Layer-by-Layer Assembly for Nanoarchitectonics

Chapter 1.4

Layer-by-Layer Assembly for Nanoarchitectonics A.C. Santos*,†, I. Pereira*,†, C. Ferreira*, F. Veiga*,†, R. Fakhrullin‡

*Department of Pharmaceutical Technology, Faculty of Pharmacy, University of Coimbra, Coimbra, Portugal, †REQUIMTE/LAQV, Department of Pharmaceutical Technology, Faculty of Pharmacy, University of Coimbra, Coimbra, Portugal, ‡Institute of Fundamental Medicine and Biology, Kazan Federal University, Kazan, Republic of Tatarstan, Russian Federation

1. Introduction Nanotechnology constitutes as an innovative solution to tackle a set of issues not previously expected to be solvable, providing an enormous range of opportunities for life sciences applications, and hence has gathered an increasing interest from the scientific community [1]. In this context, the manufacturing technique is a key aspect to successfully formulate promising functional nanomaterials for biomedical applications [2]. For macroscale and microscale materials, these structures follow the same fabrication principle. However, the extension and reduction to nanoscale dimensions via top-down techniques may be unreliable due to disturbances based upon statistical distribution, components’ mutual interactions, and thermal fluctuations [2, 3]. This issue was overcome by the recent concept of nanoarchitectonics, as proposed by Masakasu Aono, which sees the arrangement of nanomaterials as a whole system of mechanisms founded on the organization of building blocks rather than the combination of distinct processes [3]. Nanoarchitectonics enables the fabrication of functional nanomaterials with the coordination of diverse interactions capable of guiding the structure’s configuration. Furthermore, this concept supports the creation of new functionalities in accordance to interactions among single components and may establish a guide for novel structures design [3]. The concept of nanoarchitectonics encompasses several approaches, e.g., the deposition of a wide diversity of functional building blocks onto a substrate through layer-by-layer (LbL) technique [4–22]. LbL is based on the staggered deposition of generally two spontaneous interacting components, usually polyelectrolytes with opposite charges, upon a substrate with a washing step between each deposition, whereby each layer deposition step causes a charge inversion at the film’s surface (Fig. 1) [2, 22]. By this means, altering the charge density or Advanced Supramolecular Nanoarchitectonics. https://doi.org/10.1016/B978-0-12-813341-5.00005-X © 2019 Elsevier Inc. All rights reserved.

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FIG. 1  Sequential deposition of polycations (blue) and polyanions (red) onto a planar substrate with surface charge inversion by an immersive layer-by-layer assembly technique: (1) immersion of the planar substrate in a polyanion solution; (2) removal of loosely bound polyelectrolytes through a washing step; (3) Immersion of the substrate in a polycation solution; (4) removal of loosely bound polyelectrolytes through a washing step. Schematic presentation of: (A) planar substrate; (B) deposition of polyanion layer; (C) deposition of polycation layer; (D) multilayer film produced through layer-by-layer technique.

the number of coating layers are strategies capable of producing materials with different composition and surface charge. The use of planar substrates as substrates for LbL coating is based on a simple procedure, in which the substrate is immersed in a polymer solution followed by washing steps. On the other hand, particulate substrates require their dispersion in solution, pelleting, and centrifugation as the washing steps [23, 24]. Although the consecutive layers interact via ionic and electrostatic interactions, this is not a prerequisite. Recent studies have reported other successful interactions, such as covalent linkage (Fig. 2), hydrogen bonds, and biological-specific interactions [2, 25]. LbL technique is, thereby, a flexible technique incorporating several features, including the deposition methodology, the number of layers and film thickness, the nature of the interactions between layer components and the produced functional materials, offering support to achieve the desired functional properties or specific targeting, as well as control over drug release and diffusive properties [2, 22, 26]. The drug release may occur by the exposition to external conditions that affect the integrity of the film structure, namely by stimuli-response to changes in the temperature or in the pH value, natural biodegradation or illumination by light. Furthermore, LbL does not require long-lasting nor expensive procedures, thus gaining a great deal of interest in the research community [2]. The study of the LbL technique applied to microscale-sized particles was first carried out by Sukhorukov et al. by using two distinct approaches regarding differing concentrations of polyelectrolytes [26]. Firstly, it was conducted with the deposition of oversaturated solutions of polyelectrolytes followed by centrifugation-based washings to remove loosely bound polyelectrolytes, and secondly by using just a sufficient concentration to achieve saturation, designed as a washless approach. This study has shown the charge and concentration values of polyelectrolytes as critical features to ensure the successful layer growth. According to the results, the charge must be strong enough to avoid the partial removal of adsorbed layers upon the following depositions. Regarding the latter critical aspect, the concentration has to be sufficient to provide saturation

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FIG. 2  Transmission electron microscopy images of (A) silica nanoparticles, (B) silica nanoparticles used as substrate for layer-by-layer assembly of isotactic (it-) and syndiotactic (st-) poly(methyl methacrylate) (PMMA) stereocomplex films, (C) obtained it-PMMA/st-PMMA capsules and (D) obtained it-PMMA capsules by selective extraction of st-PMMA. The images are reproduced with permission from Kida, T., M. Mouri, K. Kondo, M. Akashi, Controlled release using a polymer stereocomplex capsule through the selective extraction and incorporation of one capsule shell component. Langmuir 28(43) (2012) 15378–15384, copyright American Chemical Society.

c­ onditions. Moreover, the researchers were able to prepare hollow polyelectrolyte capsules by dissolving the colloidal core, while maintaining the integrity of the polyelectrolyte coating film [26]. In order to be considered a successful drug delivery system for low ­water-soluble drugs, LbL assembly needs to improve the dissolution rate of its encapsulated drug, thus increasing the bioavailability. The specific surface area of microscale LbL particles is not sufficient to ensure a satisfactory dissolution rate for poor soluble materials, a drawback that led to the development of LbL nanoparticles, including stimuli-responsive and multicomponent nanoscale drug delivery systems. LbL nanoparticles can considerably enhance the efficacy of low water-soluble drugs by increasing their saturation solubility and, therefore, their dissolution and bioavailability. Moreover, LbL nanoparticles may be used via intravenous administration due to their enhanced surface interfacial tension and minor particle size, extending the scope of applications relatively to microscale particles [2, 27, 28]. The coating of both hydrophilic and low water-soluble drugs using LbL ­assembly has been successfully performed and several advantages of this technique have been reported [29, 30]. LbL assembly is expected to possess high loading capacity and durability on blood circulation, ensure the protection of

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the encapsulated material against hazard conditions, enable prolonged and controlled release, allow tuning the solubility according to the solubility properties of the outer layer, evidence selective permeability and provide specific targeting. Given the promising architectures of the LbL technique, a great deal of effort has been put forth in the development of LbL assembly with novel materials and procedures for several applications in a wide variety of scientific fields [2]. Throughout the past few decades, numerous published articles have described the use of LbL assembly with a wide range of substrates and layer materials, including organic or inorganic composites (Fig. 2), proteins, DNA, biomedical implants, colloids, and even biological cells [31]. Nowadays, LbL assembly also comprises innovative and unconventional assemblies such as inorganic-organic hybrid assembly, stereocomplexation, three-dimensional (3D) printing or bioprinting, additive and subtractive nanolithography, and multilevel/multicomponent assembly [24]. These outcomes support the exponential evolution of LbL assembly. The versatility of LbL assembly represents a strong advantage for the architecture of complex nanomaterials. Another interesting point is that LbL films on different substrates may have almost equal surface functionalities [31]. The ability to use such diverse substrates, some of which require different processing conditions, particularly biological materials, gave rise to the development of LbL assembly of several technologies that may be organized into five categories: immersive, spray, electromagnetic, spin, and fluidic assembly [32–37]. Furthermore, LbL assembly of nanomaterials progressively hinder the use of centrifugation to remove the excess polyelectrolytes, thus compelling the development of washless LbL methodologies [38]. From several studies, researchers realized that the chosen technique did not only influence the process properties, e.g., scalability or duration period, but also the physical and chemical characteristics of the formed LbL film, in terms of homogeneity, thickness, and layer organization. In spite of this progress, experts highlight the need for further research focused on the comparison of assembly techniques to better understand the best features of each technology and support the development of novel automated systems that facilitate the scale-up of LbL assembly [37]. Considering the wide spectrum of applications that LbL assembly presents, this chapter aims to cover a comprehensive discussion of different LbL nanoarchitectonics, particularly LbL assembled capsules, LbL coated gelatin nanostructures, LbL coated drug-core nanoparticles and LbL nanomaterials-based coated cells.

