Insulating and Semiconducting Polymeric Free-Standing Nanomembranes with Biomedical Applications

In recent decades, polymers have experienced a radical evolution: from being used as inexpensive materials in the manufacturing of simple appliances to be designed as nanostructured devices with important applications in many leading fields, such as biomedicine at the nanoscale. Within this context, polymeric free-standing nanomembranes - self-supported quasi-2D structures with a thickness ranging from ∼10 to a few hundreds of nanometers and an aspect ratio of size and thickness greater than 106- are emerging as versatile elements for applications as varied as overlapping therapy, burn wound infection treatment, antimicrobial platforms, scaffolds for tissue engineering, drug-loading and delivery systems, biosensors, etc. Although at first, a little over a decade ago, materials for the fabrication of free-standing nanosheets were limited to biopolymers and insulating polymers that were biodegradable, during the last five years the use of electroactive conducting polymers has been attracting much attention because of their extraordinary advantages in the biomedical field. In this context, a systematic review of current research on polymeric free-standing nanomembranes for biomedical applications is presented. Moreover, further discussion on the future developments of some of these exciting areas of study and their principal challenges is presented in the conclusion section.

5 simplicity and the high degree of control over the FsNM thickness. Furthermore, the precise guidance exerted over the chemical composition of the layers results in a tremendous versatility when designing FsNM. 23 Regarding the SAM technique, Stroock et al. 21 extensively described the synthesis of polymeric FsNM with 10-15 nm in thickness and well-defined lateral size and shape.
On the other hand, spin-coating ( Figure 1b) is another interesting approach that allows the preparation of single-or multi-layered FsNM in a few steps. In this case, the optimization of the spin-coating parameters (e.g. spinning speed and time, and the solution concentration) leads to ultra-thin films with controlled features, thickness and homogeneity. In this procedure, which seems to be the most versatile and easy-going, the liquid polymeric solution is spin-coated onto a solid substrate previously coated with a sacrificial layer. Hence, by dissolving the sacrificial layer in an appropriated solvent, the FsNM is detached from the substrate and released into a liquid environment where it can be then easily handled with syringes or pipettes. Most of the FsNM described in this review were fabricated using a combination of both the LbL assembly and spin-coating procedures.
advantages but also some limitations (i.e. lack of mechanical integrity, absence of biodegradability and hydrophobicity). 37,[40][41][42] ECP blending with conventional insulating polymers or biopolymers is the most popular approach followed to solve these drawbacks. [43][44][45][46][47][48] An alternative and also effective strategy, that was recently reviewed by Albersson and co-workers, 43 is the synthesis of new biodegradable polymers containing conducting oligomers. Due to the growing interest in this class of new biomaterials, the palette of degradable and electrically conductive polymers is progressively expanding to meet the demands of specific applications within the biomedical field. For example, in the last year Schmidt and co-workers reported biodegradable electroactive copolymers composed of oligoaniline-based blocks linked to polyethylene glycol or polycaprolactone blocks, which deliver anti-inflammatory drugs on the application of electrochemical stimuli. 49 In this work we use the concepts discussed in the two previous sections to review the fabrication of FsNM for biomedical applications. Firstly, we will discuss the design and development of FsNM made of biopolymers and insulating polymers. After this, we will focus on FsNM fabricated by blending conventional polymers with ECP. Finally, several concluding remarks on the achievements and future perspectives of this outstanding research field will be exposed.

Biomedical Applications of Insulating and Electrochemically
Inactive Free-Standing Nanomembranes through variations in their molecular weight. 50 Also, poly(ether ester) copolymers have been successfully employed due to their excellent properties (e.g. elasticity, toughness, strength and easy processability), which arise from the combination of both soft and hard segments along their chemical structure. 51 In regard to biopolymers, the most frequently used are collagen and polysaccharides, such alginate and chitosan, their applications being usually focused in tissue engineering. 52,53 In the biomedical context, FsNM need from specific requirements such as biocompatibility and, in some cases, also biodegradability and/or bioresorbability need be met. Furthermore, by successfully controlling the chemical composition and the fabrication process, it is possible to tune and adjust the physicochemical, mechanical, chemical and morphological properties of FsNM to promote cell-substrate interaction, which is particularly relevant in regenerative medicine (i.e. cellular organization can be directly regulated through the cellular microenvironment). 54,55 Their structure and flexibility allows FsNM to coat the surface of devices that interact with biological systems or, alternatively, FsNM can be introduced in a needle by aspiration, and then be moved and released/injected in liquid environments (e.g. finely positioned on surgical incisions or directly used for wound treatments).
Next sub-sections review the most representative examples of FsNM made of insulating polymers for advanced biomedical applications. Table 2 summarizes their main characteristics, which are described below organized by polymeric families (namely, polyester, polysaccharide and other polymeric sources).

Polyester Free-Standing Nanomembranes
Takeoka and co-workers 56  showing an excellent sealing efficacy that did not require adhesive agents. Moreover, the incision healed completely without scars and tissue adhesion. In an effort to deeply investigate the possible biomedical application of these PLA FsNM, their antiadhesive and fixative characteristics were further investigated: an intraperitoneal polypropylene overlaid mesh (IPOM) with PLA nanosheets was placed on an intact peritoneum ( Figure   4b). 57 Results evidenced that PLA FsNM are feasible to induce adhesion prophylaxis in IPOM, having a beneficial effect as an atraumatic fixation tool. In addition, it was found that nanosheets do not cause inflammatory reaction, which was attributed to the excellent biocompatibility of PLA. 57 Within this research line, in another study, Takeoka and co-workers 58 tested the feasibility of PLA FsNM as a wound dressing against burn infections in an in vivo mouse model partial-thickness injury. PLA FsNM tightly adhered onto the burn lesion on the mouse back without any adhesive agents, while adhesion to any opposing tissues/organs was not observed. Moreover, its transparency enabled the visual observation of the wound condition, and thus the infection evolution and healing process were easily monitored. In addition, PLA FsNM functioned as a barrier against Pseudomonas.
aeruginosa when it was inoculated on the wound lesion. Therapy with PLA nanosheet protected against an inflammatory response. 59 Hence, the tested system rendered an effective dressing material for the management of partial-thickness burn wounds.
