(PDF) Polysaccharide-based polyelectroly

Current Opinion in Colloid & Interface Science 15 (2010) 417–426 Contents lists available at ScienceDirect Current Opinion in Colloid & Interface Science j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c o c i s Polysaccharide-based polyelectrolyte multilayers Thomas Crouzier, Thomas Boudou, Catherine Picart ⁎ CNRS UMR 5628, LMGP and Grenoble-INP, MINATEC, 3, Parvis Louis Néel, 38016 Grenoble, France a r t i c l e i n f o a b s t r a c t Article history: In recent years, the layer-by-layer technique has grown in various ﬁelds. One of the emerging trends of bio- Received 7 April 2010 applications is the use of polysaccharides as main ﬁlm components, which stems from their intrinsic Received in revised form 7 May 2010 physical, chemical and biological properties. These allow the simple formation, by self-assembly, of new Accepted 7 May 2010 kinds of mimics of extra-cellular matrices from plant and animal tissues. These assemblies, which possess Available online 13 May 2010 speciﬁc properties arising from their hydration and internal composition, can indeed contain additional Keywords: functionalities obtained by chemical modiﬁcation of the biopolymers or ﬁlm post-processing. They can be Polyelectrolyte multilayer (or layer-by-layer) molded into different forms (ﬁlms, membranes, and capsules). Polysaccharides © 2010 Elsevier Ltd. All rights reserved. Biomimetism Coating Diffusion Internal composition Chemical modiﬁcation Mechanical properties 1. Introduction powerful model for investigating the mechanisms of diffusion and water retention, as polysaccharides-based PEM ﬁlms are amongst the Layer-by-layer deposition of polyelectrolytes on to a solid most highly hydrated. Deposition of ﬁlms on to a biodegradable core substrate has become a popular tool for producing new types of or on to a detachable substrate has made possible the formation of thin coatings with controlled architecture. The formation of polyelec- polysaccharide-based capsules or membranes. These new nanostruc- trolyte multilayer ﬁlms (PEM) is usually acknowledged to be related tured biomimetic membranes may ﬁnd applications as biodegradable to the formation of polyelectrolyte complexes in solution (also called drug delivery systems or functional patches. More recently, the “complex coacervates”). The method allows the polymers to self- formation of hydrid ﬁlms made of polyelectrolyte blends has brought assemble on a surface with a ﬁlm growth that depends on the respec- further understanding of the polyelectrolyte pairing and of the tive intrinsic properties of the polyelectrolytes as well as on the preferential incorporation of sulfated polysaccharides. Also, the experimental conditions. Although many studies focus on synthetic possibility of chemically modifying the polysaccharides and success- polyelectrolytes, which offer numerous possibilities in the variation of fully incorporating these new derivatives into PEM ﬁlms has recently buildup conditions, multilayer ﬁlms containing polysaccharides have been shown by several groups. This is a powerful tool for giving PEM attracted considerable attention in the past 12 years. These poly- ﬁlms additional functionalities and for designing multifunctional saccharides are known to be crucial for the proper formation of native architectures. Overall, understanding how these PEM ﬁlms self- plant and human tissues in terms of structural and functional organi- assemble at the nanometer scale and how is it possible to tune their zation. Their constitutive side groups such as hydroxyl, carboxyl and multiple properties (hydration, mechanical properties, bioactivity, sulfate, as well as their persistence length and the length of the spatial organization…) could be of help to us for our fundamental polymeric chain (number of saccharide units), are all important for understanding of native tissues, as well as for biomedical applications. their interactions with water and proteins. In this review, the discus- We believe that these PEMs could be used as a tool for biologists sion focuses on polysaccharide-based PEM with speciﬁc emphasis on working on matrix biology, for biophysicists but also for biomaterials progress made in the last 3 years. In the past few years, much better scientists. Indeed, the bottom-up approach of PEM ﬁlms reduces the knowledge of ﬁlm growth, hydration and internal composition has complexity of native extra-cellular matrices (ECMs), which are often been gained. Diffusion and dynamics in polysaccharide-based ﬁlms investigated in a top to bottom approach (by successive degradation have also been investigated and quantiﬁed. These ﬁlms are indeed a of the components of the matrix). PEMs can bring new insights into the interactions between polysaccharides and peptides, polypeptides, or proteins in a reconstituted thin matrix. Also, polysaccharide-based ⁎ Corresponding author. Tel.: + 33 0 4 56 52 93 11; fax: + 33 0 4 56 52 93 01. PEM ﬁlms are a new kind of coating whose properties can be ﬁnely E-mail address:

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(C. Picart). tuned and, in this sense, be employed for systematic studies of the 1359-0294/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.cocis.2010.05.007 418 T. Crouzier et al. / Current Opinion in Colloid & Interface Science 15 (2010) 417–426 inﬂuence of several adjustable parameters. Finally, thanks to their be involved in the regulation of their bioactivity [8]. In particular, high versatility and ease of deposition on to various kinds of support afﬁnities with several morphogens and growth factors, such as the materials, polysaccharide-based PEM ﬁlms will probably be a great basic ﬁbroblast growth factor (bFGF) and bone morphogenetic opportunity for biomaterials scientists to modify the surface of growth factors (BMPs) [9] have been reported with heparin (HEP). biomaterials selectively, while maintaining their bulk properties These molecules are indeed key players in the maintenance of tissues unmodiﬁed. There is no doubt that fundamental understanding of and affect numerous cellular processes including cell proliferation and these fascinating assemblies will serve many different aspects of differentiation. The ECM thus