2.  Layer-by-Layer Nanoarchitectonics 2.1  Layer-by-Layer Assembled Capsules The principle of engineering LbL assembled capsules [26] stands generally on the deposition of a LbL capsule upon the core, followed by the core dissolution and expulsion from the interior of the intact multilayer film, resulting in hollow LbL capsules (Fig. 3). When preparing LbL assembled capsules one must pay

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FIG. 3  Fabrication of layer-by-layer assembled capsules using sacrificial cores: (1) substrate particle; (2) deposition of the first polyelectrolyte layer; (3) deposition of polyelectrolyte layer of opposing charge; (4) deposition of nanoparticles; (5) alternate deposition of negative and positive polyelectrolyte layers; and (6) core dissolution, while maintaining the integrity of the multilayer film.

careful attention to avoid critical aspects such as polydispersity, irregular shell coverage, and core solidification. The formed hollow capsules have been used to encapsulate molecules or nanoparticles of interest, confer protection, target specific sites, and enable controlled release (e.g., the reduced release rate of furosemide by 50–300 times) [9, 39]. In accordance with these relevant properties, several studies have shown good prospects regarding the use of LbL assembled capsules as drug delivery systems and nanoreactors. For instance, Shah et al. created a biocompatible and biomimetic environment for the stimulation of bone tissue growth and proliferation by forming a multilayer film composed by poly(acrylic acid) (PAA), as the negatively charged layer, and a complexation of chitosan and hydroxyapatite—an inorganic component of human hard tissue—as the positively charged layer [40]. This LbL assembled capsule was posteriorly capped with poly(β-amino ester), and it was used to encapsulate recombinant human bone morphogenic protein-2 (rhBMP-2)—an osteoinductive protein. In  vivo, poly(β-amino ester) is progressively degraded by hydrolytic means, thus releasing controlled quantities of rhBMP-2 over a period of time. Additionally, hydroxyapatite is dissolved, which causes the release of calcium and phosphate ions to the surrounding environment. More evidence of the immense potential of LbL assembled capsules is reflected in a study conducted by Kreft et al., in which they were able to produce a sensor system by encapsulating a pH-responsive fluophore—SNARF-1-dextran conjugate—which expresses red and green colorations in basic or acidic conditions, respectively [41]. This type of sensor may be used to assess intracellular pH variations, thus becoming a promising diagnostic tool for quite a few illnesses, especially for fighting cancer. In the use of LbL assembled capsules as nanoreactors, researchers were able to successfully perform reactions of different natures in the interior of these nanomaterials, such as chemical reactions by pH variation; reduction of silver through photocatalytic reaction; and biomimetic calcium carbonate synthesis via enzymatic reaction [42].

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These nanomaterials have gained a great deal of interest due to their versatility in terms of components, capsule particle size, thickness, and tunable permeability, making it possible to easily modulate drug release (Fig. 4) [42]. In 1998, Donath et  al. were able to control the thickness of each capsule by varying the number of film layers of poly(allylamine hydrochloride) (PAH) and poly(sodium styrenesulfonate) (PSS) [39]. Proceeding to the characterization of the formed LbL assembled capsules, the research group noticed that they presented distinct properties—permeability, stability, and selectivity—from ­liposomes and hollow core-shell particles, materials that are also used to encapsulate molecules of interest. The encapsulation of a wide variety of materials within LbL assembled capsules may be achieved by two main strategies: Encapsulation during the construction of the multilayer film; or encapsulation following the core dissolution, which foresees the possibility to tune the capsules permeability. The permeability of LbL assembled capsules suffers variations due to conformational changes of functional polymers in the multilayer film, but it may also be tuned by layer thickness [10, 43]. Déjugnat et al. studied the influence of pH variation on the

FIG.  4  (A) Schematic presentation of functional LbL assembled capsules; (B) dark field optical microscopy of a LbL assembled polyelectrolyte microcapsule; (C) atomic force microscopy (peak force tapping mode) demonstrating the morphology of LbL-assembled microcapsules; TEM images of poly(sodium styrenesulfate)/poly(diallyldimethyl ammonium chloride) capsules (D) before thermal treatment and (E) after thermal treatment, with loaded dextran. The images are reproduced from A.G. Skirtach, A.M. Javier, O. Kreft, Laser-induced release of encapsulated materials inside living cells, Angew. Chem. Int. Ed. 45 (28) (2006) 4612–4617, with permission from Wiley. Anisotropic magnetically modified LbL polyelectrolyte microcapsules assembled using calcium carbonate microcrystals as sacrificial cores: (F) spindle-shaped microcapsule templated on aragonite microcrystal; (G) two cubic-shaped LbL microcapsules templated on calcite microcrystals (note the half-dissolved calcite cores visible inside the magnetite-polyelectrolyte shells).

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permeability of LbL capsules in order to modulate the uptake and release of molecules of interest [44]. With this aim, the research group prepared hollow ­capsules composed by pH-responsive weak polyelectrolytes (PSS and PAH). At a demarcated pH grade, the charges of these polymers were found to be stable and well balanced, maintaining the capsules’ original structure. An increase in the pH value caused the imbalance of charges, triggering the higher flexibility of the capsule, the swelling and the enhancement of its permeability. Along the study, it was also demonstrated that this modification was reversible, as following the addition of the acidic solution, the LbL capsules shrunk to their original size, diminishing their permeability. The possibility of changing and reversing the permeability makes pH-responsive capsules a promising material for controlled release drug delivery systems [44]. Moreover, Mauser et al. also explored pH-responsive LbL assembled capsules using poly(4-­vinylpyridine) (P4VP) and poly(methacrylic acid) (PMA) and obtained similar results, in which the permeability rise led to the encapsulation of dextran [45]. In 2004, Gao et al. were able to encapsulate PSS/poly(diallyldimethyl ammonium chloride) (PDADMAC) capsules (Fig. 6) with dextran, a biomacromolecule, by using a swelling-­shrinking technique [46]. The core removal procedure at a low pH led to a swollen state of PSS/PDADMAC capsules, increasing their permeability and enabling the encapsulation of dextran. After incubation with a solution containing dextran, the capsules were rinsed with sodium chloride solution, causing the shrinking of the capsules, thus imprisoning the loaded dextran. Overall, this technique provided a greater loading efficiency, as the loaded amount of dextran in the capsules submitted to the shrinking procedure was almost 7 times higher than in the control capsules, which were not washed with the salt solution [46]. To better understand the influence of different salts in the permeability of LbL assembled capsules, Georgieva et al. incubated PSS/PAH capsules in solutions of carbonates, phosphates, and chlorides in a concentration range from 0.05 to 1.7 M [47]. It was shown that moderate concentrations of salt solutions were sufficient to influence the permeability of LbL assembled capsules, whereby carbonates have presented stronger effects [47]. The permeability can also be modified by variation of ionic forces, e.g., PSS/PAH capsules shifted from “close” to “open” conformations after the addition of ethanol to a water solution, enabling the encapsulation of urease [43]. It is important to note that in the latter case, the electrostatic interaction between the polyelectrolytes was weakened enough to cause changes in the film’s structure even though the capsule maintained its size [43]. The loading efficiency of macromolecules may be enhanced by photoinduced procedures, which reduce the LbL capsules’ permeability, thus entrapping the loaded material. It is possible to successfully perform this technique by using photoresponsive polymers, e.g., adding photoactive azobenzene and polyelectrolytes [48]. The thermal behavior of LbL assembled capsules was also studied, and results shown that higher temperatures lead to permeability reduction and entrapment of the loaded molecules (Fig. 7) [49]. The encapsulation strategies described above show a great potential for drug loading into LbL capsules. Nevertheless, it is