More recently, the same group obtained PLA nanosheets by combining a spincoating-assisted multi-layering process of PVA and PLA with a peeling step. Therefore, after the spin-coating and by dissolution of the sacrificial PVA layers between PLA layers in distilled water, a number of PLA FsNM which corresponded to the number of multi-layering processes of PVA and PLA were obtained. 59 The thickness of the resulting FsNM, which were transparent and extremely flexible, was determined to be 60 ± 6 nm when spin-coating a PLA solution (10 mg/mL) at 4000 rpm for 20 s. Later, these nanosheets were fragmented to form a stable suspension, and then reconstructed to form a single continuous film that attached to various interfaces without the need of adhesive agents ( Figure 5). The new patched film was applied as a physical barrier against burn wound infection with P. aeruginosa. Both in vitro and in vivo assays evidenced that the patched film exhibited excellent barrier ability to prevent infection during the treatment of burns for 3 days.
With the aim of designing a novel patch for bone or tendon repair and healing, biocompatibility was evaluated using C2C12 cultured cells. 61 Results evidenced early differentiation with the fusion of cells into firmly adherent myotubes, proving that such flexible nanofilms behaved as good bioactive matrices for cell anchoring, spreading and proliferation.
The effect of an underlying substrate on the interaction between cells and PLA FsNM was recently investigated. 62 For this purpose, polyester-based nanosheets (with thickness values from 29 ± 1 nm to 703 ± 4.4 nm, depending on the PLA concentration during the spin-coating process) were also fabricated by spin-coating and PVA acted as the sacrificial layer. After PLA FsNM releasing in a liquid environment, they were collected on stainless steel mesh (PLA-mesh), and subsequently used for cell adhesion studies using rat cardiomyocytes cells (H9c2). Results were compared to the ones obtained on a control interface: SiO 2 substrates coated with an ultra-thin layer of PLA (PLA-substrate). Although topological and mechanical properties of PLA FsNM did not influence the cell viability after 24h of culture, cells did geometrically sense the stiffness of the underlying material, thus affecting the adhesion geometry. Briefly, PLA-mesh induced an anisotropic adhesion of H9c2 cells along the metal wire, while H9c2 cells on PLA-substrate adhered isotropically, independently of the nanosheet thickness.
Accordingly, cells distinguished the increase in the nanosheet stiffness and preferentially adhered onto the rigid interface. Interestingly, cellular anisotropy decreased by increasing the thickness of the PLA FsNM deposited onto the mesh because the nanosheet stiffness was more homogeneous throughout the surface. Considering the huge difference between the Young's modulus of PLA FsNM (from 3.5 ± 1.3 to 7-10 GPa depending on the thickness) and the metal substrate (hundreds of GPa), it was concluded that cell adhesion was mechanically regulated by the stiffness of the underlying substrate when the thickness of the PLA FsNM was in the order of tens of nanometers.
PLA FsNM have also been modified and/or functionalized by means of collagen deposition, magnetic particles entrapment and drug loading. In a recent study, Niwa et al. 63 prepared PLA nanosheets modifying only one surface with collagen to endow different discrete functions to each side of the nanosheet: anti-adhesive and pro-healing properties. For this purpose, PLA FsNM were prepared by spin-coating using conditions similar to those previously described (thickness: 59.5 ± 9.5 nm). A collagen layer was subsequently deposited on the surface following two approaches: solvent casting or spincoating. The latter strategy resulted in an ultra-thin collagen coating (thickness of 5-10 nm) more homogeneous and hydrophilic than that obtained by solvent casting, which exhibited a thickness of ∼120 nm. The disorganized collagen fibrils formed on PLA nanosheets when covered using the spin-coated method induced a hydrophilic microenvironment that improved cell adhesion and spreading, in comparison to that obtained by solvent casting. Besides, fine networks of actin filaments were clearly identified in cells cultured on the former biointerface, as opposed to the latter system.
As a first step to develop magnetic FsNM to be remotely controlled and manipulated, Taccola et al. 64 embedded superparamagnetic iron oxide nanoparticles (SPION) into PLA nanosheets by spin-coating (5-20 mg/mL PLA solution in chloroform and 1-15 mg/mL SPION colloidal solution at 3000 rpm for 20 s using a PVA sacrificial layer). Magnetic composites were coloured with different degrees of intensity depending on the nanoparticles load, and the inclusion of SPION in the polymeric matrix did not alter their magnetic behaviour, yielding FsNM with high saturation magnetization and magnetic susceptibility. For nanosheets with high concentration of SPION, the magnetic response increased because of the formation of clusters, albeit their presence did not alter the integrity and stability of the FsNM. Results were good enough to promote further investigation in this area, suggesting that controllable supports with magnetic properties could be fabricated in a near future for biomedical applications.
FsNM with unidirectional drug delivery ability have the potential to improve therapeutic efficacy, while minimizing undesirable side effects. In this sense, Sun and coworkers 65 fabricated robust and flexible FsNM by sandwiching drug-containing polyelectrolyte multilayer films between a capping and a barrier layer of PLGA. The drug-containing films were prepared by the LbL assembly of chemically cross-linked poly(allylamine hydrochloride)−dextran (PAH-D) microgel and hyaluronic acid (HA), which can load methotrexate (MTX), a negatively charged cancer-inhibiting drug. The PLGA barrier layer, which was obtained by spin-coating, prevents MTX release, and the PLGA capping layer regulates the unidirectional MTX release kinetics in a precisely controlled manner. In vitro cancer treatments evidenced that MTX released from the FsNM preferentially inhibited the proliferation of HeLa cells. The versatility of LbL assembled polymeric films as drug carriers allows the loading of a wide variety of drugs and bioactive agents. Thus, the design of highly efficient unidirectional drug-delivery systems with minimizing side effects to normal tissues has a great potential for clinical applications.
