and matrix production due to increased hydrophobicity, leading to the formation of large aggregates. Consequently, a higher gel porosity occurred at 37 C, which comprised a favorable cell environment (Ohya and Matsuda, 2005). 4.2. PELG–PEG–PELG triblock copolymers New types of thermoresponsive gelling systems based on polypeptide poly(g-ethyl-l-glutamate)-poly(ethylene glycol)-poly (g-ethyl-l-glutamate) triblock copolymers (PELG–PEG–PELG) were tested for local and prolonged delivery of anticancer drugs. These hydrogels obtained from aqueous polypeptide solutions demonstrated much lower critical gelation concentration than the traditional hydrogels. According to biocompatibility studies, the in situ-formed hydrogels lasted for 21 days in the subcutaneous tissue. Furthermore, these formulations were applied as injectable implants with incorporated paclitaxel (PTX) in order to assess in situ antitumor activity. The results revealed that the PTXincorporated hydrogels could efficiently suppress tumor growth, while the damage to other organs was not registered. As a conclusion, these polypeptide-based thermoresponsive hydrogels possibly have the potential to serve as an effective vehicle for local antitumor drug delivery (Cheng et al., 2013). 5. Synthetic thermoresponsive polymers 5.1. N-isopropylacrylamide copolymers Poly(N-isopropylacrylamide copolymers) (pNiPAAm) is a synthetic, thermoresponsive polymer with a sol–gel transition temperature of about 32 C in aqueous solution. The LCST of pNiPAAm solution is strongly dependent on polymer molecular weight and structural architecture as well as on the presence of additives like salts, surfactants, or co-solvents (Liu et al., 2004b). Among many thermoresponsive polymers, pNiPAAm is probably the most extensively studied mainly due to its water solubility at room temperature (Fig. 1e). Above the LCST, the solution becomes opaque and transforms into a gel, primary due to hydrophobic interactions and coil-to-globule transition (Kulkarni and Aloorkar, 2010). Namely, at temperatures below LCST, hydrogen bonds between the amide groups of polymer and water molecules occur. Upon heating above the LCST, the hydrophobic interactions between polymer chains become predominant, hydrogen bonds are broken and the polymer dehydrates. The phase transition
temperature of pNiPAAm solution can be controlled by shifting the hydrophilic/hydrophobic balance by copolymerization of pNiPAAm with other monomers. For instance, an increase in hydrophobicity (i.e., incorporation of butyl methacrylate) or in molecular weight results in LCST decrease. Conversely, the incorporation of hydrophilic monomers (i.e., acrylic acid or hydroxyethyl methacrylate) promotes the formation of hydrogen bonds with thermoresponsive units, which increases the LCST (Kim et al., 2009). The swelling and release profiles of pNiPAAm hydrogels with regard to physicochemical properties of the embedded drugs have been evaluated by Coughlan et al. (Coughlan et al., 2004). The swelling of hydrogels with hydrophobic drugs was decreased, whereas the systems with hydrophilic drugs incorporated demonstrated enhanced swelling. In addition, a solubility-dependent drug release was observed for hydrophobic drugs, but hydrophilic drugs exhibited a molecular weight-dependent drug release. As mentioned above, hydrogels based on pNiPAAm and its copolymers are the most extensively investigated thermoresponsive systems used in drug delivery (Li et al., 2008; Liu et al., 2008, 2007; Nakayama et al., 2006; Sousa et al., 2010), cell encapsulation and delivery (Garbern et al., 2010; Na et al., 2007, 2006), and cell culture surfaces (Hatakeyama et al., 2006; Nakayama et al., 2012). In order to achieve thermoresponsive hydrogels with improved properties, a lot of work has been done on NiPAAm copolymers. The examples include NiPAAm copolymers with acrylic (AA) and propylacrilic acid (PAA), as well as with poly(ethylene oxide) (PEO) and poly(L-glutamic acid). The poly(NiPAAm-co-AA) solutions were tested as cell- and drug-delivery vehicles and also as cell matrices in a refillable bioartificial pancreas (Chae et al., 2001). Thermoresponsive pNiPAAm solutions were proposed as an extracellular matrix for islets of Langerhans placed in an immunoprotecting pouch installed in diabetic patients. It was shown that the copolymer, which formed a gel, efficiently immobilized rat islets without impairing insulin secretion, providing prolonged insulin secretion. (Vernon et al., 1999). The thermoresponsive p(NiPAAm-co-AA) hydrogel was studied as a vehicle for chondrocytes, dexamethasone, ascorbate as a differentiation factor, and transforming growth factor b3 (TGF-b3). This hydrogel was implanted subcutaneously in mice and significant collagen II expression as well as proteoglycan and polysaccharide production were observed after 8 weeks (Na et al., 2006). Poly(NiPAAm-co-AA) was also synthesized and copolymerized with ethyl acrylate using interpenetrating polymer network (IPN) technology (Liu et al., 2006). An interpenetrating polymer network consists of physically interconnected hydrophilic and hydrophobic network, while individual components retain their original properties. In addition, poly(NiPAAm-co-PAA) copolymers exhibit both temperature- and pH-responsive behavior (Yin et al., 2006). Thus, hydrogels based on poly(NiPAAm-co-PAA) can be useful for drug delivery applications in which temperature and local pH differences can both act as stimuli (Li et al., 2008). These thermoand pH-responsive properties definitely open up new opportunities in intelligent drug delivery systems. Recently, polymeric nanofibers from a blend of polyvinyl alcohol (PVA) and pNiPAAm were prepared for the topical delivery of levotiroxine (Azarbayjani et al., 2010). The authors concluded that the nanofibers were able to release levotiroxine in a sustained fashion, through diffusion and erosion mechanisms, providing the appropriate drug concentration for a long time and promoting the drug accumulation in the superficial skin layers. Thermoreversible systems based on triblock linear and star copolymers derived from linear and multi-arm PEG as the water-soluble central block and pNiPAAm as the thermoresponsive terminal blocks were also developed (Lin and Cheng, 2001). Injectable aqueous solutions were present at room
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Table 2 Basic physico-chemical characteristics of PEO–PPO–PEO copolymers considering chemical composition of three-block copolymer chains, average molecular weight, cloud point, representing sol–gel transformation temperature and HLB (Bromberg and Ron, 1998). Copolymera
Composition
Average MW
MPPO
PEO (wt%)
CPb ( C)
HLBc
L64 F68 F88 P103 P104 P105 F108 F127 L122
EO13PO30EO13 EO76PO29EO76 EO103PO39EO103 EO17PO60EO17 EO27PO61EO27 EO37PO56EO37 EO132PO50EO132 EO100PO65EO100 EO12PO67EO12
2900 8400 11,400 4950 5900 6500 14,600 12,600 5000
1740 1680 2280 3465 3540 3250 2920 3780 3600
50 80 80 30 40 50 80 70 20
58 >100 >100 86 81 91 >100 >100 19
12–18 >24 >24 7–12 12–18 12–18 >24 18–23 1–7
a b c
L, F, and P indicate liquid, flakes, and paste, respectively. Cloud point in aqueous 1 wt% solution. Hydrophilic–lipophilic balance.