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i­ mportant to note that these techniques require the addition of specific substances and may possible cause structural changes due to osmotic shock. Several studies report the production of LbL assembled capsules using many materials, including natural and synthetic polyelectrolytes, inorganic and organic composites, and even DNA [23, 40, 50]. In particular, researchers have employed DNA as a polyanion in LbL assembled films, due to its anionic nature [51]. Petrov et al. successfully performed a different approach to encapsulate proteins into LbL assembled capsules by exploring the adsorption capacity of calcium carbonate particles [52]. The proteins were coprecipitated with calcium carbonate during its formation from the mixture of calcium chloride and sodium carbonate solutions. The produced particles were posteriorly used as substrates for LbL assembly. Following the construction of the LbL film, the core of calcium carbonate was dissolved, resulting in the protein release within the LbL assembled capsules. The authors highlight that this approach may be used for a wide variety of macromolecules of interest [52]. Each layer component provides different functionalities to the capsules, and it is possible to produce multifunctional materials by adding functional components in the construction of the multilayer film [53]. This property of LbL assembled capsules is a key aspect to perform triggered and controlled drug release [51]. Aiming to further develop LbL nanoarchitectonics, various researchers have performed studies exploring the influence of the added components on the initiation of drug release. These studies report stimulus of a diverse nature, such as ultrasound, electromagnetic fields, and laser radiation, among others [51, 54, 55]. As most of LbL assembled capsules are based on electrostatic interactions, the pH value is an obvious triggering agent to control drug release initiation and has been explored in numerous studies [41, 51]. These parameters are promising for intracellular drug delivery, given the difference between intracellular and extracellular pH values, 5.2 and 7.4, respectively [51]. Another strategy to perform intracellular drug delivery involves disulfide reduction, as it was explored by Haynie et al. [56] and Zelikin et al. [57], in separate studies. This technique stands on the principle of the intracellular having a more reductive environment compared to the extracellular media. Given this, disulfide-­stabilized LbL assembled capsules could be specifically disintegrated by disulfide reduction in the intracellular environment [56]. For remote activation of release, infrared laser radiation does not need invasive techniques to initiate release and has shown great potential. For example, Skirtach et al. were able to trigger release from capsules without jeopardizing the cell’s integrity and viability [58]. In order to tune the release initiation after a specific incubation time, De Geest et al. produced capsules consisting of a semipermeable membrane involving a degradable microgel core [59, 60]. Once the core begins dissolving, the capsules swell until the pressure exceeds the tensile strength, causing the capsules to explode and expel their content. Researchers noticed that by increasing the crosslink density of the microgel, the degradation rate of the core decreased, making it possible to control the time of the capsule explosion. Experts believe that this strategy may

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be further studied and applied in a unique vaccination shot rather than booster injections to create immunity, given that several LbL assembled capsules with different timepoints were exploded at various antigen pulses [51]. The mentioned examples demonstrate that different properties may be tailored to tune and/or trigger drug release, taking into consideration the intended timepoint or specific site of release. Furthermore, additional types of stimuli-responsive capsules have been exploited, e.g., glucose-responsive capsules, whose release is activated by the increase of glucose levels above critical levels [60, 61].

2.2  Layer-by-Layer Coated Gelatin Nanostructures Gelatin is a natural biopolymer originated by hydrolysis or by thermal or enzymatic degradation of structural animal collagen protein [62, 63]. Gelatin has a generally regarded as safe (GRAS) status and, for that reason, has a wide range of applications, especially in the field of targeted drug delivery. This is also due to its protein structure, which shows a variety of different accessible functional groups that can be modified with targeting ligands [63]. Gelatin’s affordable price combined with its biodegradable and biocompatible nature strengthens the use of this biopolymer as a primary excipient in the production of new technological nanostructured materials, such as gelatin nanoparticles, so-called nanospheres, characterized by the soft gel-like interior and can be widely applied in drug and gene delivery [62, 63]. The LbL assembly technique was employed by Shutuva et al. [20] to modify the surface of gelatin nanoparticles with the purpose of modulating the cellular uptake and increasing the colloidal stability and the release period of natural polyphenols with known anticancer properties. Gelatin type A (GelA) and gelatin type B (GelB) nanoparticles were prepared by a modified two-step dissolving technique. A LbL shell with 20 nm of thickness starting with synthetic polymers (negatively charged) and finalized with a natural polymer layer was assembled on the positively charge surface of the gelatin nanoparticles. The multilayered gelatin nanoparticles shell was composed of the following polyelectrolytes: PSS/PAH, polyglutamic acid/poly-l-lysine (PGA/PLL), dextran sulfate/protamine sulfate (DexS/ProtS) and carboxymethyl cellulose/Gel A (CMC/GelA). It was shown that the polyelectrolyte multilayer does not cause significant variations in the particle size, which remained ca. 200 nm, or in the shape of the epigallocatechin gallate (EGCG), tannic acid, curcumin, and theaflavin-adsorbed gelatin nanoparticles. Nanoparticles prepared with GelA showed a higher polyphenol loading capacity in comparison to GelB nanoparticles, due to different amino acid contents that can modify the hydrophobicity of the nanoparticles and affect polyphenol adsorption. The encapsulation values are higher for high molecular weight polyphenols, which show a substantial number of phenolic groups. Therefore, theaflavin, that holds a high molecular weight, evidenced an encapsulation efficiency value near to 70%, while the encapsulation values for the other tested polyphenols were found to be below 30%. Nevertheless, the

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adsorption of natural polyphenols in gelatin nanoparticles surrounded by a LbL shell retained the antioxidant properties of these phytochemicals, as proved by the ABTS cation radical assay. Moreover, it was confirmed that when encased in the gelatin nanoparticles, particularly, EGCG interacts with the hepatocyte growth factor (HGF)-induced intracellular signaling in the breast cancer cell line MBA-MD-231 maintaining the same anticancer properties as when administered alone. Thus, polyphenol-loaded gelatin nanoparticles coated with a LbL shell have a strong potential for cancer therapy use. Recently, cadmium selenide (CdSe) quantum dots (QDs), semiconductor and photoluminescent (1–10 nm) nanoparticles were assembled by the LbL technique in the surface of gelatin nanoparticles to increase QD photostability and biocompatibility [65]. These gelatin nanoparticles were developed by desolvation and crosslinking and, thereupon, in the positively charged surface of the gelatin nanospheres, a LbL shell composed of a polyanion, PSS, and a polycation, polydiallyldimethyl ammonium chloride (PDAC) was assembled. The surface of the gelatin nanoparticles was coated with a first layer of PSS, followed by PDAC and, lastly, by QDs, with the PSS/PDAC/QDs coating cycle repeated four times to achieve four layers of QDs. The thickness of the LbL shell was 20±5 nm. This work was focused on the protective functions of the LbL shell to avoid the proton-induced etching, which reduces the semiconductivity, photoluminescence, and lifetime of QDs when subjected to acidic pH. The QD photoquenching in acidic environments limits their application as imaging agents of biological cells that survive in low pH value such as endosomes, lysosomes and, specially, cancer cells. The core-shell QDs-gelatin nanoparticles (0.4 mg/mL) were incubated in the NIH/3T3 mouse fibroblast cell line for 24 h and, by using fluorescent confocal microscopy, it was observed that the large diameter (480±40 nm) of the QDs-gelatin nanoparticles impairs the internalization of the particles by passive endocytosis, meaning that those nanostructures might only be internalized by phagocytic cells (e.g., macrophages). Nevertheless, the last layer composed of PSS promotes cell adhesion and may maintain the nanoparticles in in vivo circulation for longer periods. These QDs-gelatin nanoparticles assembled by the LbL technique are biocompatible, therefore, nontoxic to normal mice fibroblast cells (below 5 mg/mL) and could be applied not only as fluorescent imaging agents, but also as drug or gene delivery systems in acidic environments or microenvironments, such as, neoplasms, constituting a potential therapeutic solution for cancer. Gelatin is a versatile material and recent work underlines the applications of this natural material combined with the LbL assembling technique in tissue engineering and regenerative medicine [66]. In this study, a freestanding nanomembrane, using solidified gelatin as a substrate, was produced. The LbL technique was applied to the production of a biomimetic material, an ultrathin 3D chitosan-alginate nanomembrane intended to mimic the extracellular matrix. Chitosan and alginate were the biopolymers chosen to construct the nanomembranes, due to their inherent biocompatibility and noncytotoxicity demonstrated