work as a storage reservoir, by locally research, from fundamentals to applications. concentrating them, presenting them at the cell-surface and protect- ing them from enzymatic degradation, while releasing them at the 2. Why using polysaccharides in PEM ﬁlms? appropriate time [10]. In addition, speciﬁc cell-surface receptors for polysaccharides have 2.1. Deﬁnitions been identiﬁed. For instance, in the case of HA, CD44 or RhAMM are known to be involved in HA-mediated cell adhesion, proliferation, and Polysaccharides are a family of carbohydrates that play funda- survival [11]. mental roles in many biological contexts. Their structure is made of Finally, due to their high level of hydration, HA, chondroitin sulfate A sugar rings linked by glycosidic bonds and various side functions. Two (CSA) or alginate (ALG) are also known for their protein-repellent elements are of utmost importance in the chemistry of polysacchar- properties [12]. This effect was attributed to the presence of the ides. Firstly, the glycosidic bonds can be the target of glycoside hydration shell around the polysaccharides, and precluded the protein/ hydrolase enzymes and can thus be biodegraded relatively easily. polyelectrolyte interactions. Secondly, the side groups can directly affect the polysaccharide's charge density, hydration and chemical reactivity, and can also be 2.3. Advantages and limitations of polysaccharides responsible for the formation of secondary structures. When charges are present, polysaccharides behave like polyelectrolytes. The nega- The exceptional structural and functional properties of polysacchar- tive charges are carboxylic groups (COO−) with pKa around 3–5 or ides reviewed above are the rationale for their use as biomaterials as sulfate groups (SO−3 ) with a pKa of around 0.5–1.5 [1]. The positively well as for surface modiﬁcation of biomedical devices. Being natural charged groups are ammonium groups (NH+ 3 ) with a pKa of around 7– constituents, they are ideal building blocks for creating systems 10 [2]. PEM ﬁlms self-assemble thanks to interactions between the mimicking the structural and biochemical properties of the in vivo negative and positive groups and thanks to the entropic gain asso- cellular environment. Also, these polymers are naturally degraded by ciated with these associations. There are two main types of poly- different kinds of enzymes present in vivo giving polysaccharides a great saccharides: those present in plant cells, cellulose being the main advantage over synthetic polyelectrolytes. For instance, (CHI/HA) ﬁlm polysaccharide on earth, and those present in animal tissues, with biodegradation in intro and in vivo has already been evidenced [13]. glycogen the most important for energy storage. These superior advantages for biomedical applications as compared to synthetic polyelectrolytes explain why they have been increasingly 2.2. Structure and functional roles of polysaccharides employed as ﬁlm constituents since the early 2000s. However, certain difﬁculties are encountered when working with Polysaccharides play many different roles in vivo. First, they are polysaccharides. First, although the commercial availability of poly- essential structural components of both plant and animal cells, and saccharides has increased with the increasing demand, in particular for contribute to their unique self-assembling properties. For instance, HA, wide variations in quality from one provider to another and from cellulose can self-assemble into microﬁbrils that in turn associate into batch-to-batch can be an issue. Most polysaccharides are extracted larger ﬁbers through hydrogen interactions. Chitin is the main struc- and puriﬁed from natural tissues and are thus dependent on natural tural element of the crustacean exoskeleton (crab, shrimp, etc…) and variations. Polydispersity is another concern, which renders system- cell walls of fungi. In human connective tissues, large quantities of atic studies on the effect of molecular weight variations in a limited glycosaminoglycans (GAG), which are long unbranched polysacchar- range quite difﬁcult. An exception should be noted for HA, for which ides composed of a repeating disaccharide unit (hexose linked to a there has been recent developments in production technology, such as hexosamine), are found in the extra-cellular matrix. Chondroitin recombinant production [14]. In addition, the commercial availability sulfate (CS) is the most prevalent, together with heparin sulfate (HS) and quality (monodispersity) of HA lots have greatly improved in the and hyaluronan (HA). Of note, hyaluronan is the only non-sulfated past few years [15]. Chemical modiﬁcation can be a real challenge GAG and heparin has the highest negative charge density of any because of the high hydration shell and poor solubility in organic known biological molecule. HA and CS are responsible for the unique solvents. In addition, the variety of reactive groups present on the hydration and mechanical properties of synovial ﬂuid, cartilage and different polysaccharides (OH, COO−) is limited. Their ﬂexibility is also tendons. HA and CS are indeed highly hydrated polymers surrounded lower than that of synthetic polyelectrolytes as they have a higher by respectively ∼ 20 and ∼30 water molecules per disaccharide unit in persistence length. Due to their lower solubility, pH and ionic strength interaction through hydrogen bonds [3,4]. (I) can only be varied to a lower extent than for their synthetic Importantly, these polysaccharides are part of the pericellular coat counterparts. Thus, the overall degree of freedom in the choice of (also called glycocalyx). This coat, which can be up to several µm in buildup conditions seems more limited. The advantages and limita- thickness [5], plays a major role in the interactions between a cell and tions of using polysccahrides in PEM ﬁlms for biomedical applications its environment by mediating cellular adhesion and the diffusion of are summarized in Table 1. biomacromolecules such as growth factors [6]. Second, polysaccharides have a key functional role. Starch and 2.4. Polysaccharides used in PEM ﬁlms glycogen are energy storage molecules. Glucose is soluble in water, hydrophilic and takes up