temperature, but they formed relatively strong elastic gels at body temperature due to hydrophobic interactions of NiPAAm oligomer blocks. These copolymer solutions produce gels via physical crosslinking among pNiPAAm aggregates, while diblock copolymers gel by a micelle packing mechanism. 5.2. PEO/PPO/PEO triblock copolymers and derivatives The copolymer blocks based on poly(ethylene oxide)-b-poly (propylene oxide)-b-poly(ethylene oxide) (PEO–PPO–PEO) sequences (Fig. 1f) constitute a family of triple blocks with the commercial name Pluronics1. They were introduced in the late 1950s and are an important group of synthetic polymers exhibiting thermoresponsive property in concentrated aqueous solutions. The PEO–PPO–PEO triblock copolymers (poloxamers) cover a large number of liquids, pastes, and solids, and include polymers with various molecular weights and PEO/PPO block ratios (Table 2). As seen in Table 2, the cloud point of 1 wt% solution varies from 19 C, for the L122 copolymer with low PEO content and low HLB, to above 100 C, for poloxamers with high PEO content. Although most poloxamers listed in Table 2 have a LCST well above normal body temperature, they actually demonstrate gelation at 37 C in concentrated solutions. Although the gelation of poloxamers has been extensively investigated, the real mechanism still remains a controversial issue. The formation of micelles in aqueous poloxamer solutions as a result of PPO block dehydration has been proven by various experimental techniques (Schillén et al., 1993; Wanka et al., 1990; Zhou and Chu, 1988). Namely, these copolymers form micelles above critical micelle concentration (1 mg/ml), which equilibrate with poloxamer unimers at low temperature (Alexandridis and Alan Hatton, 1995). Further, below a critical micelle temperature, both ethylene and propylene oxide blocks are hydrated, and PPO is relatively soluble in water. As the temperature increases (Fig. 6),
PPO chains become less soluble, and micellization becomes more important. As the number of unassociated unimers in solution is reduced, the micelle volume fraction (Fm) is increased. At a definite point, micelles come into contact and no longer move, forming various liquid crystalline phases (cubic 3D-lattice, hexagonal 2D-lattice, and lamellar bilayers). When Fm > 0.53, the formulation turns into a gel due to the micelle packing (Mortensen and Pedersen, 1993). Hence, packing of micelles and micelle entanglements may be the mechanisms of poloxamer gelation in aqueous solution as a response to the temperature increase (Cabana et al., 1997). On the other hand, the reason for gel–sol transition at high temperatures is still unknown and could be possibly related to the shrinkage of the PEO corona of the micelles and the interactions between PEO chains and PPO core (Li et al., 1997). Among more than 30 different non-ionic surface active agents of the poloxamer family, Poloxamer 407 (P407), also known under the registered trademark of Pluronic F1271 (BASF laboratories, Wyandotte, MI, USA), has probably been used most extensively in drug delivery studies. P407 has a molecular weight of about 12,600 (9840–14,600), with a PEO/PPO ratio of 2:1 containing about 70 wt % of ethylene oxide, which contributes to its hydrophilicity (Table 2). It has been reported to have a low viscosity below 4 C, whereas forms a gel at concentrations higher than 20 wt% at 25 C. P407 is more soluble in cold than in hot water due to the presence of hydrogen bonds at low temperatures (Escobar-Chavez et al., 2006). The P407 aqueous solution in the concentrations between 20 and 30 wt% shows reversible gelation upon heating. Namely, at low temperature (
Contents lists available at ScienceDirect
International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
Thermoresponsive polymers: Insights into decisive hydrogel characteristics, mechanisms of gelation, and promising biomedical applications Maja Radivojša Matanovi c, Julijana Kristl, Pegi Ahlin Grabnar * University of Ljubljana, Faculty of Pharmacy, Ašker9 ceva 7, Ljubljana 1000, Slovenia
A R T I C L E I N F O
A B S T R A C T
Article history: Received 14 March 2014 Received in revised form 9 June 2014 Accepted 16 June 2014 Available online 17 June 2014
Thermally induced gelling systems have gained enormous attention over the last decade. They consist of hydrophilic homopolymers or block copolymers in water that present a sol at room temperature and form a gel after administration into the body. This article reviews the main types of thermoresponsive polymers, with special focus on decisive hydrogel characteristics, mechanisms of gelation, and biocompatibility. Promising biomedical applications are described with a focus on injectable formulations, which include solubilization of small hydrophobic drugs, controlled release, delivery of labile biopharmaceutics, such as proteins and genes, cell encapsulation, and tissue regeneration. Furthermore, combinations of thermoresponsive hydrogels and various nanocarriers as promising systems for sustained drug delivery are discussed through selected examples from the literature. Finally, there is a brief overview of current progress in nano-sized systems incorporating thermoresponsive properties. ã 2014 Elsevier B.V. All rights reserved.