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in previous studies. In water, chitosan is a polycation and alginate is a ­polyanion, creating the required conditions for the application of the LbL assembly technique. Briefly, first the gelatin substrate was coated with a chitosan layer followed by alginate, with this chitosan/alginate bilayer deposition repeated 7 times. In order to favor cell adhesion on the surface of the membrane, one more layer of chitosan was assembled (a total of 15 layers, or 7.5 bilayers, assigned as: (chitosan/alginate)7-chitosan). Thereupon, these gelatin gels were moved into a medium at a temperature of 37°C, which triggered their melting and the freestanding chitosan/alginate nanomembrane with ca. 3750–7500 nm was released. The produced membrane was used to seed bone marrow stem cells (BMSCs) and the in vitro findings indicated that the nanomembranes promoted BMSCs adhesion and proliferation. The biocompatibility of the nanomembrane was also tested in  vivo with mice, and it was proved that the nanofilm may accelerate cell migration to the mice skin burn wounds, enhancing the survival of the transplanted cells and reducing the time of re-epidermalization. The nanofabrication process of the chitosan/alginate nanomembranes is, thereby, found to be simple, affordable, and might be applied to promote the process of wound healing or to facilitate stem cell delivery. This study reports and emphasizes an innovative approach to combine the use of gelatin and the LbL assembly technology and extends the applications of this association to additional research fields.

2.3  Layer-by-Layer Coated Drug-Core Nanoparticles Another interesting and promising application of LbL consists in the design of multilayered nanoshells through the LbL assembly upon pure low soluble drugbased nanocores. In fact, low soluble drugs evidence restricted bioavailability fundamentally due to their low solubility in water, which strongly impairs an effective drug delivery and their clinical application [67]. This limitation is surpassed by encapsulation using nanoparticles, particularly with a particle size below 200 nm, a dimension that allows for higher accumulation at the required tumoral targets, owing to an enhanced permeation and retention effect [68]. Additionally, LbL assembly is simply implemented by the use of multilayer films endowed with targeted molecules or ligands directed to particular cell surface receptors [69]. The production of LbL-coated drug nanosuspensions is initiated by the formation of well-dispersed template drug-based nanocores, which is followed by the assembly of the LbL nanoarchitecture at the surface of the previous by the establishment of electroestatic interactions. The intrinsic surface charge of the selected drug plays a key role in the initial production of drug nanocores. In fact, some low soluble drugs present enough intrinsic surface charge to be able to confer the deposition of the first polyelectrolyte coating layer, which is accomplished during or shortly after the first size-reduction stage. However, in the case of drugs with insufficient surface intrinsic charge, ionic stabilizers are frequently applied to supply surface charge, establishing strong electrostatic

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i­ nteractions with a pure drug nanoparticles surface. The following stage consists in the LbL coating process, which is carried out progressively upon the surface of drug nanocores, through the alternated deposition of positively charged polyelectrolytes (polycations) and negatively charged polyelectrolytes (polyanions) up to the nominated number of polyelectrolyte layers [70]. In order to obtain LbL self-assembly nanoparticles, two generic experimental manufacturing approaches exist to obtain a high level of drug d­ ispersion capable of producing initial drug nanocores, precisely the top-down and ­bottom-up approaches. Top-down approaches concern particle size drug reduction procedures to the nano range, enabling the direct drug nanoencapsulation, comprising sonication-assisted disintegration [70] and wet media milling [71] of a coarse drug material in an aqueous or nonaqueous liquid. Bottom-up approaches consist of the drug nanoprecipitation by the addition of a cosolvent [38, 70, 72], drug nanoprecipitation by pH change [73], solvent evaporation/ emulsification [74, 75], and spray drying [76]. Differences exist when comparing both of the previous approaches, regarding fundamentally the features of formed drug nanocores, which strongly vary according to the selected approach. Top-down approaches do not use harsh solvents, although a strong energy input is imposed and a significant heat is generated during the process, hindering the application of thermolabile materials [77]. Such approaches have shown to be more efficient in the production of ca. 200 nm-sized nanoparticles, contrasting with bottom-up approaches, which are more effective in the obtainment of ca. 100 nm-sized nanoparticles. However, bottom-up approaches produce lower product yields, and require the use of organic solvents, which have environmental and safety issues [70, 78, 79]. On the basis of the foregoing considerations, a thorough selection and optimization of the drug nanocores preparation approach should be carried out towards obtaining the optimal characteristics of the nanoparticles, particularly regarding the particle size, the surface charge, and the colloidal and chemical inherent stabilities. After the formation of low soluble drug nanocores takes place, the application of the LbL assembly is aimed at the preparation of LbL-coated drugnanocore-based nanoparticles. The LbL assembly consists in the alternated deposition of two or more polyelectrolytes upon the surface of the substrate, the drug nanocores [80]. Each polyelectrolyte layer involves the adsorption of a nonstoichiometric excess of polyelectrolyte charge in relation to the previous assembled layer of the LbL nanoarchitecture, which allows the formation of a receptive surface for the following layer deposition [81, 82]. This process should be carried out in conditions capable of assuring both the lowest solubility and the highest stability of the encapsulating drug, in order to maximize the encapsulation efficiency [72]. Additionally, the required number of polyelectrolyte pair bilayers depends on the purpose of the nanoparticles application, however two or three polyelectrolyte coating bilayers are considered enough to provide good colloidal stability. Further number of bilayers are required for particular advanced characteristics, for example, using targeting processes.