space, whereas it is insoluble in the form of In the past 10 years, there have been many attempts to assemble starch and can be stored much more compactly. In starch, the glucose polysaccharides into PEM ﬁlms. As mentioned above, most poly- molecules are bound by the easily hydrolysable alpha bond, in order saccharides are negatively charged and are thus used as polyanion to render the sugar reserve rapidly available [7]. In animal reserves, constituents, unless they are chemically modiﬁed to render them similar structures and bonds are found in glycogen. In the ECM of polycationic. This was indeed the case for amide modiﬁed HA [16] and tissues, HS is known to interact with a large range of proteins and to for quaternized chitosan [17]. Plant derivatives such as carboxymethyl T. Crouzier et al. / Current Opinion in Colloid & Interface Science 15 (2010) 417–426 419 Table 1 Comparison between the advantages and disadvantages of synthetic polyelectrolytes and polysaccharides used for the buildup of multilayer ﬁlms in the context of biomedical and biomaterials research. Synthetic polyelectrolytes Polysaccharides Advantages • Large choice of chemistries, structure and charge densities • Natural polyelecrolytes (biomimetism) • Flexibility • Interesting structural properties: interactions with water, self-assembly, hydrogel formation • Large working range of pH and ionic strength • Functional properties: speciﬁc cell receptors, interactions with bioactive molecules (growth • Easy chemical modiﬁcations factors…), present in the pericellular coat • Abundant and usually cheap • Biodegradability and biocompatibility (for most of them) • Available with highly controlled quality Limitations • Most often non-biodegradable • Limited availability with well deﬁned properties (purity, polydispersity); often puriﬁed from • Potentially harmful degradation products natural tissues • No particular bioactivity • Chemical modiﬁcations can be particularly difﬁcult due to the poor reactivity of the group, low charge density and poor solubility in solvents (need for “biofriendly” processes) • Limited pH and ionic strength working range due to solubility issues cellulose [18], pectin [19] and its derivatives polygalacturinic acid and associated with exothermic complexation, whereas the exponential furcelan [20] have been successfully used. Animal-derived polysac- growth regime relates to endothermic complexation. [34] charides such as HA [21], CS [22], HEP [23] and mucin [24] are also Simulations are also only in the early stages. According to Holm et increasingly investigated for their ability to form PEM ﬁlms with al. in their recent review [35], no simulation study has been able to speciﬁc properties. The choice of a polycationic polysaccharide is very reproduce the exponential growth of PEMs. The interesting predic- limited. In fact, only chitosan (a de-acetylated form of chitin) is tions made by Hoda and Larson [36] about exponential growth require currently available and used in PEM ﬁlms. Thanks to its numerous a test via numerical simulations. One of the reasons for the lack of interesting properties, including wide availability, biocompatibility, results may be that, so far, numerical studies have never explored the wound-healing and antibacterial properties [25], chitosan is probably inﬂuence of a mismatch on the degree of charges between two dif- by far the most widely used polysaccharide in LbL ﬁlms. ferent polyelectrolytes. As indicated earlier and as will be shown As an alternative to chitosan, the cationic polypeptide PLL has been below, this is, however, quite probably often the case for exponen- widely used in combination with polysaccharides in PEM ﬁlm [21,26]. tially growing ﬁlms. Poly(L-lysine) (PLL) can be considered as a model protein as lysine is, together with arginine, the major positively charged amino-acid found in proteins. To a certain extent, such polypeptides can mimic the natural 3.1. Hydration and swellability polysaccharide–protein interactions occurring in vivo, for instance proteoglycan assemblies. Nonetheless, other synthetic cationic poly- Usually, the known properties of polysaccharides in solution will electrolytes such as poly(ethylene imine) (PEI) [18] and poly(allylamine directly inﬂuence the properties of the PEM ﬁlms formed. Hydration is hydrochloride) (PAH) [27] are often used as polycations. a striking example. Neutron scattering is a major technique to study the distribution of salt ions and water in synthetic polyelectrolyte 3. Film growth, hydration and internal composition multilayer ﬁlms [31]. However, due to the inherent difﬁculty in drying the polysaccharide-based ﬁlms without changing their structure and Polysaccharide-based PEM ﬁlms were at the origin of the discovery due to the fact that deuterated polysaccharides are difﬁcult to prepare, of a new growth mode, namely the exponential growth of ﬁlm there are to our knowledge no data available on polysaccharide-based thickness based on the diffusion of at least one of the polyelectrolyte PEM ﬁlms probed by neutrons. For such ﬁlms, hydration is usually species in the ﬁlms. Polysaccharide multilayer ﬁlms containing ALG probed by comparing hydrated adsorbed masses measured by Quartz and HA in combination with PLL (PLL/ALG) [26] and (PLL/HA) [21] Cristal Microbalance with Dissipation monitoring (QCM-D) and dried were the ﬁrst exponentially growing ﬁlms to be reported. These masses measured by optical methods such as Surface Plasmon greatly contrasted with the widely studied and linearly growing poly Resonance (SPR), ellipsometry or Fourier Transform Infrared Spec- (styrene sulfonate)/poly(allylamine hydrochloride) (PSS/PAH) syn- troscopy in Attenuated Total Reﬂection mode (ATR–FTIR). Water thetic ﬁlms. This type of growth was initially mostly observed in ﬁlms content is reported to be very high, ranging from 70 to 90% in (PLL/ based on polysaccharides and polyaminoacids [21,26,28] but it is pectin) ﬁlms [19,37], 60% in (PLL/CSA), ∼90% in (PLL/HA) ﬁlms [38], nowadays widely recognized that many different types of systems, 80% for (CHI/HA) [39] and for starch based ﬁlms [40], 93% for (PLL/ including synthetic PEM ﬁlms, can grow exponentially [29]. Polyelec- furcellaran) [20] and over 