Keywords: Thermosensitive hydrogels In situ forming systems Controlled release Protein delivery Nanocarriers Tissue regeneration
Contents 1. 2.
3.
4.
5.
6. 7.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Thermoresponsive polymers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Decisive characteristics of thermoresponsive hydrogels . . . Mechanisms of gelation . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Biocompatibility and biodegradability of thermoresponsive 2.3. 2.4. Drug-release mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . Thermoresponsive polymers based on polysaccharides . . . . . . . . . Cellulose derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Chitosan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Xyloglucan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Thermoresponsive polymers based on polypeptides . . . . . . . . . . . Gelatin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. PELG–PEG–PELG triblock copolymers . . . . . . . . . . . . . . . . . 4.2. Synthetic thermoresponsive polymers . . . . . . . . . . . . . . . . . . . . . . 5.1. N-isopropylacrylamide copolymers . . . . . . . . . . . . . . . . . . . PEO/PPO/PEO triblock copolymers and derivatives . . . . . . . 5.2. 5.3. PEO/PLGA/PEO triblock copolymers . . . . . . . . . . . . . . . . . . . PCL–PEO–PCL triblock copolymers . . . . . . . . . . . . . . . . . . . 5.4. Thermoresponsive systems and nanocarriers . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
* Corresponding author. Tel.: +386 147 69621; fax: +386 142 58031. E-mail address: [email protected] (P.A. Grabnar). http://dx.doi.org/10.1016/j.ijpharm.2014.06.029 0378-5173/ ã 2014 Elsevier B.V. All rights reserved.
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1. Introduction Treating a disease with multiple dosing strategies and using conventional drug formulations has many drawbacks. These include fluctuation in drug plasma concentration, various side effects due to the high plasma level of a drug, and ineffective treatment because of the low drug concentration. Another important limitation of multiple dosing regimens is poor patient compliance, especially in diseases that require daily injections for years. Therefore, for effective treatment, it is desirable to maintain drug plasma level within the therapeutic concentration range for as long as the treatment requires. Today researchers are interested in novel drug delivery systems used to direct drugs to the specific site of action and to achieve a controlled release of drug with preferable release kinetics (He et al., 2008). Considering this, there has been growing interest in developing in situ forming or gelling systems as appropriate matrices suitable for local and prolonged drug and gene delivery, but also for cell encapsulation and tissue engineering (Jankovi c et al., 2013). These formulations include low-viscous fluids which can be injected into the body prior to gelling or solidifying. In addition, they represent “user-friendly” therapeutics and diagnostics. A prepared polymer solution can gel after photopolymerization (Burkoth and Anseth, 2000), chemical crosslinking (Ossipov and Hilborn, 2006), or ionic crosslinking (Kuo and Ma, 2001). In situ gelling systems that do not require aggressive and complex preparing procedures have gained growing attention. They are called “smart materials” because they respond to change in temperature, pH, or ionic strength (Peppas et al., 2000). Thermally responsive hydrogels have been extensively investigated because of their simple application and low adverse effects on tissues compared to other stimuli (Ruel-Gariépy and Leroux, 2004). Above a certain concentration, they represent a sol at ambient temperature, whereas show gelation in a body (Lai et al., 2014). Their thermoresponsive and biodegradable properties make them potential candidates for drug delivery, cell therapy, as well for tissue engineering. Thermoresponsive systems for drug delivery are generally classified into hydrogels, interpenetrating networks, micelles, and polymerosomes. Among the most studied are hydrogels, which are insoluble matrices of hydrophilic homopolymers or block copolymer networks that swell in water or physiological fluids (Klouda and Mikos, 2008). Network architecture could be enabled by physical interactions or by chemical crosslinking. Interpenetrating
263
networks are constructed of two chemically linked polymeric conjugates that interact by physical entanglements, providing unique properties to the hydrogel. Micelles are constructed from amphiphilic block copolymers that self-assemble in a solution (Rezaei et al., 2012). Polymerosomes are also formed by selfassembling of block copolymers, but they have a hydrophilic core and a hydrophobic corona. Therefore, they have been used for the delivery of hydrophilic drugs (Ward and Georgiou, 2011). This article reviews thermoresponsive polymers that form in situ gelling systems in aqueous solutions in response to temperature change. The main topics addressed are types and characteristics of polymers, their gelation mechanisms, and promising biomedical uses. Furthermore, the combination of thermoresponsive hydrogels and various nanocarriers, as an advanced system for sustained drug delivery, is discussed through selected examples from the literature. Finally, there is a brief overview of current progress in nanocarrier systems incorporated with thermoresponsive properties. 2. Thermoresponsive polymers As shown in Table 1, many polymers exhibit a thermoresponsive phase transition property. They can be divided into two major classes based on their origin: naturally occurring polymers and synthetic materials. Cellulose, chitosan, xyloglucan, and gelatin, along with their derivatives, are some examples of natural polymers. Synthetic materials include poly-N-isopropylacrylamide (pNiPAAm), poly(ethylene oxide)-b-poly(propylene oxide)-b-poly(ethylene oxide) (PEO/PPO/PEO) block copolymers, poly(ethylene oxide)-b-poly(D,L-lactic acid-co-glycolic acid)-bpoly(ethylene oxide) (PEO/PLGA/PEO) triblock copolymers, and amphiphilic triblock copolymers, composed of PEO and polye-caprolactone (PCL) (PEO/PCL/PEO). The structures of selected thermoresponsive polymers are presented in Fig. 1. Interest in thermoresponsive polymers has rapidly grown, although the set of polymer structures capable of responding at an applicationspecific temperature has not grown at the same rate. However, new thermoresponsive polymer compositions are continually being developed with a focus on better biocompatibility and biodegradability. A greater role in the next generation of smart thermosensitive materials is expected (Hennink and van Nostrum, 2012; Roy et al., 2013). Polymers that are able to respond to changes close to body temperature are further discussed in Sections 3 and 4.