Finally, Okamura et al. 66 prepared disk-shaped PLGA nanosheets and modified their surfaces with the dodecapeptide H12 (HLGGAKQAGDV), which is a fibrinogen γ-chain carboxy-terminal sequence (γ400-411) that specifically recognizes the active form of glycoprotein IIb/IIIa on activated platelets. Under flow conditions, H12-PLGA FsNM were found to interact with activated platelets on a collagen surface faster than H12-PLGA microparticles did. In addition, only FsNM induced 2D spreading of platelet thrombi on collagen-immobilized plates. These results suggested that PGLA FsNM might be a suitable candidate as novel platelet substitutes and, also, an alternative to human platelet concentrates infused to treat bleeding in patients with severe thrombocytopenia. 66

Polysaccharide Free-Standing Nanomembranes
Polysaccharides are a class of biological macromolecules that participate in a wide range of biochemical and biomechanical functions. Because of their unique properties, these biopolymers are currently playing an important role in materials science. The structure and function of polysaccharides, and their nanoscale assembly for biomedical materials were recently reviewed by Boddohi and Kipper. 67 However, this section will draw the attention to those studies that used polysaccharide as the main polymeric source to develop and design FsNM. As in the previous section, biomedical applications and promising results will be highlighted.
In 2007, Fujie, Okamura and Takeoka 68 constructed what they called a "nanoadhesive plaster", a multilayered FsNM (thickness: 30.2 ± 4.3 nm) made of chitosan and sodium alginate (Na-alginate) that was deposited onto a PVA-silicone rubber substrate using the spin-coating assisted LbL approach. More specifically, the layers of each polyelectrolyte were spin-coated on the silicone rubber (millimetre scale thickness: 1.0 mm) previously covered with a sacrificial PVA layer (microscale thickness: 1.2 µm). As a result, the nano-adhesive plaster consisted on the superposition of three different types of free-standing sheets (Figure 6a). Chitosan and Na-alginate contain amino and carboxylic groups, respectively, as cationic and anionic polyelectrolytes at ambient pH, which enormously facilitated their assembly in the FsNM. The elastic modulus, which was determined by the bulge test, was found to be 1.3 GPa. The good adhesiveness and flexibility of the nano-adhesive plaster was evidenced by transferring it, after modification with a luminescent pigment for ease of visibility, not only onto the skin of a human arm 68 (Figures 6b-6d) but also onto a tissue (rat cecum) surface. 69 In a subsequent study, the same group proved the potential biomedical application of these this adhesive, robust and flexible nano-adhesive plasters made of chitosan and Na-alginate in a surgical intervention. 70 Thus, a FsNM with a thickness of 75 nm repaired an in vivo visceral pleura defect induced on beagle dogs without any loss in the respiratory function and without significant inflammatory response. In addition, the influence of the thickness in the mechanical properties of polysaccharide FsNM was demonstrated, which enabled an easy modulation of the nanosheet features.
Besides, the therapeutic effect of the nano-adhesive plaster on murine cecal puncture was evaluated (Figure 7). 71 The sealing effect of the multilayered chitosan and Naalginate-based FsNM inhibited effectively the bacterial infection, and increased the survival rate of the individuals. Interestingly, the treatment with the polysaccharide nanosheet provoked less inflammatory response than the suture. 71 Despite the promising results, a small percentage of bacteria were able to pass thorough the FsNM. To overcome this aspect, an antibiotic-loaded polysaccharide nanosheet was developed. 72 In this new approach, an antibiotic (tetracycline, abbreviated TC) was sandwiched between a new PVA layer (named PVAc in Figure 8), which acted as a protector, and the LbL assembled polysaccharide platform (total thickness: 177 nm). The resulting FsNM, which comprehended three functional layers (Figure 8), exhibited an important anti-microbial effect and a relatively low inflammatory tissue response. 72 As a consequence, in vivo studies using overlapping therapy with these new PVA / TC / polysaccharide FsNM showed a significantly increase in mouse survival after cecal puncture. Later research evidenced that TC-loaded chitosan and Na-alginate multilayered FsNM exhibited a potential antibacterial activity when covering mice dorsal skin artificially burnt and infected with P. aeruginosa. 73 All mice treated with the TC-loaded polysaccharide FsNM survived, whereas mice treated with TC-unloaded nanosheets and mice left untreated displayed increased rates of mortality due to bacterial infection. Moreover, TC-loaded FsNM prevented not only local inflammation but also systematic inflammation.
Hagisawa et al. 74 described a novel therapy in which the overlapping of several chitosan / Na-alginate FsNM with a thickness of 75 nm (i.e. multioverlapping therapy) sealed and stopped a massive venous hemorrhage on rabbits. Before in vivo tests, the mechanical durability of the nanosheets and its hydrostatic pressure resistance was evaluated. Four pieces of overlapping nanosheets were able to stand 80 ± 6 mm Hg of pressure. In regard to its degradability, polysaccharide nanosheets experience a decrease in their thickness in degradation conditions (phosphate-buffered saline, PBS, at 37 ºC).
Nevertheless, they retained more than 70% of the thickness after seven days. Positively, after one month, no inflammatory tissue reaction was detected around the FsNM attachment and findings revealed a complete wound healing. The multioverlapping therapy would represent a great advantage for surgical operation, especially in trauma patients with bleeding from large veins.
Otani et al. 75 have reported the therapeutic use of bilayered polysaccharide FsNM made of chitosan and Na alginate as arachnoid plaster in a microneurosurgery environment because its semi-absorbent and potent physical adhesive strength features.
These authors observed that the application of overlapping FsNM without using chemical bonding agents completely avoided cerebrospinal fluid leakages in the cerebral cortex.
There was evidence of a relation between the number of overlaid nanosheets and the reinforcement effect: the more layers displayed, the more it improved. Besides, after six months, no inflammatory infiltration was detected.