90% for (CHI/mucin) ﬁlms [41]. In relation trolyte diffusion was found to be a key feature of this type of growth. to this high hydration, ﬁlm refractive indices are usually low (∼ 1.38– Evidenced for the ﬁrst time on (PLL/HA) ﬁlms [21,29], some chains 1.40) for polysaccharide-based ﬁlms [42]. Hydration is a critical labeled with ﬂuorescein isothyocyanate (FITC) were found to remain parameter for PEM ﬁlms, impacting ﬁlm thickness, swellability and free in the ﬁlms. These were able to diffuse toward the upper part of diffusion of the ﬁlm's components. Film hydration can vary during the ﬁlms upon deposition of the HA layer. A signiﬁcant contribution to ﬁlm buildup. Indeed, when two oppositely charged polymers complex ﬁlm growth thus came from these free chains that were able to interact together, the counterion–polymer and water–polymer bonds are with the incoming HA chains. Watery and very thick (PLL/HA) ﬁlms disrupted, which can lead to dehydration of the multilayer ﬁlm [19]. then became a model for deeper understanding of these phenomena. Several chemical side groups appear to interact with water molecules Later studies brought a better view of the diffusion process, in via hydrogen bonds. For instance, the ester groups present in pectin particular on its limitation to a certain zone [30]. Better understanding are known to be good acceptors of hydrogen bonds and can thus trap of the different growth mechanisms is also emerging. [31] For instance, water molecules in proximity to the polymer. Films made from pectin Porcel et al. showed that a transition from exponential to linear growth with decreasing ester content have been found to be less hydrated and occurs at a certain level in ﬁlm buildup. [30] It also appears that, even thus thinner and more dense [19]. Acetamido groups present on CS for synthetic polyelectrolyte ﬁlms, exponential growth becomes and HA may also be partially responsible for the high hydration of dominant when NaCl concentrations increase [32] or when temper- (PLL/CSA) and (PLL/HA) ﬁlms. Interestingly, although HA and CSA ature is increased [33]. Interestingly, isothermal titration microcalori- have differently charged chemical functions and charge densities, metry investigations indicate that the linear growth regime is they both have the acetamido group and both yield thick, hydrated 420 T. Crouzier et al. / Current Opinion in Colloid & Interface Science 15 (2010) 417–426 ﬁlms in combination with PLL [38]. Heparin, with a similar structure 4. Physical and chemical parameters and molecular weight: but without this acetamido group, yields thin, dense ﬁlms. inﬂuence on ﬁlm growth The ﬁlm swelling properties (i.e. their ability to change volume and thickness as the environmental conditions are changed, such as pH, pH and ionic strength are two important parameters that can be ionic strength, hydration) has been found to depend on buildup varied to modulate ﬁlm thickness. Their systematic inﬂuence on ﬁlm conditions. In the initial studies on HA-based PEM ﬁlms, the buildup growth has been studied mostly for PEM ﬁlms based on synthetic was performed in physiological conditions with no intermediate polyelectrolytes [47]. As a general rule, ﬁlm thickness increases when drying stems. The ﬁlms are thus already highly hydrated and do not the pH is close to the pKa of the polyelectrolytes and when I is further swell in physiological solution (pH 6.5–7.5 and ionic strength increased. Only few systematic studies are speciﬁcally dedicated to of 0.1–0.15 M NaCl). The ﬁlm was found to shrink to ∼ 50% upon polysaccharide-based PEM ﬁlms. For polysaccharides, the working dehydration in ethanol baths of increased concentration [43]. Film ranges for pH and I are usually limited to 3–10 for pH and 10− 4 M to cross-linking also induced a slight ﬁlm swelling (∼10%) as cross- 1 M for I respectively, due to their solubility limits. Very interestingly, linking is performed at a slightly lower pH (5.5) [44]. On the contrary, it appears that such studies have mostly been performed on CHI- when the ﬁlms are built by alternate dipping in the polyelectrolyte containing ﬁlms in association with different polyanions. Of note, the solution followed by intermediate drying steps, the swelling of the pH working range for CHI is usually limited to pH below 5.5 due to its ﬁlm between the dried and wet states can be very high (hundreds low pKb (close to 6). On the contrary, for HEP (the only polysaccharide of %). According to Barrett et al. [45] who used intermediate drying being a strong polyelectrolyte), its total charge is almost independent steps, the swelling of (PAH/HA) ﬁlms shows a high dependence on the on the pH (a single COO− group versus 2 to 3 sulfate groups). In a assembly solution pH. The swelling ratio varied between 2 at physio- pioneering work, Lvov et al. showed that the thickness of (CHI/PSS) logical pH (pH = 7) to more than 8 at very acidic pH (pH = 2) and was ﬁlms increased with I and that the adsorption kinetics depended on I more intermediate at basic pH (pH = 10) with a swelling ratio of [48]. Later on, Richert et al. [49] showed for (CHI/HA) ﬁlms that ﬁlm about 5 (i.e. 500%). thickness increased with I over the range 10−4 M to 0.15 M NaCl, as higher I did not lead to proper PEM ﬁlm formation. Post-assembly 3.2. Internal composition stability was also checked for such ﬁlms [50]. Radeva et al. [51] evidenced, while maintaining the pH of CHI Film hydration is related to the afﬁnity between the charged constant at 4, that the pH of the carboxymethyl cellulose (CMC) groups of the two polyelectrolytes. A polyelectrolyte couple with high solution signiﬁcantly inﬂuenced ﬁlm growth: ﬁlm thickness was afﬁnity will chase water during complexation and form very dense higher when buildup pH was close to the pKa of CMC. Recently, and highly cross-linked networks, resulting in ﬁlms close to a “glassy Boddohi and Kuiper [52] showed for (CHI/HEP) ﬁlms that ﬁlm state” (i.e. “frozen” chains without mobility) [46]. The interactions thickness increased with ionic strength in the 0.1 to 0.5 M NaCl range. between the polyelectrolytes in terms of stoichiometry and afﬁnity In addition, the inﬂuence of pH (from 4.6 to 5.8) was most prominent are thus critical parameters. A