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Traditional LbL assembly on colloidal dispersion makes use of intermediate polyelectrolyte washings or purification steps, specifically by the use of centrifugation and filtration procedures in order to remove the polyelectrolytes excesses between sequential layers adsorption. Pharmaceutical technology formulation progress has led the transition of drug formulations based on microparticles to nanoparticles, which impose greater demands for the separation of nanoparticles, namely longer periods and speed of centrifugation. In c­ onsequence of these boundaries, the washless LbL process was introduced as an alternative for centrifugation and filtration processes, significantly reducing the duration of the LbL assembly [38, 70, 78]. This way, the LbL assembly process may be optimized for the LbL thickness, morphology, stability, and permeation of the LbL films, among other features [80]. This technology holds remarkable advantages compared to conventional coating procedures, including: the ease of the process and equipment; the coating adequacy for the majority of surfaces, comprising nanoparticles; the flexible application to irregularly shaped and -sized surfaces; the achievement of stable LbL films; and the control over the optimized and required LbL film thickness. This innovative LbL technology upon the surface of nanocores introduces the distinct pharmaceutical technological gain of modulation of the encapsulated drug release pattern. With respect to the oral route, it is intended to retain drug integrity, assure colloidal stability, and decrease the drug dissolution rate under low pH stomach conditions [70, 83], particularly for drugs with associated gastric secondary effects [73, 84, 85]. A controlled drug release pattern achieved by the use of the LbL coatings at the surface could control the drug systemic absorption, and consequently retain the drug therapeutic concentrations for long periods. In the same way, the LbL coatings may be applied to control the drug dissolution and absorption regarding additional administration routes, e.g., the pulmonary route, together with the capability of locally drug release, e.g., for topical administration. These attributes assume particular relevance in the case of anticancer drugs intended for the intravenous route, as the extension of the circulation bloodstream period constitutes a condition for the drug passive accumulation in tumors [86, 87]. Diverse investigations have been carried out towards this objective by using chemotherapeutics, as paclitaxel and camptothencin nanocores coated with LbL films, which required PEGylation to avoid opsonization and clearance by the mononuclear phagocyte system (MPS) [38, 72, 88–90]. LbL-coated nanoparticles evidence a diffusion-controlled drug release mechanism, which is constituted by two stages: An initial diffusion of solvent molecules across the LbL film, followed by the dissolution and diffusion of drug molecules to the exterior of the LbL film. The drug release rate is influenced by the LbL film permeability, which is, in turn, dictated by the LbL film thickness and by the particle size and concentration of the pores formed between the multilayers [30, 91]. The LbL film thickness, the particle size, and the concentration of the pores may be controlled by modifying the natural permeability of the LbL

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film, by changing the type and the number of assembled polyelectrolyte layers and the assembly media [70, 92]; as well as by modifying the permeability of the LbL film by stimuli application, which consists in the change of medium after the LbL coating process [93]. The application of the LbL technology at the surface of drug nanocores promotes the enhancement of the chemical stability and the colloidal stability of those nanosuspensions. In terms of chemical stability, the presence of the LbL film provides a protection for enzymatic and chemical degradations. For instance, the LbL coating of camptothecin nanocores avoided the neutral and alkaline hydrolysis of the active lactone form into the inactive carboxylate form, thus maintaining the chemical stability of the encapsulated camptothecin for a long period. In one study, the camptothecin lactone form was preserved at pH 7.4 by the encapsulation into LbL nanoparticles, enabling a triple action of the encapsulated camptothecin in CRL2303 glioblastoma cells [39, 72]. In addition, the LbL coating technology promotes the enhancement of the colloidal or physical stability of the nanosuspensions, as a consequence of the LbL-charged layers’ presence at the surface of low-soluble drug nanocores [78, 94, 95]. This occurs by avoiding the nanoparticle growth conferred by electrostatic stabilization, and by offering resistance for dissolution, antagonizing respectively, the aggregation and Ostwald ripening processes [72, 74]. For example, LbL nanoparticles of paclitaxel, furosemide, and isoxyl were stable for an extensive period of 120 days, in contrast to free-drug forms which evidenced a particle size enhancement into the microscale after 5 days [74]. In addition, the use of PEG in the external layer or in the inner layers of the LbL film was shown to enhance the colloidal stability through steric repulsion, which assumes particular interest for the nanoparticles stabilization in physiologically relevant media [71, 72]. The LbL film architectures, without considering the encapsulated drugs, showed to be noncytotoxic and additionally to be independent of the film thickness in a large range of concentrations [70, 71, 73, 84]. In an investigation carried out to evaluate the cytotoxicity of polyelectrolyte PAH evidenced a higher toxicity than the polyanion poly(acrylic acid) (PAA) on rat smooth muscle A7r5 and human osteosarcoma U-2 OS cells. Interestingly, besides the toxicity of both polyelectrolytes, their combined use in the construction of LbL films eliminated their isolated toxic effects. This occurred due to the establishment of stable polyvalent interpolyelectrolyte interactions, capable of hindering additional phenomena as cell uptake or interactions with membranes [96]. In addition, LbL nanoparticles have shown to not exert hemolytic activity in contact with red blood cells [71, 97], suggesting they may be safe nanostructures. Additional relevant capabilities are efficiently introduced by these structures, and the functionalization of the LbL films enabled very promising results regarding targeting capacities to specific tissues, especially to tumors. Targeted tumor-specific mAb 2C5-LbL tamoxifen and paclitaxel-loaded LbL nanoparticles induced greater cytotoxicity values in human epithelial breast cancer

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MCF-7 and BT-20 cancer cells in comparison to the nontargeted nanostructures [18]. In another study, camptothecin-loaded LbL nanoparticles caused necrosis contrary to the free camptothecin, clearly depicting the sustained drug release promoted by LbL nanoparticles, with a significantly superior inhibition effect in the rat brain glioblastoma cells growth [72]. Superior cytotoxicity effects were also reported in MCF-7 cells using docetaxel-loaded LbL nanoparticles in relation to the free drug, particularly by the use of positively charged nanoparticles, which were found to have an improved cellular uptake [97]. Other investigation in MCF-7 breast cancer cells supported the potential of the LbL formulations in the metastic breast cancer fighting after verifying the capacity of paclitaxelloaded LbL nanoparticles in blocking the MCF-7 cells at the G2/M phase [89]. Moreover, a reduced uptake of LbL-coated nanoparticles by phagocytic cells was demonstrated, which emphasizes the high potential of the in vivo application of these nanoparticles [98]. Superior LbL architectures are already reported in the literature, particularly by the design of multidrug- loading nanoparticles, which enable the drugs temporally colocalization in tumor cells with optimum synergy, reducing possible differences in the pharmacokinetics of both drugs and in the systemic toxicity. Recently, paclitaxel-loaded LbL nanoparticles with coencapsulation of lapanitib into the LbL film led to enhanced cell death of resistant ovary adenocarcinoma OVCAR-3 cells when compared to the exposition with the paclitaxel-loaded LbL nanoparticles [99]. Lastly, to the best of our knowledge, just one in vivo study exists regarding the application of LbL-coated low soluble drug nanocores. This study implies the intravenous administration of PEGylated LbL-coated paclitaxel nanoparticles in subcutaneous HT-29 tumor xenografts-bearing mice, whose results demonstrated a rapid clearance with an accumulation in the mononuclear phagocyte system organs. Such output was explained by in vivo destabilization processes, particularly by the shedding of the PEGylated LbL film from the surface of ­paclitaxel nanocores exerted by the serum components. The authors emphasize the need for application of superior interaction forces, by means of, for example, covalent bonds, rather than singly electrostatic interactions. An additional stronger interactions interplay would confer superior stealth properties with adapted characteristics for in vivo administration [71].