In addition to chitosan and Na-alginate, proteoglycan HA has also been used to fabricate polysaccharide FsNM. For instance, Fujie et al. 76 prepared multilayered nanosheets of HA and collagen on a water-soluble sacrificial supporting substrate using the LbL assembly method. The thickness of these FsNM was found to grow exponentially with the number of HA and collagen layers. This was attributed to the fact that the amount of HA adsorbed in the LbL structure is less than that of collagen. 77 Therefore, the polyion pairs mediated by the electrostatic interaction between collagen and HA molecules induced a nonlinear growth in the LbL system. The mechanical properties of these FsNM, as determined by the bulge test, were found to depend on the fibrous or non-fibrous structure of collagen layers. Thus, the elastic modulus of nanosheets made with non-fibrous collagen and a high content of HA (thickness: 62 ± 7 nm) was 4.3 ± 0.6 GPa, which is a value comparably smaller than that of the previously reported chitosan / Na-alginate nanosheet (9.6 GPa for a thickness of 75 nm 70 ). This low elastic modulus was attributed to the hydrophilic and moisture-sensitive nature of HA molecules. In contrast, the elastic modulus of FsNM made with fibrous collagen (thickness: 42 ± 4), which had a low content of HA, increased to 12.5 ± 1.5 GPa, thus evidencing a greater mechanical durability. Collagen structures in bone and skin have an elastic modulus of 17.2 and 4 GPa, respectively. Therefore, FsNM made of HA and fibrous or non-fibrous collagen efficiently imitate the mechanical properties of these tissues. Consistently, FsNM made with non-fibrous collagen exhibited a surface with softer elastic properties than those observed in FsNM with fibrous collagen. Besides, cell adhesion studies using NIH-3T3 cells showed that HA / non-fibrous collagen FsNM gave lower cell adhesive elongation in comparison to nanosheets with fibrous collagen and a low content of HA. On the basis of this study, authors concluded that cell adhesive properties can be tuned by changing the structural components of the nanosheets (i.e. the content of polysaccharide and collagen fibrils), 76 thus opening a new door to the production of novel engineered scaffolds for regenerative medicine as well as cell biology.
An innovative and audacious strategy has been recently reported by Chen et al. 78 who prepared cell-polymeric nanocomposites for the fabrication of FsNM lined with cells ( Figure 9). First, poly(N-isopropylacrylamide) (PNIPAM), which is a temperatureresponsive polymer, was grafted onto glass-slides. After this, cells were cultured on the resulting hydrophobic layer. Once 80-90% cell confluence was achieved, the LbL process was conducted on the surface of the cell sheet. However, gelatin, which is a natural biocompatible polyelectrolyte, was previously deposited as cell-contacting layer to keep a high cell viability during the deposition process. 79,80 Thus, this gelatin layer is of great importance during the LbL self-assembly step on the cell-sheet surface. 79,80 Specifically, three alternating charged polysaccharide bilayers of chitosan and Na-alginate, (chitosan/Na-alginate) 3 , were assembled onto the gelatin-coated cells cultured on the PNIPAM-grafted surfaces. The deposition of gelatin, chitosan and Na-alginate layers, which are extra cellular matrix (ECM) related components, on the cell sheet resulted in a cell adhesive surface that efficiently interacted with cells. 81 The free standing cell/gelatin/(chitosan/Na-alginate) 3 film was peeled off from the PNIPAM-grafted surface upon temperature changes. By this strategy, the assembly of cell sheets with ultra-thin ECM components to form FsNM can be potentially used to fabricate complex artificial soft tissues to substitute some elements in native tissues.

Other Polymeric Sources for Free-Standing Nanomembranes
Poly(ethylene oxide) (PEO) is a thermoplastic biocompatible material widely used in biomedical applications, as for example scaffolds, drug delivery systems and sensor devices. [82][83][84] To extend their list applications, the LbL technique was used to fabricate FsNM made of PEO. 85 This was achieved by creating solid-state hydrogen bonded assemblies that allowed the incorporation of stable interdigitated layers of PEO and poly(acrylic acid) (PAA) at the nanometer length scale (the thickness of each PEO/PAA bilayer was 80 nm). Free-standing films containing 100 bilayers were transparent, smooth to the touch and exhibited elastomeric properties during handling. Although these were not explicitly proved with experiments, Lutkenthaus et al. 85 suggested many biomedical applications for these hydrogen bonded assemblies, as for example biosubstrates, drug delivery devices and pH-sensitive sensors.
Ono and Decher 86 reported the use of pH-responsive self-standing polyelectrolyte multilayer membranes, which were fabricated by spraying polymer solutions, for biomedical applications. Concretely, a silicon wafer substrate was covered by a sacrificial multilayered film made of PAA and poly(ethylene glycol) (PEG), which disintegrated by a pH change releasing a target membrane. In that work, the released membrane consisted of a simple polyelectrolyte multilayered film of poly(allylaminehydrochloride) (PAH) and poly(sodium-4-styrenesulfonate) (PSS), which was initially constructed on the top of the PAA/PEG pH-responsive film. The pH response mechanism is based on the alteration of the hydrogen bonds established between PAA and PEG when the carboxylic acid groups present in PAA transform into carboxylate ions. From a biomedical point of view, the advantages of the PAA/PEG pH-responsive film, which were obtained with thickness values ranging from 55 to several hundreds of nanometers and areas of a few square centimetres, rely on the fact that PAA and PEG are biocompatible, biotolerated or bioinert. Accordingly, their release would not lead to adverse effects in the bioenvironment, and thus could be used in therapeutic devices and aids.