question arises from these facts: how for the intermediate ionic strength (0.2 M NaCl), with thicker ﬁlms does polyelectrolyte chemistry, and in particular charge density, affect formed at the highest pH (i.e. closer to the pKb of chitosan). the ﬁlm composition and ionic pairing of the polymers? The effect of temperature has been studied little. Kankare et al. For polysaccharide-based ﬁlms, the question was assessed by two observed for (PLL/HA) ﬁlms that temperature had an important effect different approaches. Ring et al. investigated systematic changes in on ﬁlm growth. Their data suggested a change in diffusion rate with a the degree of de-acetylation of pectin in (PLL/pectin) ﬁlms [19,37]. temperature rise from 0.5 °C to 55 °C. The ratio of PLL/pectin monomers was slightly below 1, between 0.83 Overall, although less studied, polysaccharide-based PEM ﬁlms and 0.97 depending on the degree of pectin de-esteriﬁcation. In our appear to roughly follow the trends observed for synthetic PEM ﬁlms group, we chose a series of structurally similar polysaccharides that with respect to their dependence on pH, I and temperature. For the bear an increasing charge density. To this end, HA, CSA and HEP, with reasons mentioned above, systematic studies on the effect of molec- their increasing sulfate contents (from 0 to 2.5) and charge density, ular weight are more tedious than for synthetic polyelectrolytes, were selected. The internal composition for (PLL/HA), (PLL/CSA), because of their higher polydispersity. We have already reported the (PLL/HEP), and (CHI/HA) were probed by FTIR [38] [39]. The PLL/ effect of polyelectrolyte molecular weight variations [42], where no polyanion monomer ratio was around 0.5 for all the ﬁlms investigat- clear common trend could be deduced from the different studies. ed. Although the polycationic/polyanionic ratios were different in the two studies, many similarities emerged. First, the composition ratio 5. Diffusion and dynamics in polysaccharide-based ﬁlms did not seem to drastically depend on the charge density of the polyelectrolytes. It seems that sterical hindrance is an important Diffusion of one of the ﬁlm's components has in many cases been driver for polysaccharide assembly, which is in a certain way inde- related to exponential ﬁlm growth. The ﬁrst visual proof of PLL-FITC pendent of the charge position or density. Second, because of this diffusion in a (PLL/HA) ﬁlm [29] was obtained from confocal laser approximately constant ratio, the charge is directly inﬂuenced by the scanning microscopy (CLSM). Later on, CHI-FITC was also found to diffuse charged density of the polyelectrolytes used. Thus (PLL/pectin), (PLL/ in (CHI/HA) ﬁlms [49]. Several studies then focused on measuring HA) and (CHI/HA) ﬁlms were found to be positively charged, (PLL/ diffusion coefﬁcients in order to obtain better insight into the structure CSA) is almost neutral whereas (PLL/HEP) ﬁlms are negatively and dynamics of the ﬁlm. Diffusion can either be measured “in plane” by charged. This excess of charges is compensated by mobile counterions ﬂuorescence recovery after photobleaching (FRAP, using a ﬂuorescence to ensure the overall electroneutrality of the ﬁlm. Third, growth mode microscope) or by FRAPP (Fluorescence Recovery After Pattern Photo- was affected by charge density, more linear growth being observed for bleaching) or “out of plane” for diffusion in the z-direction. In the latter highly charged polysaccharides (highly de-acetylated pectin or case, Fluorescence Resonance Energy Transfer (FRET) between a heparin), whereas growth was exponential for the less charged ﬂuorescently labeled polyelectrolyte embedded in the ﬁlm and an polysaccharides (HA, CSA, highly acetylated pectin). Of note, ionic added ﬂuorescent layer of the same polyelectrolyte can be used. The pair pairing in polysaccharide ﬁlms has been investigated very little. In our of dyes must be carefully chosen so that energy transfer can occur. recent work, we showed that sulfate groups are much more likely to Alternately, CLSM can also be used but it is limited to thick ﬁlms (a few interact with the ammonium groups of the polycation than the car- µm in thickness). Evanescent wave techniques such as Fourier Transform boxylic groups [38]. This must be related to the preferential incor- Infrared Spectroscopy in Attenuated Total Reﬂection mode (ATR–FTIR) poration observed in blended ﬁlms (see paragraph 7). are also an appropriate tool but the z-resolution is limited by the T. Crouzier et al. / Current Opinion in Colloid & Interface Science 15 (2010) 417–426 421 penetration depth of the evanescence wave (typically 500 to 900 nm dealt with the study of the conﬁnement of ferricyanide (Fe(CN)6)3− depending on the type of crystal used). ions into (PAH/ALG) and (PAH/CMC) ﬁlms. Their loading was higher The “in plane” diffusion of PLL-FITC in (PLL/HA) ﬁlms measured by when the surface charge of the ﬁlm was positive [57]. On the contrary, FRAP–CLSM was found to be 0.1 µm2/s with 40% of the PLL chains positively charged hexa-ammine ruthenium ions Ru(NH3)3+ 6 could mobile [53]. A better view of PLL-FITC dynamics was recently obtained not penetrate the ﬁlms, which suggested that PAH was in great excess by FRAPP. The diffusion behavior was found to be different for PLL in such ﬁlms. Of note, a similar trend concerning the presence of a chains deposited on top of the ﬁlm or PLL chains embedded in the ﬁlm, great excess of polycations, i.e. a signiﬁcant non stoichiometry, was even under just one HA layer. For embedded chains, two populations also found in other polysaccharide multilayer ﬁlms containing PLL were found: a mobile one with a diffusion coefﬁcient D of the order of [38]. In a later work by Anzai et al. [58], a signiﬁcant effect of the type 0.1 µm2/s and a population that appears immobile (D b 0.001 µm2/s). of polycation (whether PEI or PDAMAC) was found on the redox For chains deposited on top of the multilayer, a third and rapidly properties of the (FeCN3−6 ) ions. diffused population appeared (D = 1 µm2/s). These results showed that there are different types of diffusive PLL chains in ﬁlms. 6. Microcapsules and membranes based on polysaccharide In a FRAPP study by Von Klizing et