Table 1 Thermoresponsive polymers with the decisive hydrogel characteristics. Thermoresponsive polymers
Naturally occurring polymers
Poly(N-isopropylacryl amide) (PNiPAAm)
Gelatine MC HPMC Chitosan/ polyol salt Xyloglucan PNiPAAm-coAA PNiPAAm-coPEO
Gelation mechanism
Gelation concent. (wt%)
Gelation Storage modulus temp. at LCST ( C) (kPa)
References
Coil to triple helix transition Hydrophobic interactions
3 1–10
16
30–35
0.1–0.3
a b
Aqueous solution of gelatin gells in response to decrease in temperature (possesses UCST). The viscosity (h) of MC-based gel.
264
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Fig. 1. Chemical formulas of selected thermoresponsive polymers.
2.1. Decisive characteristics of thermoresponsive hydrogels The decisive characteristics of thermoresponsive hydrogels are presented in Table 1. The ideal system is a solution that represents a free-flowing liquid at room temperature, while gel after administration into a body (Ruel-Gariépy and Leroux, 2004). The structure of thermoresponsive polymers reflects a fine balance between the hydrophobic and hydrophilic groups. Due to this balance, a small change in the temperature in the aqueous polymer solution can create a new adjustment of the hydrophobic and hydrophilic interactions among the polymer blocks and water molecules
(Bajpai et al., 2008). The common feature of these polymers is the critical solution temperature (CST), which is defined as the temperature at which the polymer solution undergoes phase separation. Below the lower critical solution temperature (LCST), the polymers are soluble, and above the LCST they become hydrophobic and insoluble, causing the gel formation. This is actually a crucial process for the application of thermoresponsive gels in drug delivery (Bromberg and Ron, 1998). In contrast, a polymer solutions that gel upon cooling have an upper critical solution temperature (UCST) (Peppas et al., 2000). Generally speaking, the decisive characteristics of gels being
M.R. Matanovic et al. / International Journal of Pharmaceutics 472 (2014) 262–275
thermoreversible are LCST, gelation polymer concentration, and viscosity parameter after gelation. It is important to point out that both gelation temperature and viscosity of thermoreversible gels are highly influenced by polymer concentration. As presented in Fig. 2a, with an increase in polymer concentration, the sol–gel transition temperature decreases as hydrophobic polymer interactions occur faster (Jeong et al., 1999). In addition, the effect of the polymer concentration on the LMWH and storage modulus (G0 ) value is shown in Fig. 2b. As obvious, at the beginning, G0 is low but increases drastically with temperature increase as a result of the gelation. Once the gel is formed, G0 becomes independent of the temperature. Further, the highest G0 is obtained with the most concentrated polymer solution. Once more, the transition temperature decreases with increasing concentration, and the curve is displaced to the left (Ricci et al., 2002). The thermoresponsiveness of hydrogels can be experimentally verified by various techniques which include spectrometry, differential scanning calorimetry, and rheology. 2.2. Mechanisms of gelation Various processes are responsible for thermally induced gelation of aqueous polymer solutions. The thermogelation mechanisms include coil-to-helix transition, hydrophobic interactions, micelle packing, and entanglements, as well as micellar growth combined with hydrophobic interactions (Klouda and Mikos, 2008). The common characteristic of thermosensitive hydrophilic homopolymers is the presence of hydrophobic groups such as methyl, ethyl, and propyl groups (Qiu and Park, 2001). The interactions that take place in an aqueous polymer solution are: polymer–polymer, polymer–water, and water–water (Klouda and Mikos, 2008). As mentioned above, below the LCST the polymers are soluble, and above the LCST they become increasingly hydrophobic, forming the hydrogel. At LCST, the scattering of light in the solution is increased due to collapse and aggregation of polymer chains. Subsequently, at cloud point, the two phases appear: collapsed gel and the water expelled from the gel. From the thermodynamic perspective, this abrupt change in solubility in response to the change in temperature is governed by the free energy of mixing (Schild, 1992). A change in environmental temperature results in negative free energy of the system, which makes polymer–water association unfavorable, allowing interactions between polymer chains and between water molecules. This negative free energy (DG) of association is attributed to the higher entropy term (DS) with respect to the increase in the enthalpy term (DH) in the thermodynamic relation: DG = DH TDS. The
265
governing water–water interactions increase the entropy term (DS). Subsequently, as DG is negative polymer chains associate. These hydrogen (water–water) and hydrophobic (polymer–polymer) molecular interactions depend on temperature, and thus, determine the sol–gel transition (Klouda and Mikos, 2008; RuelGariépy and Leroux, 2004). At the sol–gel transition point, the solvated polymers quickly dehydrate and change to a more hydrophobic structure. On the other hand, some amphiphilic block copolymers, which undergo self-assembly in solution, exhibit micelle packing and thermogelation ability due to the polymer–polymer interactions (Mortensen and Pedersen, 1993). Thermoresponsive amphiphilic copolymers can form micelles or hydrogels in an aqueous environment at different temperatures, depending on their concentration. Several amphiphilic biodegradable copolymers, which contain hydrophobic segments such as poly(e-caprolactone) (PCL), poly(D,L-lactide), poly(glycolide) (PGA), and their copolymers, poly(lactide-co-glycolide) (PLGA), and hydrophilic segments such as polyethylene glycol (PEG), in di- or tri-block copolymers, have been used (Lai et al., 2014). The thermosensitivity of these micelles can be modulated by altering the concentration in aqueous solution or the composition of the copolymers. In addition, those block copolymers constructed of a hydrophobic core and hydrophilic corona have been mostly used for the delivery of poorly water-soluble or lipophilic drugs. As typical micelle size is between 20 and 100 nm, the renal clearance and rapid uptake by the reticuloendothelial system are avoided, and thus, the sustained drug delivery is provided. Finally, it is important to mention that all these mechanisms include reversible physical linkage of the polymers, so that gel–sol transition is enabled after the gelling stimulus is removed. 2.3. Biocompatibility and biodegradability of thermoresponsive polymers For most applications of biomaterials, their contact with cells and tissues via surfaces is inevitable (Drotleff et al., 2004). According to that, hydrogels intended for biomedical applications are required to have acceptable biocompatibility and biodegradability. Indeed, their hydrophilic surface has low interfacial free energy in contact with body fluids, which results in a low tendency for proteins and cells to adhere to these surfaces. (Hennink and van Nostrum, 2012). The characteristic of hydrogels with various chemical compositions and structures rely not only on the preparation methods but also on the monomers used in their synthesis. Numerous crosslinking methods routinely used for hydrogel preparation, both the
Fig. 2. (a) The phase diagram of PEG–PLGA–PEG triblock copolymer aqueous solution, gelation temperature vs. polymer concentration. Sol (flow) to gel (no flow) transition temperature was measured by the test tube inverting method increasing 2 C/step. Ref. (Jeong et al., 1999); (b) curves of the sol–gel transition temperature for Poloxamer 407 and lidocaine 2% solution shown as changes in elastic modulus (G' (Pa), at a frequency of 1 rad s1) at different polymer concentrations: (&) 20, () 25, and (*) 30%. Ref. (Ricci et al., 2002).