In another example, the preparation of PEG-terminal FsNM was based on the extensively cross-linking of aromatic 4'-nitro-1,1'-biphenol-4-thiol (NBPT) self-assembled monolayers (SAM) deposited onto a gold support by exposure to low energy electrons, thus resulting in a mechanical and thermally stable monolayer. 87 Simultaneously, the terminal nitro groups of the NBPT molecules were converted to reactive amine moieties to which epoxy functionalized PEG chains were subsequently coupled. As a result, these films exhibited protein repelling properties, which in turn ensure the lack of protein denaturing when they are used as support in transmission electron microscopy studies. 87 Meyerbröker and Zharnikov developed highly elastic, hydrophilic and ultra-thin membranes consisting entirely of PEG. 88 This was achieved by preparing an ultra-thin stable PEG-hydrogel precursor film composed of a mixture of epoxy-and amineterminated PEG moieties, which was deposited onto a supported sacrificial layer (i.e. a 100 nm gold film evaporated onto a silicon substrate). Then, the complementary terminal groups underwent chemical crosslinking. 89 Afterwards, the PEG/gold bilayer was separated from the silicon support and the sacrificial layer was dissolved releasing the PEG FsNM, which could be transferred onto a grid or any other arbitrary substrate. These nanosheets exhibited sufficient mechanical stability, and also high flexibility (extraordinary low Young's modulus of only ∼2 MPa). Such behavior is characteristic of elastomers, 90 and it was never observed before in nanomembranes. Indeed, the only analogous systems are polyisoprenes and polyisobutenes nanomembranes, which are not biocompatible, prepared using Langmuir-Blogett technology. 17 On the basis of their properties, PEG nanosheets are potentially useful as a highly sensitive support and sensor element for biological samples.
In another work, porous multilayered films of PAH and PAA were prepared using the LbL dipping technique, nanopores being subsequently created with a pH treatment. 11 The porosity induced by pH adjustment resulted in a significant increase in the thickness.
For example, the thickness of films made of 8 PAH/PAA bilayers before and after the creation of nanopores was 101 to and 253 nm, respectively. Ketoprofen and cytochalasin D, which represent different types of drugs that can be entrapped in these films, were successfully loaded and showed zero order release kinetics over a long period of time.
The amount of drug loaded and released could be tuned by varying the number of bilayers in the porous regions of the films, whereas variations of the pore size controlled the release flux of a given drug. . On the other hand, sheet-and tube-like FsNM of PAA/PAH were prepared by exfoliating PAA/PAH multilayered films from substrates in an aqueous acid solution containing Cu 2+ . 91 Initially, multilayered films were prepared by LbL deposition onto silicon wafer, quartz or glass tubes substrates. Their ion-triggered exfoliation was achieved by breaking the electrostatic interactions between the PAA layer and the underlying substrate. However, it should be noted that, although PAA/PAH bilayers were nanometric, ion-triggered exfoliation was applied to films with 15 bilayers of submicrometric thickness (∼0.8 µm) and, therefore, these cannot be considered as nanomembranes. The developed technology was proposed for the fabrication of smallcaliber artificial blood vessels.
Gui et al. 82 fabricated LbL multilayer films made of poly(diallyldimethylammonium) (PDDA) and poly(4-vinylpyridine propylsulfobetaine) (P4VPPS), a zwitterionic polysulfobetaine, in an acid aqueous solution at pH 2 with 0.5 M NaCl. The average growth rate was estimated to be ∼29.0 nm per PDDA/P4VPPS bilayer. These films were pH-dependent, thus disintegrating in alkali aqueous solution, especially at pH ≥ 12, which suggested a potential application as sacrificial layers for the release of other polymeric films. This was proved by depositing a multilayered film of PDDA and poly(sodium 4styrenesulfonate) (PSS) on the top of a 10-bilayered PDDA/P4VPPS sacrificial film.
After treatment with alkali aqueous solution at pH 12, the PDDA/PSS film was released keeping its integrity in air. Previously, Dubas et al. 93 had reported the fabrication of PDDA/PSS FsNM using PDDA/PAA LbL multilayer films as sacrificial layers.
Kohri et al. 94 presented a new approach for the fabrication of smooth and stable FsNM composed of a polymer brush, poly(2-hydroxyethyl methacrylate) (PHEMA), supported on a ultra-thin (i.e. ∼6 nm) colourless polydopamine (PDA) layer. This was achieved through the surface-initiated atom radical polymerization (ATRP) of 2hydroxyethyl methacrylate (HEMA) on the PDA layer, which resulted in optically transparent and colourless free-standing PHEMA brush films with tailored thickness of 16-75 nm. Previous to this process, the PDA ultra-thin film was placed onto a silicon or glass substrate covered by a cellulose acetate sacrificial layer (thickness: ∼115 nm). The scheme of the synthetic design is displayed in Figure 10. Although no application was explicitly tested, authors suggested that PHEMA FsNM can be used as multi-stimuli responsive sensors after functionalizing their surface. 94 Surface-functionalized FsNM were produced grafting poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC) brushes by the ATRP of 2-methacryloyloxyethyl phosphorylcholine (MPC) on nanosheets made of PAH, PAA and PS, which were prepared by the spin-coating assisted LbL technique (thickness of 85 ± 2 nm). 95 The properties of PMPC-nanosheets (physiological stability, surface wettability and antibiofouling response) were regulated by several parameters, such as the thermal crosslinking of the FsNM, the grafted amount of MPC and the thickness of PMPC brushes (ca. 11 nm). PMCP-nanosheets were easily peeled, transferred using tweezers with the aid of a water-soluble PVA sacrificial layer, cut into any shape by scissors, and patterned with a needle. Nanosheets patched on cell culture substrates exhibited antibiofouling properties such as anti-coagulant behaviour of human blood cells as well as the potential to microscopically pattern murine fibroblasts cells. The overall of the results provided an important physicochemical insight into the remarkable biological response of surface-functionalized nanosheets, which represent not only a powerful tool for biomedical applications but also an alternative to conventional micropatterned techniques used in bionanotechnology. Accordingly, the protein micropatterned FsNM system was proposed as an interesting tool for cell-directed culture in muscular tissue engineering.