al. [54], lateral diffusion multilayer ﬁlms coefﬁcient measurements on poly(dimethyldiallyl ammonium chlo- ride) (PDDA)/HA ﬁlms using PAH-FITC as the probing layer were PEM ﬁlms can be deposited on a non planar template, such as a estimated at 5 × 10− 4 µm2/s, i.e. much below the values given in the microparticle, to create polyelectrolyte microcapsules. To this end, the aforementioned studies. These discrepancies may be explained by a core must be degraded after ﬁlm deposition. Free-standing ﬁlms (i.e. more dense structure for PDDA/HA ﬁlms as compared to PLL/HA or by membranes) can also be prepared by detachment of the ﬁlm from the the fact that the PDDA/HA ﬁlms were dried after buildup, which may supporting substrate. In both fabrication processes, the detachment or increase their ordering. However, further experiments are needed to dissolution steps have already been performed in rather harsh test these hypotheses. conditions (HF acid or concentrated HCl) in the case of synthetic In a recent work, Tilton et al. probed the dynamics of chitosan in polyelectrolytes [59]. This may explain why only few examples of (CHI/HEP) ﬁlms [55]. Using different ﬂuorescently labeled chitosan, polyelectrolyte microcaspules containing polysaccharides have been they measured the “out of plane” (z-direction) diffusion using FRET. shown, as alternative strategies for core dissolution needed to be By modeling their experimental data of FRET efﬁciency, they deduced established. One of the ﬁrst types of microcapsule consisted of (CHI/ values of 1 to 8 × 10− 8 µm2/s depending on the salt concentration and chitosan sulfate) [60]. DeGeest et al. have already succeeded in pH of the CHI (higher for higher I and pH). Crouzier et al. [38] recently preparing capsules containing dextran sulfate [61]. As interactions reported values of 3 × 10− 3 µm2/s for the lateral diffusion of PLL-FITC in between the polyelectrolytes are usually weaker in the case of (PLL/HEP) ﬁlms (∼80 nm in thickness), which was 20 times less than polysaccharides, there is now a search for milder conditions for core in HA-based ﬁlms. Here again, further studies are required to fully dissolution in aqueous conditions, and in particular for lowering the understand the origin of such a high (105 fold) difference between osmotic pressure that is produced during core dissolution. To this end, these studies on quite similar ﬁlms. biodegradable cores such as CaCO3 are preferred [62,63], as well as Diffusion of proteins or of smaller molecules such as ions can be other kinds of template such as microgels [64]. LbL assemblies based used for electrochemistry studies on redox molecules. For example, on HA have only recently been the basis for microcapsule formation myoglobin was loaded into (CHI/HA) ﬁlms by means of post-diffusion. from a CaCO3 template [62]. The conditions for core dissolution were The inﬂuence of several factors, such as layer number, the pH of the carefully investigated and it was found that EDTA or citric acid are loading solution, as well as its ionic strength, were systematically successful chelating agents. However, in some cases, chemical cross- studied (Fig. 1) [56]. The cyclic voltammetric peak pair of the linking prior to core degradation is necessary [62,64] in order to myoglobin FeIII/FeII redox couple for (CHI/HA)n-myoglobin ﬁlms on strengthen the PEM ﬁlm. pyrolytic graphite electrodes was used to investigate the loading Interestingly, a considerable effect of the molecular weight of HA behavior of (CHI/HA) ﬁlms with regard to myoglobin. Another report as well as on the HA solution concentration was observed in the case of PAH/HA microcapsules. This was attributed to the diffusion of polyelectrolyte chains into the porous CaCO3 pores (60 nm in diameter) for low MW HA. Higher HA concentrations led to a more dense and entangled structure that was more favorable for capsule formation. PEM membranes based on polysaccharides are also rare. For the same reason as for capsule preparation, conditions that are too harsh can lead to ﬁlm rupture. This was indeed observed for (PLL/HA) ﬁlms after dissolution of the polystyrene substrate in THF (tetrahydrofuran) [65]. An alternative strategy was to chemically cross-link the ﬁlms prior to their detachment in 0.1 M NaOH. In this case, the membranes were homogeneous. Kotov et al. investigated the mechanical proper- ties of membranes made of CHI and montmorillonite (MTM), as CHI possesses higher strength than PDADMAC [59]. They successfully prepared such membranes, which were however found to exhibit much lower mechanical properties than their PDADMAC counterparts. These authors attributed the decrease in mechanical properties to the lower ﬂexibility polymer (persistence length of ∼ 5 nm for CHI) resulting in poor interfacial adhesion with the clay. Fig. 1. Inﬂuence of the number of layer pairs (n) of (CHI/HA)n-myoglobin ﬁlms on (a) the maximum surface concentration of electroactive myoglobin (Γmax) measured by 7. Hybrid ﬁlms cyclic voltametry in pH 7.0 buffers at tmax and 0.2 V s− 1, (b) the surface concentration of myoglobin (Γmax) measured by QCM at the same tmax, and (c) the ratio of Γ*max/Γmax. (From Lu and Hu, J. Phys. Chem. B, 110, 23710–23718, 2006) ; copyright American Polyelectrolyte blends (reviewed by Quinn et al. [66]) have Chemical Society 2006. recently emerged as a new tool for modulating ﬁlm thickness, ﬁlm 422 T. Crouzier et al. / Current Opinion in Colloid & Interface Science 15 (2010) 417–426 morphology and secondary structure, degradation rates, protein are hydrogel-like ﬁlms with elastic behavior, but [(CHI/HA)3/CHI/PAA]n adsorption [67] or even mechanical properties. A limited number of exhibited a ﬁlm bulk that resembled Newtonian ﬂuid. studies deal with polysaccharides as one of the components in the More generally, highly diffusive ﬁlms can be combined with blend. Quantitative FTIR spectroscopy measurements using character- “blocking layers” or barriers by depositing dense ﬁlms such as (PSS/ istic peaks for each polyelectrolyte allow their mass fraction in the ﬁlm PAH), [73] or degradable polymer layers consisting in poly(lactic-co- to be determined. When HA was mixed with either PSS (a strong glycolic acid) [74]. Such ﬁlms are particularly