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Fig. 3. Drug release from a thermosensitive polymer micelle upon temperature increase (Nakayama et al., 2006).
chemical and physical approach, can be found in the literature (Hennink and van Nostrum, 2012; Roy et al., 2013). However, the use of physically crosslinked gels is more favorable due to the fact that crosslinking agents are not required for their preparation (Hennink and van Nostrum, 2012). Today much newer synthesized thermoresponsive polymers are available because they have undergone extensive biocompatibility examinations (Roy et al., 2013). As biodegradability is a desirable hydrogel property, weak bonds are, therefore, frequently introduced in their structure. The labile bonds can be broken under physiological conditions usually by hydrolysis, which means that the compounds formed can either be metabolized into harmless products or can be excreted by the renal filtration process. Biodegradable properties are also considered preferable for micelle-forming block copolymers because polymers obtained after degradation of the hydrophobic block are expected to be quickly excreted by the kidneys if the molecular weight of the thermoresponsive block is below the critical value (approx. 40,000) (Nakayama et al., 2006). Regarding the description above, it is clear that the attention should be paid to a variety of material design principles and a number of material classes used for thermoresponsive polymer synthesis. Interdisciplinary research between polymer chemistry and biomedical science has yielded several promising thermoresponsive systems for different application routes. Biomaterials are materials that show a marked compatibility with the biological environment or are able to replace or restore biological function or deliver therapeutic substances. Mimicking the biological environment could also be a matter of shaping a material at the micrometer and nanometer scale (Drotleff et al., 2004). However, certain drawbacks prevent their broader application, such as low cell adhesion and local acidity. An acidic environment due to the formation of acids by polymer degradation is deleterious to encapsulated bioactive proteins or cells and may induce in vivo inflammation in the surrounding tissue (Lai et al., 2014). Therefore, precise knowledge of the degradation and metabolism of polymers and excretion of metabolites is necessary, even before the testing is started. To monitor thermoresponsive polymers in a laboratory environment or an in vivo environment, there are a number of standardized methods to follow today, such as in vitro degradation and pH value change of hydrogel, in vitro biocompatibility, in vitro and in vivo drug release, gelling time, degree of swelling, degree of crosslinking, viscosity along with the heating process, sol–gel transition, morphology, and others.
In general, drug-release mechanisms from hydrogels are classified as: (1) diffusion-controlled, (2) erosion-controlled, (3) swelling-controlled, and (4) chemically controlled (Hamidi et al., 2008). Delivery systems with diffusion-controlled drug release are known as reservoir or matrix type systems. Drug diffusion out of a hydrogel primarily depends on the mesh sizes within the gel matrix, but also on hydrodynamic radius of the drug molecule. Typical mesh sizes reported for hydrogels in their swollen state range from 5 to 100 nm, and thus, larger than most small-molecule drugs. Consequently, the diffusion of small drugs is not considerably sustained, whereas macromolecules such as oligonucleotides and protein drugs have a prolonged release (Kristl et al., 1991). In cases in which the hydrodynamic radius of a drug molecule is larger than hydrogel pores, erosion or swelling will be the driving mechanism for release. In erosion-controlled hydrogels, the release is conducted by the surface or bulk erosion. Another interesting issue that has to be addressed is controlled drug release from thermoresponsive polymeric micelles. They consist of block copolymers that in a solution form micelles with a hydrophobic core and a thermoresponsive outer block. Drug molecules are embedded into the inner core, while the outer shell has a thermoresponsive ability (Fig. 3). These polymeric micelles show dramatic thermoresponsive on/off switching behavior for drug release according to the thermally induced structural changes in the micellar shell structure (Fig. 4). At temperatures below the polymer LCST, micelles are constructed of the hydrophilic outer shell and the insoluble inner core. At temperatures above the LCST, the hydrated shell shrinks and the drug is released by diffusion. Moreover, the drug release is quit by simply cooling below the LCST and accelerated again upon another heating. Hence, in this case, the drug release is governed by the change in temperature and the rate of drug diffusion (Chung et al., 1999; Nakayama et al., 2006).
2.4. Drug-release mechanisms During the development phase of a new delivery system, it is very important to know the transport processes in the gelled matrix. If injectable fluid consisting of a thermoresponsive polymer is introduced into the body and changes its structure from sol to gel, then a prolonged release is expected.
Fig. 4. On/off switching behaviour of drug release from thermoresponsive polymeric micelles, according to the change in temperature (Chung et al., 1999).