PMMA was also employed for the preparation of FsNM displaying phase separation morphology. This was achieved by spin-coating a polymer mixture solution of PMMA and polystyrene (PS) onto a PVA-coated substrate. 97 Due to the intrinsic immiscibility of PMMA and PS, rapid quenching during the spin-coating process induced their phase separation. In order to obtain porous nanosheets, PS regions were dissolved by immersing the PMMA-PS FsNM into cyclohexane. The thickness of these films, which ranged from 38.8 ± 1.1 to 110.2 ± 2.0 nm, and the diameter of the pore, which varied between 64.4 ± 9.3 and 187.2 ± 33.9 nm, were controlled through the PMMA:PS ratio and the spincoating conditions. Furthermore, authors demonstrated that when the thickness of the ultra-thin films is comparable to the dimensional scale of the phase separation domains, it is possible to prepare perforated FsNM with nanopores in the range of tens of nanometers. 97 The availability of these perforated FsNM is proposed to be of immense value in many biomedical applications, as for example cell culture devices, high flux biosensors and drug delivery systems.
Flexible PS FsNM for directing cellular organization were prepared by Fujie et al. 98 by combining spin-coating and microcontact printing methodologies. The PS nanosheet thickness, which ranged from tens to hundreds of nanometers, was controlled through the concentration of the polymer solution used during the spin-coating process. In this work, In an earlier study, Tsukruk and co-workers 4,99 prepared hybrid organic-inorganic FsNM with extraordinary sensitivity and unique auto-recovering ability. These nanosheets (thickness of 25-70 nm) were prepared by spin-assisted LbL assembly of PAH and PSS on a sacrificial substrate. The most innovative aspect of these FsNM is the intercalation of a central layer containing gold nanoparticles (diameter of 12.7 nm) sandwiched between PAH/PSS bilayers. Thus, the general formula of these nanosheets can be described as: (PAH/PSS) n PAH-Au-(PAH/PSS) n-PAH, were n was varied between 3 and 11. The thickness of the system with a gold central layer was ∼20 nm higher than that of films without gold nanoparticles, independently of n. The mechanical properties of these hybrid FsNM (elastic modulus of 30-40 GPa, ultimate strain of about 2% and ultimate tensile strength higher than 100 MPa) were found to surpass those reported for a much thicker (i.e. submicrometer scale) nanoparticle-containing free-standing LbL films. 99 Moreover, the overall of the examined properties suggested a wide variety of prospective applications, 4 those related with the development of chemical and temperature micro-array sensors being particularly relevant in the biomedical field.
Metallic nanoparticles were also used to fabricate a superhydrophobic/hydrophilic asymmetric FsNM using the LbL approach on a Teflon substrate. 100 In this case, the layer assembly was achieved between a poly(ethyleneimine)-Ag + complex (PEI-Ag) at pH 9 and PAA at pH 3.2. The incorporated silver ions were reduced to silver nanoparticles during the thermal treatment applied to promote the cross-linking by imide bonds formation. As a result, silver loaded films displayed asymmetric wettability: the top surface was superhydrophobic, while the bottom one was hydrophilic. On the one hand, the superhydrophobic side limited the bacterial adhesion and exhibited self-cleaning properties; on the other, the hydrophilic side delivered bactericidal silver ions. These silver-functionalized hybrid films can be of great potential in open wound patches or in the barrier, separation, transportation or drug delivery field, although exceeding the nanoscale (thickness ranging between 2 and 20 µm, depending on the number of layers).
That aspect was overcome by Hammond and coworkers, 101 who designed contact-killing ionically cross-linked LbL ultra-thin films using N,N-dodecyl,methyl-polyethylenimine (DMLPEI), which has microbicide activity, layered with PAA (thickness < 100 nm for films with a number of bilayers ranging from 2 to 20). A pH treatment was applied during the assembly process to rearrange the microbicide polycation chains. Thus, at acid pH, the amount of positive charges on the surface available to interact with the bacterial cell membrane is higher than at neutral pH. Consistently, films as thin as 10 nm made at pH 3 were more lethal to both airborne and waterborne bacteria than films made at pH 7.
Baxamusa et al. 102 proposed the fabrication of FsNM without using any sacrificial layer for their release from the substrate. More specifically, these authors proposed the direct delamination of ultra-thin films made of poly(vinyl formal) (PVF) resin, PS or PMMA. Delamination of a thin film from its substrate spontaneously occurs when the strain energy (G v ) in the film exceeds the interfacial energy resisting separation (γ). In practice, this occurs when the film thickness (L) satisfies the following condition: 103 where υ f is the Poisson's ratio of the film, γ is the difference in interfacial energy between the laminated and delaminated state, E is the Young's modulus of the film, and ε is the strain mismatch between the film and the substrate. Delamination and capture on wire supports of extraordinarily thin and large polymeric thin films have been enhanced by decreasing the interfacial energy between the film and its deposition substrate through electrostatic adsorption of a cationic polyelectrolyte. By this procedure and using polydiallyldimethylammonium chloride (PDAC) as polyelectrolyte, the minimum delaminated film thickness for PVF, PS and PMMA was found to be 8, 12 and 15 nm, respectively. The characteristics of this methodology, which can be extrapolated to many types of polymers, make the fabrication of FsNM for biomedical applications a potential scalable process.
Lastly, extraordinarily thin FsNM (thickness < 5 nm) were obtained by spatially confined polymerization of a unique and elaborate 2D supramolecular system composed of two liquid-crystalline lamellar bilayer membranes made of self-assembled nonionic surfactant, dodecylglyceryl itaconate (DGI), which was sandwiched by a water layer ( Figure 12). 104 Nanosheets are achieved using a simple free-radical polymerization under UV radiation with high yield and in large quantity. The covalently bonded twomolecular-thick sheets exhibited a high mechanical strength and thermal stability.
Moreover, an important characteristic of these ultra-thin sheets is the high-density of functional groups exposed to the outer surfaces. Post-functionalization of the hydroxyl groups at the head of DGI located on the outer surfaces of these nanomembranes, opens the door to many practical applications in the biomedical field. intense interest within the biomedical field. [121][122][123][124][125][126] In this application, ECP act as interfaces between bio-substrates and inorganic electrodes favouring lowered impedance between the electrode and electrolyte interface. Accordingly, as ECP-based biosensors consist of organic films (or even hydrogels) supported on inorganic electrodes rather than in selfsupported organic membranes and, therefore, their discussion has not included in this review.