interesting for drug synthetic polyelectrolyte) [68] or HEP (a strong polysaccharide) [69] delivery applications where different reservoirs (the diffusive part) and combined with PLL as the polycation, a preferential incorporation would serve as storage for delivering different molecules embedded of the strong polyelectrolyte was always observed. Of note, a very steep in separate compartments in the ﬁlm. Indeed, the highly hydrated and increase in the HA effective mass content in the ﬁlm was evidenced weekly coupled polysaccharide-based PEM ﬁlms such as (PLL/HA) are when the HA mass fraction in solution was within the 95–100% range. suitable for the incorporation of liposomes, which themselves can When a sulfated polysaccharide (β-1,3 glycan sulfate (GlyS)) was potentially be loaded with bioactive compounds. Recently, liposomes mixed with ALG (carboxylic groups), a similar trend was observed: ﬁlled with an AgNO3 solution were successfully incorporated in (PLL/ GlyS was found to insert preferentially into the ﬁlm [70] and it almost HA) ﬁlms to form antibacterial coatings. [75]. totally exchanged the ALG when brought into contact with a (PLL/ALG) multilayer (Fig. 2). The preferential incorporation was explained by the 8. New functionalities obtained by chemical modiﬁcation of higher charge density of GlyS polymers over ALG and by the presence of the polysaccharides sulfate groups in GlyS that interact more strongly with the ammonium groups of PLL than the carboxylic groups present on the ALG chains. As described previously, the properties and functionalities of PEM Isothermal titration calorimetry (ITC) experiments, which allowed the ﬁlms depend greatly on the chemistry of the polysaccharides used. It enthalpic contribution of the complex formation to be measured, is thus possible to modulate or add functionalities to a ﬁlm by supported this assumption. Interactions between PLL and HA are chemically modifying its constituents. In the last ﬁve years, several endothermic while those between PLL and HEP are exothermic [69]. polysaccharide modiﬁcations have been developed to provide them The details of polyelectrolyte arrangements within blend ﬁlms are still with new functionalities [76]. unresolved and difﬁcult to investigate. Several questions remain unanswered. For instance, why are the (PLL/HA-HEP) blend ﬁlms 8.1. Grafting of photo-sensitive groups very thin (∼100 nm) whereas they contain a small but non negligible % of HA? How are the HA chains organized in the ﬁlms? Although the grafting of photo-reactive groups on to synthetic Interesting information and new ﬁlm properties can be gained from polyelectrolytes and their subsequent use for LbL ﬁlm formation has another strategy, which makes use of adding synthetic polyelectrolyte been developed for several years [77,78], this chemical modiﬁcation has layers on top or in-between polysaccharide layers. For instance, only recently been applied to natural polysaccharides for PEM Francius et al. ﬁrst deposited soft (PLL/HA) layers and capped them formation. Of note, thick polysaccharide-based hydrogels formed by with hard PSS/PAH ones [71]. A considerable stiffening of the multilayer photo-cross-linking are already relatively common [79]. Pozos Vasquez was observed by using the Atomic Force Microscopy (AFM) indentation et al. presented an alternative chemistry for synthesizing vinylbenzene technique (see paragraph 8). Salomäki and Kankare hybridized (CHI/ (VB)-grafted hyaluronan derivatives (HA-VB) [80] via an ester linkage. HA) layers with synthetic polyelectrolyte PAA, added at different steps These anionic derivatives were assembled with cationic PLL to prepare in the buildup [72]. They found that the exponentially growing ﬁlms photo-cross-linkable PEM ﬁlms. Shining UV light on to ﬁlms will induce could be tuned to linear by codepositing PAA in multilayers. PAA was the formation of covalent bonds between the photo-reactive groups, shown to destroy the soft diffuse matrix formed by CHI/HA. and thus crosslink the ﬁlm. The extent of the cross-linking and ﬁlm Interestingly, (CHI/HA/CHI/PAA)n ﬁlms resembled (CHI/HA), which stiffness was found to depend on the degree of VB grafting, as evidenced by nano-mechanical measurements. The synthesis of photo-cross- linkable polysaccharides is of particular interest for the future development of spatially patterned polysaccharide-based ﬁlms. 8.2. Grafting of hydrophobic groups Delivery of poorly soluble (highly hydrophobic) drugs is a challenge in the ﬁeld of drug delivery. Whereas post-diffusion has already been used for loading small drugs [81], peptides [82] or growth factors [83], this method is not appropriate for loading high amounts of hydropho- bic compounds. To explore the potentiality of PEM ﬁlms as delivery reservoirs for hydrophobic molecules, Guyomard et al. synthesized anionic amphiphilic polysaccharides of varying hydrophobicity obtained by grafting alkyl chains on to carboxymethyl pullulan (CMP) [18,84]. As the backbone of CMP is highly hydrophilic, the hydropho- bicity of these amphiphilic derivatives resulted only from the grafted alkyl groups and led to the formation of hydrophobic nanodomains in the ﬁlms. Such ﬁlms were probed for their ability to trap a hydrophobic dye, Nile Red (NR) (considered here as model hydrophobic drug). The authors demonstrated that the maximal amount of dye loaded in the Fig. 2. Apparent mass fraction y of GlyS in the PEI-[GlyS(x)-ALG(1−x)/PLL]7 ﬁlms as a ﬁlms depended on the degree of grafting of the CMP derivatives. Later function of x, the mass fraction of GlyS in the blend solution. The optimal value of y was on, such ﬁlms were used to deliver a non-water soluble natural determined by ﬁtting to the experimental IR absorbance spectra. The dashed line antibacterial peptide, gramicidin A, and was shown to effectively kill a represents the ideal situation for which the mass fraction of GlyS in the ﬁlm would be equal to its mass fraction in the blend. The error bars correspond to the width of the ﬂat gram-positive bacterium, E. faecalis [85]. region of the minimum in the b ξ2N vs. y curves. (From Ball et al., Langmuir 25:3593– Recently, alkylamino hydrazide derivatives of HA were similarly 3600, 2009, reproduced with