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3. Thermoresponsive polymers based on polysaccharides 3.1. Cellulose derivatives Originally, cellulose is a natural polysaccharide, insoluble in water. However, substitution of the hydroxyl groups with some hydrophobic segments renders the cellulose water-soluble. Certain cellulose derivatives show thermogelation properties in aqueous solutions (Calejo et al., 2012). In the concentrations 1–10%, aqueous solutions of such derivatives are liquid at low temperature, whereas yield gels when the temperatures is elevated. Methylcellulose (MC; Fig. 1a) and hydroxypropyl methylcellulose (HPMC; Fig. 1b) represent these polymers. MC exhibits thermoreversible gelation in aqueous solution, gelling at temperatures between 40 and 50 C, while HPMC shows a phase transition between 75 and 90 C (Sarkar, 1979). The thermogelation mechanism of aqueous MC and HPMC solutions is attributed to hydrophobic interactions among polymer chains containing methoxy groups, and therefore, the gelation is highly affected by substitution at the hydroxyl group of cellulose. Upon heating, polymer–polymer interactions become dominant, causing the gel formation. The important aspect is that phase transition temperatures of cellulose derivatives can be modified by some chemical and physical alterations. In addition, by reducing the hydroxypropyl molar substitution of HPMC, its transition temperature can be lowered to about 40 C (Sarkar, 1979). MC has been grafted with synthetic pNiPAAm in various ratios to produce fast reversibly thermogelling hydrogels and enhance the mechanical strength of the hydrogel (Liu et al., 2004a). Furthermore, recently a gel combined of low-molecular-weight MC and poloxamer micelles has been developed for subcutaneous and prolonged delivery of docetaxel. These mixtures could form a gel at temperatures between 15 and 40 C, depending on the drug concentration. The combined system released docetaxel for more than 30 days; moreover, the docetaxel stability was maintained (Kim et al., 2012). Another attractive approach towards a thermoresponsive MC systems is functionalization of MC with protein laminin, and thus, creating a bioactive scaffold for neural tissue engineering (Stabenfeldt et al., 2006). 3.2. Chitosan Chitosan is a linear polysaccharide composed of randomly distributed b-(1,4)-linked D-glucosamine and N-acetyl-D-glucosamine (Fig. 1c), which has been used extensively in drug delivery. It is biocompatible, biodegradable, and considered non-toxic. Chitosan is a hydrophilic, cationic polymer with gelation ability (Kristl et al., 1993). Gelation appears at body temperature when the pH of chitosan solution is increased to 7.2. Chitosan-based thermoresponsive gelling systems have also been investigated. Chenite et al. developed a thermosensitive neutral liquid formulation based on chitosan/polyol salt combinations (Chenite et al., 2000). Namely, pH-responsive chitosan solutions were transformed into thermoresponsive gelling systems after addition of polyol salts such as b-glycerophosphate. At pH 7, the solution was a clear liquid at ambient temperature, whereas exhibited gelation around 37 C. The dominant gelation mechanism of chitosan/b-glycerophosphate solution is assigned to hydrophobic interactions of chitosan chains that can be improved by the structuring glycerol–water interactions upon heating. A chitosanbased thermoresponsive gelling system was proposed for local application of the hydrophobic drug paclitaxel in order to prevent tumor recurrence. In vitro release profiles displayed prolonged paclitaxel delivery for over a month (Ruel-Gariépy et al., 2004). Furthermore, Bhattarai et al. embed hydrophilic poly(ethylene glycol) (PEG) into chitosan, the water solubility of chitosan was
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enhanced, and gelation occurred at physiological pH values (Bhattarai et al., 2005a). This approach was also applied in the development of a advanced system for the controlled albumin release (Bhattarai et al., 2005b). A thermoresponsive chitosanbased hydrogel for sustained release of insulin has been thoroughly investigated in vitro. According to the obtained results, the insulin was embedded into the aqueous gel regions, showing a sustained release over a period of 2 weeks. Additionally, the amount of released insulin increased with an increasing proportion of b-glycerophosphate (Kempe et al., 2008). Generally speaking, the chitosan b-glycerophosphate thermogelling systems seem to be an attractive delivery system for peptides and proteins. When it comes to tissue engineering applications, various thermogelling chitosan systems have been studied as potential cell carriers. A hydrogel based on watersoluble chitosan and copolymer of NiPAAm was investigated for chondrogenic differentiation of human mesenchymal stem cells both in vitro and in vivo (Cho et al., 2004). Moreover, Crompton et al. concluded that polylysine modified chitosan-glycerophosphate salt hydrogel could be an excellent candidate for neural tissue engineering (Crompton et al., 2007). In addition, Yan et al. developed chitosan-based injectable hydrogels modified with glycerophosphate and hydroxyethyl cellulose whose biocompatibility for culturing mouse mesenchymal stem cells was investigated. It was concluded that this system can be used as an interesting injectable in situ forming scaffold that represents a delivery system for biologically active therapeutics (Yan et al., 2010). 3.3. Xyloglucan Xyloglucan (Fig. 1d) exhibits thermoresponsive behavior when more than 35% of its galactose residues are removed. For treated xyloglucan, a lower and upper transition temperature from sol to gel and from gel to sol, respectively, were found. When temperature is increased, hydrophobic parts aggregate to minimize the hydrophobic surface area contacting the water, and thus, causing gelation (Hoare and Kohane, 2008). Xyloglucan gel is produced from solutions with concentrations in a range from 1 to 2% (Miyazaki et al., 1998), but strong hydrogels were only obtained at 3 wt% at 37 C (Nisbet et al., 2006). Even though rheological and morphological data of xyloglucan hydrogels are scant, this cytocompatible polysaccharide has been studied as a delivery vehicle for a number of applications (Ruel-Gariépy and Leroux, 2004). More recently, Chen et al. tested properties of xyloglucan hydrogel as biomedical sustained-release carriers. According to rat experiments, the results indicated the potential application of xyloglucan hydrogel as carriers for the oral drug delivery system of paracetamol. It was non-toxic and had remarkable water-retaining capacity (Chen et al., 2012). 4. Thermoresponsive polymers based on polypeptides 4.1. Gelatin Typical natural polymers showing thermally induced phase transition are gelatin and carrageenan. When the temperature is raised above 30 C, they adopt a random coil conformation at sol state. Upon lowering the temperature below 25 C, gelatin solution solidifies, and a continuous network is formed by partial helix formation (Fig. 5; Joly-Duhamel et al., 2002). As biomedical applications require the opposite behavior, gelatin has been grafted with other polymers that exhibit thermogelation upon heating. The main advantage of gelatin is its biodegradability and biocompatibility. Moreover, it is available for easy modifications at the amino acid level. A proper mixture of gelatin and
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Fig. 5. Thermoreversible gelation of gelatine and carrageenan caused by coil-triple helix transitions (Bromberg and Ron, 1998).