Biomedical Applications of Free-Standing Nanomembranes with Electroactive Conducting Polymers
Nevertheless, one of the major drawbacks to obtain free-standing nanosheets Although both forms of PAni did not induce any sensitization and skin irritation, the two materials but specially the PAni hydrochloride exhibited considerable cytotoxicity.
Polymer purification via re-protonation ↔ de-protonation cycles led to a significant reduction in cytotoxicity, suggesting that the negative effects were provoked by low molecular weight reaction residues or by oligomers rather than by the own PAni molecules. Subcutaneous implantation of PAni emeraldine base films into animal models showed inflammation response and fibrous encapsulation. 128,129 In opposition, PPy exhibited good biocompatibility. Wang et al. 130  FsNM obtained using this procedure were composed of a unique ECP domain with a thickness of ∼21 nm, widths of 2-6 µm, and lengths greater than 10 µm (Figures 14b and   14c). The electrical conductivity of these nanosheets was determined to be 30.6 S/cm, thus one order of magnitude higher than the value displayed by spherical PPy nanoparticles (diameters 30-50 nm) prepared by emulsion polymerization (2.9 S/cm).
Besides, the feasibility of PPy FsNM to perform as HCl and NH 3 vapour detectors was evaluated: the system exhibited high sensitivity and a fast response with respect to PPy nanoparticles, which was attributed to the increased surface area and porosity of the former nanostructure. Despite the fact that the application studied for PPy FsNM was not biologically related, the methodology used is of relevant importance to successfully obtain FsNM.
More recently, Qi et al. 151  months, revealing a very noticeable stability in air. Although no specific application was examined, potential applications were mentioned (e.g. biosensors and artificial muscles).
On the other hand, the number of studies devoted to PAni-based nanosheets, which mainly consist of PAni/inorganic hybrid composites, is very scarce and their application in the biomedical field is practically inexistent. For example, very recently, it was reported the fabrication of layered PAni/graphene/PAni nanosheets (the thickness of PAni and graphene layers was of 3.7 and 8.9 nm, respectively), which exhibited excellent gravimetric capacitance. 153 This sandwiched structure was essentially oriented towards applications in energy storage devices, solar cells, semiconducting devices, etc. Niu et al. 154 used a "skeleton/skin" strategy for the preparation of free-standing, thin and flexible single-walled carbon nanotube (SWCNT)/PAni hybrid films by a simple in situ electrochemical polymerization method. In this approach, directly grown SWCNT films with a continuous reticulate structure acted as the template, whereas PAni layers acted as the skin. The resulting hybrid films displayed a much higher conductivity compared to that of SWCNT/PAni composite films based on the post-deposition of the SWCNT film.
Flexible, thin and lightweight supercapacitors were fabricated using SWCNT/PAni hybrid films. Although the applications of PAni-based nanomembranes 153,154 were not related with biomedicine, the above described properties may be useful for the fabrication of energy storage components for biomedical equipment.
Regarding to PTh and its derivatives, Greco et al. 155 reported the preparation of FsNM made of PEDOT and PSS complexes (PEDOT/PSS), where PSS acted as the dopant agent. In this study, the Supporting Layer method, which enables the release and recovering of the free-standing nanosheet, was used ( Figure 15). Firstly, a layer of watersoluble PVA is deposited as the sacrificial layer on a substrate (PDMS) by spin-coating.
Then, the desired nanosheet is supported on that sacrificial layer. Later, once the bilayered film is dried, it is peeled off from the substrate. The thickness of those FsNM, which was controlled through the rotation speed, ranged from ∼100 nm (rotation speed of PEDOT/PSS was also employed by Greco et al. 160 to prepare and characterize robust wrinkled conductive surfaces. This was achieved by following the simple two-step approach (metal deposition and subsequent heating) developed for the fabrication of nanowrinkles on shape-memory polymer sheets. 161 Specifically, the ECP nanosheet (thickness ranging from 53.6 ± 1.2 to 120.9 ± 1.5nm) was deposited onto a thermo- have been prepared by spin-coating. For this system, the remarkable influence of the film-air interface in the thermal properties was examined by comparing the response of nanosheets and bulk P3TMA powder. 163 Although P3TMA, which is soluble in tetrahydrofuran, chloroform and dimethylformamide, among a few others, does not form free-standing films, 164 the understanding of its properties is of great interest because, as it will be discussed in the next sub-section, P3TMA has been combined with conventional biodegradable polymers to fabricate different types of electroactive FsNM with biomedical applications. Interestingly, the glass transition temperature determined using microcantilevers coated with ultra-thin P3TMA films resulted 5.2 ºC higher than that obtained for bulk powder samples. Moreover, ultra-thin films showed nanospherical aggregates of small (∼40 nm) size, while powder bulk samples presented a micrometric granular morphology ( Figure 17).

Nanomembranes
As mentioned above, blending of ECPs with insulating polymers is the most commonly followed approach to overcome the poor mechanical integrity of organic semiconductors. Accordingly, free-standing membranes have been fabricated by solvent casting mixtures of ECP with conventional insulating polymers, such as PVA 144 and nylon 66. 165 However, in all cases, such membranes were of micrometric thickness and their potential use (e.g. optical pH sensors 144 and conductive coatings 165 ) was not related to the biomedical field, even though they were prepared by combining ECP with biopolymers derived from natural sources. For example, cellulose-PAni membranes of micrometric thickness, which were prepared by in situ polymerization of aniline in the presence of bacterial cellulose nanofibrils, were used as electromagnetic interference shielding materials despite the biological origin of the biopolymer. 166 Table 3  In a subsequent study, the same authors 170  Consistently, hydrolytic and enzymatic degradation increased with the P3TMA content (Figures 21a and 21b). Moreover, TPU-P3TMA blends behaved as biodegradable materials. Viability assays evidenced that, although all TPU-P3TMA compositions provided biocompatible blends, the viability of cells increased with the concentration of TPU in the composition (Figures 21c and 21d). The overall of the results allowed the authors to conclude that 40:60 TPU-P3TMA FsNM was the most appropriated system for tissue engineering applications.