2.4  Layer-by-Layer Assembly for Cell Surface Engineering Cell surface engineering is a recent area of scientific interest that aims to upgrade prokaryotic and eukaryotic cells. The modification of a cell surface has the intention of improve the health status of the cell and aims to functionalize it according to the specifications of the final application. Therefore, cell surface engineering has the potential to enhance the cell nutrition pathways and to confer protection against several scenarios, such as drastic alterations in cell chemical microenvironment (e.g., pH alteration); mechanical attack (e.g., phagocytosis),

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and radiation, particularly ultraviolet (UV) radiation [100]. Furthermore, cell surface engineering might provide the cell with new interesting functionalities, such as increasing mechanical robustness or conferring magnetic and/or electrical properties [100], with upgraded cells being designated as “cyborg cells” [101–107]. In the past few decades, cell encapsulation in hydrogels was the standard technique [108]. The cells were entangled in 3D networks of hydrated ­polymeric crosslinked materials. Nowadays, new advantageous techniques for coating biological cells are used. In this section we focus on the cell surface modification by the LbL technique, which is safe and does not require cell machinery or genetic material manipulation [100]. Nevertheless, other approaches, such as the bioinspired silicification of cells, can be employed. Recent studies report the encapsulation of yeast cells [109] and human cervix carcinoma cells (HeLa) and other mammalian cells [110] in silica shells in order to confer cell resistance against external stress factors. Living cells evidence a series of proficient features that enable their use as LbL templates and will be subsequently described: first, cells have a size in the micrometer range and exhibit behavior similarities with colloid microparticles; cells have a wide range of different sizes and shapes; they grow easily and in large numbers without associated expensive costs; they might be simply destroyed using chemical or enzymatic treatments; and, lastly, many cells have naturally negative surface charges, which benefit the sequential deposition of polyelectrolytes [111]. Furthermore, it is relevant to highlight that the LbL cell coating has no impact on the cell cycle, i.e., the division occurs normally and the offspring cells exhibit a free surface, reducing the general impact of the deposition of incompatible and possible harmful polyelectrolytes and nanomaterials [112]. In fact, the fabrication of flexible nano-based shells that endure cell division is still a challenge [108]. Cell coating using the LbL technique via electrostatic interactions or by hydrogen bonds can be applied directly or indirectly under specific physiological conditions (e.g., temperature, pH, and/or ionic strength) [108]. In the direct technique, the polyelectrolytes multilayer is assembled in the surface of individual or aggregated cells, while in the indirect technique individual or aggregated cells are encapsulated in combination with an inorganic biomaterial (e.g., hydrogels) in a liquefied or nonliquefied state, which serves as a substrate that enables the incorporation of cell adhesion substrates, such as growth factors. The LbL technique has been widely used in the surface functionalization of isolated microorganisms, such as bacteria [113] and yeasts [114], as well as in the functionalization of isolated mammalian cells, specifically, erythrocytes [115], normal and cancerous human or animal (mainly murine) cell lines [116], and stem cells [117]. Moreover, several reports in the literature describe the use of the LbL technique in the coating of cellular aggregates [118, 119] and multicellular species [120].

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Furthermore, cells, mainly bacteria and erythrocytes, have been used as templates for polymeric multilayer hollow microcapsules assembled by the LbL technique. Thereafter, the deposition of the polyelectrolyte layers in the cellular templates might be carried out by different techniques according to the cells that are used [121]. Hollow polyelectrolyte multilayer microcapsules were obtained using porous cell-containing alginate and calcium carbonate cores. After the LbL deposition of PAH and PSS in a calcium carbonate core containing ­individual green fluorescent protein (GFP) expressing Escherichia coli, the core composed of calcium carbonate was dissolved with ethylenediaminetetraacetic acid (EDTA) and hollow microcapsules were produced [122]. The authors reported that the E. coli-containing hollow capsules enhanced the bacterial growth cycle. Moreover, human cells, particularly erythrocytes, were also exploited in the fabrication of hollow capsules [123]. In this study, a multilayer of PAH and PSS was deposited in human erythrocytes, prior fixation with glutaraldehyde; afterwards it was applied a treatment with a deproteinizing agent in order to promote the solubilization of the cytoplasmatic constituents of the cell. Furthermore, chitosan/alginate hollow capsules were produced to encapsulate murine fibroblast cells (L929 cells) [124]. Poly(l-lactic acid) microparticles incorporated in the hollow capsules created cell adhesion sites, which, in association with the LbL shell, sustained the adhesion and proliferation of these anchorage-dependent cells. Notwithstanding, depending on the type of biological cell, all the hollow capsules designed from different living biological cells have the advantage to protect the interior from external influences and maintain two main cellular features, the particle size and shape, which are important in systemic drug delivery, i.e., by simplifying the carrier circulation through small capillaries, thus allowing a faster action on the target [108, 111]. A wide range of different polymers might be used in the production of a hollow capsule. After the production of hollow capsules through the LbL technique, researchers focused their attention essentially in the application of this technique to encapsulate living cells, preserving its viability and functionalities. Therefore, it becomes crucial to use biocompatible polymers that are permeable to oxygen and nutrients, which are essential for cell maintenance. Additionally, the polymer multilayers need to allow the passage of cellular metabolism byproducts [108]. The electrostatic assembly of polyelectrolytes, mainly polycations, in living cells is linked with toxic effects. In that sense, hydrogen-bonded assembly of uncharged polymer pairs such as poly(N-vinylpyrrolidone)/tannic acid may surpass the toxic effects of LbL deposition in the surface of living cells, particularly in yeast cells [125].

2.4.1  Layer-by-Layer Functionalization of Microorganisms The simple structure, easy manipulation, and the high growth rate of microorganisms (bacteria, fungi, and virus) allied with their polyvalent application in the medical, biotechnological, and industrial field led the scientific community

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to use microbial cells as templates for LbL film assembly [108]. Over time, the application of LbL in microbial cell coating was extensively studied and several different microorganisms, as well as different polymers, were applied in different deposition cycles. The cell surface polyelectrolyte deposition allows not only to modify some specific cell properties, but also confers protection against aggressive environments, preserving the microbial cells and enabling a detailed study of the microorganism physiological processes [100]. Moreover, it is also possible to study the interaction between the encapsulated microbial cells with animal models and extrapolate the results to the interaction of these upgraded microorganisms with the human body. A recent line of research combines the LbL assembly technique in microbial living cells with nanoparticles. The generalized nanomaterials deposition technique is sketched in Fig. 5A. Slightly negatively charged cells are typically first subjected to polycation solution, which forms a thin polycation nanofilm on microbial cell walls. Next, a second layer of negatively charged ­polyelectrolyte

FIG. 5  (A) A sketch demonstrating the polyelectrolyte-mediated assembly of multiwalled carbon nanotubes on live yeast cells. A similar approach can be used to immobilize virtually any nanomaterials on microbial cell walls; (B) transmission electron microscopy image of an individual yeast cell coated with PAH/PSS/PAH/carbon nanotubes/PAH/PSS; (C) transmission electron microscopy image demonstrating the monolayer of halloysite clay nanotubes secure on yeast cell with polyelectrolyte multilayers (PAH/HNTs/PAH/PSS); (D) transmission electron microscopy image of yeast cells coated with magnetic nanorods sandwiched between polyelectrolyte multilayers. Images (A) and (B) reproduced with permission from A. I. Zamaleeva, I. R. Sharipova, A. V. Porfireva, G. A. Evtugyn, R. F. Fakhrullin, Polyelectrolyte-mediated assembly of multiwalled carbon nanotubes on living yeast cells. Langmuir 26(4) (2010) 2671–2679, copyright American Chemical Society; image (C) reproduced from S. A. Konnova, I. R. Sharipova, T. A. Demina, Y. N. Osin, D. R. Yarullina, O. N. Ilinskaya, Y. M. Lvov, R. F. Fakhrullin, Biomimetic cell-mediated three-dimensional assembly of halloysite nanotubes. Chem. Commun. 49(39) (2013) 4208–4210, image (D) reproduced with permission from R. F. Fakhrullin, J. García-Alonso, V. N. Paunov, A direct technique for preparation of magnetically functionalised living yeast cells. Soft Matter 6(2) (2010) 391–397, copyright The Royal Society of Chemistry.