monomethoxy poly(ethylene glycol)-poly(D,L-lactide) produced a thermoresponsive hydrogel that underwent fast gelation upon cooling at 37 C (Yang and Kao, 2006). Gelatin grafted with poly-Nisopropylacrylamide (pNiPAAm) when the pNiPAAm:gelatin weight ratio was above 5.8 resulted in a thermoresponsive matrix analogue. Namely, it was shown that a high pNiPAAm:gelatin ratio improved the cell proliferation and matrix production due to increased hydrophobicity, leading to the formation of large aggregates. Consequently, a higher gel porosity occurred at 37 C, which comprised a favorable cell environment (Ohya and Matsuda, 2005). 4.2. PELG–PEG–PELG triblock copolymers New types of thermoresponsive gelling systems based on polypeptide poly(g-ethyl-l-glutamate)-poly(ethylene glycol)-poly (g-ethyl-l-glutamate) triblock copolymers (PELG–PEG–PELG) were tested for local and prolonged delivery of anticancer drugs. These hydrogels obtained from aqueous polypeptide solutions demonstrated much lower critical gelation concentration than the traditional hydrogels. According to biocompatibility studies, the in situ-formed hydrogels lasted for 21 days in the subcutaneous tissue. Furthermore, these formulations were applied as injectable implants with incorporated paclitaxel (PTX) in order to assess in situ antitumor activity. The results revealed that the PTXincorporated hydrogels could efficiently suppress tumor growth, while the damage to other organs was not registered. As a conclusion, these polypeptide-based thermoresponsive hydrogels possibly have the potential to serve as an effective vehicle for local antitumor drug delivery (Cheng et al., 2013). 5. Synthetic thermoresponsive polymers 5.1. N-isopropylacrylamide copolymers Poly(N-isopropylacrylamide copolymers) (pNiPAAm) is a synthetic, thermoresponsive polymer with a sol–gel transition temperature of about 32 C in aqueous solution. The LCST of pNiPAAm solution is strongly dependent on polymer molecular weight and structural architecture as well as on the presence of additives like salts, surfactants, or co-solvents (Liu et al., 2004b). Among many thermoresponsive polymers, pNiPAAm is probably the most extensively studied mainly due to its water solubility at room temperature (Fig. 1e). Above the LCST, the solution becomes opaque and transforms into a gel, primary due to hydrophobic interactions and coil-to-globule transition (Kulkarni and Aloorkar, 2010). Namely, at temperatures below LCST, hydrogen bonds between the amide groups of polymer and water molecules occur. Upon heating above the LCST, the hydrophobic interactions between polymer chains become predominant, hydrogen bonds are broken and the polymer dehydrates. The phase transition
temperature of pNiPAAm solution can be controlled by shifting the hydrophilic/hydrophobic balance by copolymerization of pNiPAAm with other monomers. For instance, an increase in hydrophobicity (i.e., incorporation of butyl methacrylate) or in molecular weight results in LCST decrease. Conversely, the incorporation of hydrophilic monomers (i.e., acrylic acid or hydroxyethyl methacrylate) promotes the formation of hydrogen bonds with thermoresponsive units, which increases the LCST (Kim et al., 2009). The swelling and release profiles of pNiPAAm hydrogels with regard to physicochemical properties of the embedded drugs have been evaluated by Coughlan et al. (Coughlan et al., 2004). The swelling of hydrogels with hydrophobic drugs was decreased, whereas the systems with hydrophilic drugs incorporated demonstrated enhanced swelling. In addition, a solubility-dependent drug release was observed for hydrophobic drugs, but hydrophilic drugs exhibited a molecular weight-dependent drug release. As mentioned above, hydrogels based on pNiPAAm and its copolymers are the most extensively investigated thermoresponsive systems used in drug delivery (Li et al., 2008; Liu et al., 2008, 2007; Nakayama et al., 2006; Sousa et al., 2010), cell encapsulation and delivery (Garbern et al., 2010; Na et al., 2007, 2006), and cell culture surfaces (Hatakeyama et al., 2006; Nakayama et al., 2012). In order to achieve thermoresponsive hydrogels with improved properties, a lot of work has been done on NiPAAm copolymers. The examples include NiPAAm copolymers with acrylic (AA) and propylacrilic acid (PAA), as well as with poly(ethylene oxide) (PEO) and poly(L-glutamic acid). The poly(NiPAAm-co-AA) solutions were tested as cell- and drug-delivery vehicles and also as cell matrices in a refillable bioartificial pancreas (Chae et al., 2001). Thermoresponsive pNiPAAm solutions were proposed as an extracellular matrix for islets of Langerhans placed in an immunoprotecting pouch installed in diabetic patients. It was shown that the copolymer, which formed a gel, efficiently immobilized rat islets without impairing insulin secretion, providing prolonged insulin secretion. (Vernon et al., 1999). The thermoresponsive p(NiPAAm-co-AA) hydrogel was studied as a vehicle for chondrocytes, dexamethasone, ascorbate as a differentiation factor, and transforming growth factor b3 (TGF-b3). This hydrogel was implanted subcutaneously in mice and significant collagen II expression as well as proteoglycan and polysaccharide production were observed after 8 weeks (Na et al., 2006). Poly(NiPAAm-co-AA) was also synthesized and copolymerized with ethyl acrylate using interpenetrating polymer network (IPN) technology (Liu et al., 2006). An interpenetrating polymer network consists of physically interconnected hydrophilic and hydrophobic network, while individual components retain their original properties. In addition, poly(NiPAAm-co-PAA) copolymers