A completely different strategy to obtain FsNM made of an ECP and an insulating polymer is the one in which both components are arranged in a bilayered configuration.
Greco et al. 172 fabricated self-supported nanosheets with patterned conductivity using PEDOT/PSS and PLA, which acted as the mechanical support layer, thus maintaining continuity and robustness. In a first step, the PEDOT/PSS layer (thickness of ∼45 nm) was spin-coated onto a substrate. Then, in a second step, and after thermal treatment, the PLA layer (thickness of ∼200 nm) was spin-coated onto the previous one. Nevertheless, n order to obtain a patterned bilayer FsNM, an intermediate step (inject patterning) was introduced just before the deposition of the PLA layer (i.e. localized over-oxidation of PEDOT/PSS nanofilm to provoke an irreversible loss of electrical conductivity at specific spots). Moreover, to enhance its electrical conductivity, which reached values of 180 S/cm, DMSO was added as a secondary doping agent. The resulting bilayered FsNM is of great interest as (bio)electrical interface and as thin floating or ultraconformable circuit.
In addition to that, the surface wettability of the bilayered FsNM was electrochemically switched through simple oxidation and reduction processes. This change was even more evident for nanosheets supported on a PS substrate. On the basis of this interesting ability, authors proposed the application of these FsNM as smart conductive biointerfaces for directing cell adhesion and differentiation. 172 A similar approach was followed by Pérez-Madrigal et al. 171 to prepare bilayered FsNM made of TPU-P3TMA and collagen. However, in this case, the role of the collagen layer was not to provide robustness, which was an intrinsic property of TPU-P3TMA FsNM, but to enhance the cellular response towards the TPU-P3TMA biointerface.
Therefore, the TPU-P3TMA/collagen bilayer was formed by incubating a spin-coated TPU-P3TMA (40:60 weight ratio) layer in a collagen solution. . Amazingly, the adsorbed collaged layer was found to form two layers ( Figure 22). The the top layer exhibited a pseudoregular honeycomb 2D network with cavities of different diameters (i.e. ranging from 194 ± 55 nm to 1.2 ± 0.7 µm) and depths ca. 73 nm, whereas, in contrast, collagen adopted a much more compact structure in the bottom layer. According to previous studies, 173,174 TPU-P3TMA/collagen biointerfaces were proposed as suitable scaffolds for biological and biomedical purposes. Similarly, a collagen layer was recently used to inhibit the cytotoxic effects of the remaining monomer leaking from a supported PTh 3 film, the resulting PTh 3 /collagen biointerface behaving as bioactive platforms. 132

Nanomembranes for Energy-Based Biomedical Applications
In the 21 st century, fuel cells have emerged as smart storage systems or alternatives energy conversion devices due to both their efficiency and the lack of pollutants emission in comparison with the coal combustion engines. 175  Electrochemically driven length variations in the film of ECP produce stress gradients across the two films interface and subsequently result in macroscopic bending, as is schematically illustrated in Figure 23. More specifically, for ECP exchanging anions with the electrolyte the film gives anticlockwise movement by oxidation (swelling) and clockwise during the reduction, while ECP exchanging cations produce clockwise movement by oxidation (shrinking) and anticlockwise by reduction of the ECP. 190 Artificial muscles based on ECP were recently reviewed by  Unfortunately, at present time all biomimetic artificial muscles based on ECP have been prepared using free-standing membranes of micrometric thickness, as occurred for fuel cell electrodes. However, the increasing evolution in biomimetic reactive devices has resulted in the opening of new technological forefronts and challenges, including the application of FsNM. 191

Conclusions and Perspectives
This review of FsNM for biomedical applications demonstrates the versatility of these simple nanostructures, in which molecules organize in a 2D configuration.
Moreover, it highlights several ideas: (i) the variety of techniques available for their preparation, (ii) the concept that the rational design of nanostructured materials can be utilized to obtain tailored properties, and (iii) the relationship between the features of the nanosheets and their application. Although the list of biomedical applications for selfsupported nanosheets is very extensive (e.g. wound dressing, patches for bone or tendon repair, scaffolds for regenerative medicine and tissue engineering, magnetically controllable bioactive platforms, drug delivery systems, platelet substitutes, devices for overlapping therapy, artificial soft tissues, biosensors, supports for biological samples, biointerfaces, coatings with microbicide properties, artificial muscles, microactuators for cell manipulation, bioelectrodes), investigation on such nanostructured organizations is still in its early stages, and many treasures still await scientific discovery. Consequently, the number of ECP studied so far, individually or combined with insulating polymers, is still limited and much more effort is necessary in this direction.

Research in
In Regarding to ECP-containing nanostructured materials, biomedical applications centred in electrophysiology is a field that needs to be further explored. One of the central goals of electrophysiology is to offer tools that can monitor and manipulate bioelectrical activities in the human brain. In particular, implantable neural prostheses aim to replace or restore lost motor functions after disease or disability. Since the brain has soft           Reproduced with permission. 98 Copyright 2013, American Chemical Society.

Advantages Disadvantages
Chemical -Large-scale production -Ease post-incorporation of other molecules to modify the ECP covalently -Many options to modify the chemical structure -The synthetic process is more complicated than the electrochemical one -Preparation of ultra-thin films becomes a difficult task Electrochemical -Ease of synthesis -Entrapment of molecules in polymer network becomes an easy process -The synthetic and doping processes occur simultaneously -Very useful to prepare ultra-thin films of controlled thickness -Removal of the films from the electrode surface is frequently a very difficult task.
-Post-covalent modification of ECP is quite complex or, even, impossible