permission, copyright ACS 2009). used to form hydrophobic nanodomains in otherwise hydrophilic (PLL/ T. Crouzier et al. / Current Opinion in Colloid & Interface Science 15 (2010) 417–426 423 HA) ﬁlms [86]. The insertion of NR into the ﬁlms after pre-complexation combined with HA. By using QCM-D, the authors found that the with the derivatives was found to depend on the alkyl chain length, on resulting ﬁlms behave as highly hydrated soft gels. This high water the degree of substitution (Fig. 3) and on the HA molecular weight. content for (PC-CHI/HA) ﬁlms reﬂects the known ability of the PC Interestingly, the effective concentration of NR in the ﬁlms was ∼5000 group to undergo hydrogen bond interactions with water molecules. times higher than the initial NR concentration in solution. The Logically, both storage and loss moduli for the (PC-CHI/HA) ﬁlms were percentage of dye released was between 10% and 60% after 8 days, about one order of magnitude smaller than those of (CHI/HA) ﬁlms. depending on the alkyl chain length of the alkyl chain grafted. Such ﬁlms Recently, two derivatives of CHI containing oppositely charged thus seem promising for the localized delivery of hydrophobic drugs at groups were synthesized by Komakowska et al. [17], one by cationic high concentrations. modiﬁcation of chitosan and the other by grafting sulfonate groups on to carboxymethyl chitosan. Thus, “one-component” chitosan-based ﬁlms 8.3. New chitosan derivatives were formed and characterized in physiological conditions (neutral pH, 0.1 M NaCl). A derivative of chitosan grafted with phosphorylcholine (PC) was recently synthesized by Winnik et al. [87] It was found to be nontoxic 9. Mechanical properties of polysaccharide-based PEM ﬁlms and soluble under physiological conditions, even with low levels of PC incorporation (∼ 20 mol% PC per glucosamine unit). This modiﬁed PC- Recently, the design of new functional surfaces with precise control CHI may be further used as a new biomaterial due to its interesting over their mechanical properties has emerged as being of utmost protein-repelling properties and biocompatibility. Like CHI, PC-CHI is importance due to the cells' sensitivity to substrate mechanical a polycation under acidic conditions and could form PEM ﬁlms when properties [88,89]. In this context, designing PEMs with tunable stiffness has become a major challenge and characterization of their viscoelastic properties is crucial. A commonly used technique consists in using an AFM to perform nano-indentation experiments in liquid [90–92]. QCM- D [93,94] and piezo-rheometry [94] have also been proposed to quantify the stiffness of such thin ﬁlms in liquid conditions. As a general rule, polysaccharide multilayer ﬁlms are much softer than their synthetic counterparts [95], but several possibilities exist for modulating their stiffness. 9.1. Inﬂuence of the structure of polyelectrolytes The ﬁrst modulation method consists in modifying the ﬁlm's internal composition and structure. Schoeler et al. and Schönhoff et al. thus investigated the buildup of ﬁlms containing PAH as the polycation and two anionic carrageenans, ι-carrageenan, which forms helical structures, and λ-carrageenan, which is in a random coil conformation [96,97]. Inspecting the morphology and internal structure of the ﬁlms revealed that the helical conformation of ι-carrageenan in solution was maintained during the multilayer buildup. Furthermore, the presence of such ordered structures resulted in thicker and smoother ﬁlms, with a Young's modulus 3 times higher than the unstructured λ- carrageenan based ones. A second strategy for stiffening PEM ﬁlms is to complexify the ﬁlm's architecture (see paragraph 7) by inserting layers of synthetic polyelectrolytes, which are more charged and more ﬂexible than polysaccharides. A considerable stiffening of the ﬁlms was observed either by deposition of synthetic (PSS/PAH) layers on top of a (PLL/HA) ﬁlm [71,98] or by insertion of PAA layers into a (CHI/HA) ﬁlm [72]. Another way of adjusting the rigidity of PEM ﬁlms consists in incorporating rigid nano-objects (nanoparticles, clays…). Inspired by inorganic–organic composite materials such as seashells and lamellar bone, this technique has already been widely used in the case of synthetic PEM but only rarely applied to polysaccharide multilayers. Podsiadlo et al. observed that a composite multilayer ﬁlm containing cationic CHI and anionic nanoparticles of montmorrillonite has a 3- fold increase in Young's modulus compared to pure CHI [59]. 9.2. Chemical cross-linking Fig. 3. (A) Nile red incorporation in the (PLL/alkylated HA-200 derivatives)18 ﬁlms followed by a ﬂuorescence microplate reader after each deposition step of the The mechanical properties of PEM ﬁlms can also be modulated by polyanion.(HA-200 means HA with a molecular weight of 200 000 g/mol; the alkylated derivates HAGCL, G being the grafting ratio and L the chain length); (B) Absorbance chemically cross-linking the polyelectrolytes. Richert et al. initially measurements at 590 ± 2.5 nm performed on the same ﬁlms. Linear ﬁts for the reported the fabrication of PEM ﬁlms based on polysaccharides and/or HA10C10 (plain lines) and HA10C10Br derivatives (branched derivatives, dotted lines) polypeptides whose rigidity was varied by cross-linking the carbox- are shown in (A) and (B). For the ﬂuorescence curves, the slopes are respectively of ylic groups of the polyanion with the amine groups of the polycation 135.3 (calculated for i ≥ 6) and 37.4 (calculated for i ≥ 10) for these derivatives; for the absorbance curves, the slopes calculated over the same range are similar with using carbodiimide chemistry [43]. Covalent amide bond formation respective values of 0.0179 and 0.0175. (From Kadi et al., Biomacromolecules 10:2875– was evidenced by FTIR. Using AFM nano-indentations, considerable 2884, 2009, copyright American Chemical Society, reproduced with permission). stiffening of both (PLL/HA) and (CHI/HA) ﬁlms was measured and the 424 T. Crouzier et al. / Current Opinion in Colloid & Interface Science 15 (2010) 417–426 Young's modulus was up to 2 orders of magnitude higher than for the [6] Evanko SP, Angello JC, Wight TN. 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