exhibit both temperature- and pH-responsive behavior (Yin et al., 2006). Thus, hydrogels based on poly(NiPAAm-co-PAA) can be useful for drug delivery applications in which temperature and local pH differences can both act as stimuli (Li et al., 2008). These thermoand pH-responsive properties definitely open up new opportunities in intelligent drug delivery systems. Recently, polymeric nanofibers from a blend of polyvinyl alcohol (PVA) and pNiPAAm were prepared for the topical delivery of levotiroxine (Azarbayjani et al., 2010). The authors concluded that the nanofibers were able to release levotiroxine in a sustained fashion, through diffusion and erosion mechanisms, providing the appropriate drug concentration for a long time and promoting the drug accumulation in the superficial skin layers. Thermoreversible systems based on triblock linear and star copolymers derived from linear and multi-arm PEG as the water-soluble central block and pNiPAAm as the thermoresponsive terminal blocks were also developed (Lin and Cheng, 2001). Injectable aqueous solutions were present at room
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Table 2 Basic physico-chemical characteristics of PEO–PPO–PEO copolymers considering chemical composition of three-block copolymer chains, average molecular weight, cloud point, representing sol–gel transformation temperature and HLB (Bromberg and Ron, 1998). Copolymera
Composition
Average MW
MPPO
PEO (wt%)
CPb ( C)
HLBc
L64 F68 F88 P103 P104 P105 F108 F127 L122
EO13PO30EO13 EO76PO29EO76 EO103PO39EO103 EO17PO60EO17 EO27PO61EO27 EO37PO56EO37 EO132PO50EO132 EO100PO65EO100 EO12PO67EO12
2900 8400 11,400 4950 5900 6500 14,600 12,600 5000
1740 1680 2280 3465 3540 3250 2920 3780 3600
50 80 80 30 40 50 80 70 20
58 >100 >100 86 81 91 >100 >100 19
12–18 >24 >24 7–12 12–18 12–18 >24 18–23 1–7
a b c
L, F, and P indicate liquid, flakes, and paste, respectively. Cloud point in aqueous 1 wt% solution. Hydrophilic–lipophilic balance.
temperature, but they formed relatively strong elastic gels at body temperature due to hydrophobic interactions of NiPAAm oligomer blocks. These copolymer solutions produce gels via physical crosslinking among pNiPAAm aggregates, while diblock copolymers gel by a micelle packing mechanism. 5.2. PEO/PPO/PEO triblock copolymers and derivatives The copolymer blocks based on poly(ethylene oxide)-b-poly (propylene oxide)-b-poly(ethylene oxide) (PEO–PPO–PEO) sequences (Fig. 1f) constitute a family of triple blocks with the commercial name Pluronics1. They were introduced in the late 1950s and are an important group of synthetic polymers exhibiting thermoresponsive property in concentrated aqueous solutions. The PEO–PPO–PEO triblock copolymers (poloxamers) cover a large number of liquids, pastes, and solids, and include polymers with various molecular weights and PEO/PPO block ratios (Table 2). As seen in Table 2, the cloud point of 1 wt% solution varies from 19 C, for the L122 copolymer with low PEO content and low HLB, to above 100 C, for poloxamers with high PEO content. Although most poloxamers listed in Table 2 have a LCST well above normal body temperature, they actually demonstrate gelation at 37 C in concentrated solutions. Although the gelation of poloxamers has been extensively investigated, the real mechanism still remains a controversial issue. The formation of micelles in aqueous poloxamer solutions as a result of PPO block dehydration has been proven by various experimental techniques (Schillén et al., 1993; Wanka et al., 1990; Zhou and Chu, 1988). Namely, these copolymers form micelles above critical micelle concentration (1 mg/ml), which equilibrate with poloxamer unimers at low temperature (Alexandridis and Alan Hatton, 1995). Further, below a critical micelle temperature, both ethylene and propylene oxide blocks are hydrated, and PPO is relatively soluble in water. As the temperature increases (Fig. 6),
PPO chains become less soluble, and micellization becomes more important. As the number of unassociated unimers in solution is reduced, the micelle volume fraction (Fm) is increased. At a definite point, micelles come into contact and no longer move, forming various liquid crystalline phases (cubic 3D-lattice, hexagonal 2D-lattice, and lamellar bilayers). When Fm > 0.53, the formulation turns into a gel due to the micelle packing (Mortensen and Pedersen, 1993). Hence, packing of micelles and micelle entanglements may be the mechanisms of poloxamer gelation in aqueous solution as a response to the temperature increase (Cabana et al., 1997). On the other hand, the reason for gel–sol transition at high temperatures is still unknown and could be possibly related to the shrinkage of the PEO corona of the micelles and the interactions between PEO chains and PPO core (Li et al., 1997). Among more than 30 different non-ionic surface active agents of the poloxamer family, Poloxamer 407 (P407), also known under the registered trademark of Pluronic F1271 (BASF laboratories, Wyandotte, MI, USA), has probably been used most extensively in drug delivery studies. P407 has a molecular weight of about 12,600 (9840–14,600), with a PEO/PPO ratio of 2:1 containing about 70 wt % of ethylene oxide, which contributes to its hydrophilicity (Table 2). It has been reported to have a low viscosity below 4 C, whereas forms a gel at concentrations higher than 20 wt% at 25 C. P407 is more soluble in cold than in hot water due to the presence of hydrogen bonds at low temperatures (Escobar-Chavez et al., 2006). The P407 aqueous solution in the concentrations between 20 and 30 wt% shows reversible gelation upon heating. Namely, at low temperature (