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is deposited, these cycles can be repeated several times. At some point, nanomaterials are sandwiched between the polymer shells. The deposition of a multilayer of PAH/PSS and bovine serum albumin/DNA (BSA/DNA) in combination with 20 nm citrate-stabilized gold and 45 nm silver nanoparticles in two species of fungi, Saccharomyces cerevisiae and Trichoderma asperellum was reported [126]. The polyelectrolyte layer protects the cell against the harmful effects that the direct deposition of nanoparticles causes to living cells. Later on, other types of nanomaterials, such as carbon nanotubes [127], halloysite clay nanotubes [112], boron nitride nanotubes [128], and magnetic nanorods [114] were used. The typical electron microscopy of yeast cells functionalised with LbL polyelectrolyte shells and nanomaterials are shown in Fig. 5B–D. The LbL assembly technique might also be used in the surface functionalization of entire living microorganisms with nanoparticles [111]. An example consists of the joint application of the LbL assembling with the deposition of gold nanoparticles with 20 nm diameter and iron oxide magnetic nanoparticles with 15 nm diameter in the surface of a nematode, Caenorhabditis elegans [120] (Fig. 6). The coating of the nematodes with the bilayer of PAH/PSS polyelectrolytes and the consequent nanoparticle deposition had no interference with the viability and reproduction of the microorganisms. Moreover, it has conferred

FIG.  6  (A) Fluorescence microscopy image of C. elegans nematode coated with (FITC-PAH/ PSS)5 multilayers; electron microscopy image of a cross-section of C. elegans nematode coated with PAH-stabilized magnetic nanoparticles (at lower (B) and higher (C) magnifications); (D) photograph demonstrating the concentration of the magnetic worms mixed with sand particles near the permanent magnet; (E) and (F) optical microscopy images of magnetically functionalized nematodes before and after magnetic separation from yeast cells. The images reproduced with permission from R. T. Minullina, Y. N. Osin, D. G. Ishmuchametova, R. F. Fakhrullin, Interfacing multicellular organisms with polyelectrolyte shells and nanoparticles: a Caenorhabtidis elegans study. Langmuir 27(12) (2011) 7708–7713, copyright American Chemical Society.

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interesting magnetic properties, which can be extended to other nematodes and may have potential imaging applications.

2.4.2  Layer-by-Layer Functionalization of Mammalian Cells The deposition of a nanocoating by the LbL technique might be able to restrain not only the passage of essential nutrients, but also the ion influx into the cell [101], destruct the cellular membranes or even interfere with enzymes, ­compromising the cell viability and the cell proliferation cycle [101, 108]. Moreover, the elastic and adhesive properties of the cell might be compromised by the polyelectrolyte coating. Therefore, the LbL assembled shell needs to act as a semipermeable membrane, which can be designed to control the molecular weight, type of interaction, number of deposited layers, and number of reactive groups per molecule [108]. In that sense, microbial cells and nematodes, due to the cell wall or cuticles features that protect the cell from mechanical or chemical stress, allied with the fact that they are autonomous systems, are more reactive to the polyelectrolyte shells than mammalian cells, so considerable physiological differences are at play when using the LbL technique in mammalian cells. The cellular membranes of mammalian cells, particularly human cells, are sensitive to the deposition of some types of polyelectrolytes, e.g., polyphosphoric acid, which can cause pore formation and subsequent cell death [108, 129]. The type of polyelectrolyte and the number of layers are predominantly responsible for the viability reduction of LbL-engineered mammalian cells. Thus, mammalian cells benefit from the use of natural biocompatible polyelectrolytes, such as polysaccharides (e.g., alginate), DNA, polyphenols, and poly(amino acids) such as PLL [108]. In a recent study, nonimmunogenic biocompatible polyelectrolytes were assembled using the LbL technique on the surface of erythrocytes with the purpose to mask the epitopes of the ABO/D (Rh) group, in order to prevent the immunity recognition and to create universally biocompatible erythrocytes to be hereafter applied in medical transfusions. First, an inner coating with four bilayers of c­hitosan-graft-phosphorylcholine and alginate were deposited, which was f­ ollowed by the assembly of two bilayers of alginate and PLL-graftpolyethylene, finishing the LbL coating with a negatively charged alginate layer, in order to prevent the agglomeration of the cells. This way, the immunocamouflage of the living red blood cells was successfully accomplished, demonstrating one of the various applications of the LbL technique [115]. Marine biocompatible polyelectrolytes, including alginate, chitosan, κ-carrageenan, ι-carrageenan, λ-carrageenan, and heparin, were used to produce nanocoatings. Those nanocoatings incorporated platelet lysate, which, in turn, is rich in growth factors that promote external cell adhesion, proliferation, and differentiation [130]. The polyelectrolyte platelet lysate-rich multilayer was assembled in human adipose derived stem cells (hASCs) using the LbL technique. hASCs cell adhesion and proliferation was promoted

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by the sulfated polysaccharide-platelet lysate multilayers demonstrating that the nanocoatings mimic the extracellular membrane. Moreover, in a recent work, platelet lysates were incorporated in the marine polysaccharides-based nanocoating of human umbilical vein endothelial cells (HUVECs) by the LbL deposition of sulfated polysaccharides. This process gave origin to stable tube-like structures, which were able to enhance the gene expression of proangiogenic factors, such as the vascular endothelial growth factor A (VEGF-A). These promising results ­emphasized once more the versatility of the LbL assembly technique. In fact, LbL offers the possibility to be combined with a cost-­effective pro-angiogenic solution to enable the formation of cell-based tubular structures in 2D/3D b­ iomaterials, which are particularly interesting for the tissue regeneration field [131]. In other research, researchers made use of LbL assembly to deposit cationic poly(dimethyldiallylammonium chloride) (PDDA), anionic PSS, 78 nm silica nanoparticles, 45 nm fluorescent nanospheres, and bovine immunoglobulin G (IgG) upon human platelets (ca. 2–3 μm). The human platelets circulate in the blood vessels and the functionalization of these cells with nanoparticles capable of incorporating drugs in association with an IgG might allow a targeted drug delivery application [6]. The enhancement of multicellular aggregates functions by the LbL deposition of polyelectrolytes has unexploited potential which only recently began to be studied [101]. In fact, it was demonstrated that it is possible to encapsulate human-isolated multicellular structures such as Langerhans islets (located in the pancreas) [132]. A polyelectrolyte multilayer composed of PAH/PSS, PDDA/ PSS, PLL/PEG, and AL was assembled on the surface of the Langerhans islets in order to avoid the immune response after the transplantation of these cellular aggregates. Promising results were obtained regarding the use of engineered cells in the treatment of type I diabetes. The transplant of Langerhans islets compared to pancreas transplantation is a slightly invasive process with a reduced morbidity risk, therefore, currently, it is considered an effective clinical option for the treatment of type I diabetes mellitus. Similar to these data, a more recent study applied the LbL technique in non-human primate Langerhans islets to reduce the immunogenicity and enable the use of these multicellular structures as an efficient treatment approach for type I diabetes. This is mainly due to the phylogenetic relationship with humans. The authors used three layers of SH-6-arm-PEG-NHS, 6-arm-PEG-catechol and linear PEG-SH to coat the multicellular islets. The combination of the engineering islets with an immunosuppressive drug protocol prolonged the survival rate in the mice xenografted model. The thin multilayered PEGylation immunocamouflaged the islets without affecting the viability and functionality [119]. The LbL technique is, in fact, a safer approach to deposit nanomaterials on the cell surface as nanomaterials are built up in each layer of polyelectrolytes without interfering with cell viability. In addition, the use of LbL to indirectly deposit nanomaterials exempts the ligand-receptor interactions, constituting

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an affordable technique [101]. It is important to highlight that, in scale, both microbial cells, microorganisms, and mammalian cells are positioned in the micrometer (10−6) range, therefore, they are not nanotemplates. However, as observed in the aforementioned cited studies, those can serve as substrates for the LbL technique, which may be associated with deposition of nanomaterials, particularly, nanoparticles, creating a new generation of multifunctional nano-­ assembled systems.

2.4.3  Alternative Techniques Based on the Deposition of Polyelectrolytes The deposition of nanomaterials, particularly nanoparticles, in cells presupposes, as previously mentioned, an indirect technique, particularly the assembly of a polyelectrolyte multilayer by the LbL technique. Nevertheless, the LbL polyelectrolyte deposition is a time-consuming technique due to the cycles of deposition and consequential washings of polyelectrolytes in excess [133]. Therefore, in order to avoid the loss of cellular viability and functionality, shortened procedures are required and, thus, other alternative approaches might be used to coat cells with nanomaterials. The indirect deposition of nanoparticles might be alternatively accomplished by the modification of the nanoparticles surface or by the pre-treatment of the cell wall (prokaryotic cells) or the cell membrane (eukaryotic cells) towards the enhancement of the affinity between the nanoparticles and the cell external face [101, 134]. For example, the electrostatic deposition of gold nanoparticles on the bacteria surface was possible due to the lysine nanoparticle coating. Lysine has high affinity to Bacillus cereus surface which favored the deposition of the nanoparticles [135]. The polycation PAH is frequently applied to stabilize nanoparticles in the surface of a wide range of different cells. For instance, in a recent study, a unicellular algae cell (Chlorella pyrenoidosa) was magnetized with a 90-nm nanolayer composed of PAH-stabilized magnetic nanoparticles with a diameter of 15 nm. These magnetic nanocoatings of the C. pyrenoidosa enabled spatial manipulation with a magnet, offering the possible application of these cells in biosensors [136]. Another example of the use of PAH as nanoparticle stabilizer consisted in the use of a LbL-based nanocoating of PAH-stabilized iron oxide magnetic nanoparticles of 18 nm in diameter in Acinetobacter bioreporters. The deposition of magnetic nanoparticles in this type of bacterial cells enabled remote control of the cells using a magnetic device. This way, the association of the polyelectrolyte with magnetic nanoparticles transforms these bacteria cells in microbiosensing robots that are able to give chemical or biological information regarding inaccessible complex environments, such as soils [137]. Furthermore, a new study performed the surface functionalization of a ­hydrocarbon-degrading bacteria Alcanivorax borkumensis, using a simple technique of deposition of magnetic nanoparticles complexed with PAH, by the formation of a magnetite coating of approximately 70–100 nm (Fig.  7).

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FIG.  7  (A) A scheme illustrating the LbL-mediated and direct approaches of cell surface ­engineering; (B) transmission electron microscopy image of A. borkumensis bacteria coated with polymer-stabilized magnetic nanoparticles; (C) atomic force microscopy nonspecific tip-sample adhesion map demonstrating the distribution of magnetic nanoparticles within the bacterial biofilm; (D) magnetically modified A. borkumensis cells acquire magnetic responsiveness: targeted movement and growth of magnetic cells on solid surface (inset shows a higher-magnification view of cells arranged on the surface). The images are reproduced with permission from S.A. Konnova, Y.M. Lvov, R.F. Fakhrullin, Nanoshell assembly for magnet-responsive oil degrading bacteria, Langmuir 32 (47) (2016) 12552–12558, copyright American Chemical Society.

This technique enabled the deposition of the nanoparticles in a single step without hindering bacterial cell proliferation. The PAH-stabilized magnetic nanoparticles allowed not only the magnetic transportation of the bacteria cells, but also the formation and discharged of the biosurfactant vesicles, which are produced by A. borkumensis to ensure the effective emulsification of oil in seawaters. In the near future, this engineered bacteria might be applied in biosensors to improve the decomposition of marine oil spills, asserting itself as an ecological solution for marine oil spills [138]. Recently, the use of different ­polycations to ­stabilize the direct deposition technique of 50 nm silver nanoparticles on the surface of Escherichia coli bacteria and Saccharomyces cerevisiae yeast was reported [133]. They used polyelectrolytes laid on PAH, PDDA, and

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poly(ethyleneimine) (PEI). This m ­ icrobial cell nanocoating assembly may be an alternative for the LbL followed by nanoparticle deposition, mainly because it is rapid and does not trigger significant viability alterations in the microbial cells. In human cells, PAH was used to directly deposit and stabilize superparamagnetic iron oxide nanoparticles onto the surface of HeLa cells without ­affecting cell viability [116]. The direct deposition of certain nanomaterials is also possible, as per an example given by Konnova et al. [139]. The absorbent properties of halloysite enabled the deposition of a 200–300 nm-thick tube magnetic nanocoating on the surface of Saccharomyces cerevisiae. The halloysite nanotubes are, therefore, a viable strategy to directly deposit magnetic nanoparticles for yeast magnetic manipulation, with the potential to be applied as adsorbents or biosensors. Nanoengineered microbial and mammalian cells have a vast range of applications in the fields of tissue engineering, drug delivery, biosensors, and those can be used as biosorbents, catalysts, or in cell-based therapies [101]. Particularly, in the tissue engineering and regenerative medicine, several research groups are focused on the use of nanocoated cells in the immunoprotection of transplants [119]. Furthermore, in tissue engineering, the use of cells with enhanced magnetic properties conferred by a nanoassembled shell enables the free structuring of the cells, which reduces the necessity of cell-supporting scaffolds and, thus, facilitates the production of artificial tissues with a morphology similar to natural tissues [1, 140]. Regarding drug delivery, an interesting progress has been carried out regarding cell-based drug delivery systems. Engineered cells might be used as biomimetic platforms to deliver specific drugs into target zones, specifically into tumor sites, owing to desired features as biocompatibility, prolonged circulation, and the determined half-life [141]. The incorporated drug in the LbL engineered cells may alter a specific metabolic or signaling pathway of the encapsulated cell, or it may interfere with the cellular metabolism of the external cells [108, 130]. The future of the drug delivery will certainly consist on the exploration of the association between these biological drug carriers and the LbL assembly technique.

3.  Conclusions and Future perspectives Nanoarchitectonics is a wide concept that includes the LbL methodology. In nanotechnology, LbL is applied in different specific systems with different applications. Throughout this chapter we have explored the wide range of reports in the area of LbL nanoarchitectonics. The principles and characteristics of LbL in the production of assembled capsules, coated gelatin nanostructures and coated drug-core nanoparticles were presented. Furthermore, a very specific topic of the use of the LbL association with nanomaterials to coat living cells was also discussed. In fact, the main strength of LbL-assembled capsules lies in their multifunctionality. Other attractive aspect of these structures is the large range of

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­ aterials that may be used. By varying the capsule’s components or the thickm ness of the multilayer film, it is possible to adjust either chemical or physical properties. Furthermore, the drug loading into the capsules does not require the use of mechanical stress or organic solvents, in contrast to some traditional drug delivery systems. From our perspective, the application of LbL-assembled capsules for intracellular drug delivery has shown great potential, although it needs further research. Up to the present, only a few in vitro studies concerning drug delivery have been performed. However, the majority of these studies have shown very positive outcomes, e.g., encapsulated cytotoxic compounds demonstrated to kill cultured tumor cells. The LbL application in the coating of cells is a quite recent line of research that holds promising perspectives. The future of the cell encapsulation will be the application of the engineered cells in cell-based therapy and cell-based drug delivery, as well as the use of new functional nanomaterials to enhance a specific property or a group of cell properties creating “cyborg” cells with a more productive living designed for a wide range of different applications. Nevertheless, it is still crucial to continue to study the toxicity of the cell nanomaterial-based shells and evaluate the risks and benefits. Finally, it is envisaged that an innovative field is rising, based on the integration of nanotechnology and biology that has shown evidence that the interface between nanomaterials and living cells has untapped potential.

Acknowledgment The work is performed according to the Russian Government Program of Competitive Growth of Kazan Federal University; funded by RFBR grant £18-34-20126 mol_a_ved and by the subsidy allocated to Kazan Federal University for the state assignment in scientific activities (#16.2822.2017/4.6).

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