Protein-based layer-by-layer films for biomedical applications

The surface engineering of biomaterials is crucial for their successful (bio)integration by the body, i.e. the colonization by the tissue-specific cell, and the prevention of fibrosis and/or bacterial colonization. Performed at room temperature in an aqueous medium, the layer-by-layer (LbL) coating method is based on the alternating deposition of macromolecules. Versatile and simple, this method allows the functionalization of surfaces with proteins, which play a crucial role in several biological mechanisms. Possessing intrinsic properties (cell adhesion, antibacterial, degradable, etc.), protein-based LbL films represent a powerful tool to control bacterial and mammalian cell fate. In this article, after a general introduction to the LbL technique, we will focus on protein-based LbL films addressing different biomedical issues/domains, such as bacterial infection, blood contacting surfaces, mammalian cell adhesion, drug and gene delivery, and bone and neural tissue engineering. We do not consider biosensing applications or electrochemical aspects using specific proteins such as enzymes.


Introduction
Biomaterials, such as implantable medical devices, are widely used in reconstructive and regenerative medicine.They are intended to be in contact with body uids and/or tissues.
Engineering the surface of such biomaterials is of vital importance for their successful (bio)integration by the body, i.e., colonization by the specic cell of the replaced tissue and/or preventing brosis and bacterial colonization.Among the surface functionalization methods, the layer-by-layer (LbL) method is simple to implement at room temperature using aqueous solutions and is potentially applicable in industry.Versatile, this method is based on the alternated deposition of oppositely charged polyelectrolytes allowing the development of biocompatible and bioactive nano or submicron lms.Since 1992 with

Muhammad Haseeb Iqbal
Muhammad Haseeb Iqbal is a postdoctoral researcher at Institute of Functional Interfaces, Karlsruhe Institute of Technology, Germany where his current research is related to protein nanoparticle-based drug delivery systems and chemical vapor deposition (CVD) polymerization.He received his PhD in Polymer Engineering (physical chemistry of polymers) from University of Strasbourg, France in 2021.During his PhD, he worked on layer-by-layer assembly of polyelectrolytes and proteins for biomedical applications.He is a recipient of "Best PhD award" in interdisciplinary category from the C'Nano, the French national competency Cluster in Nanoscience.He is co-author of 11 publications and 1 patent.

Halima Kerdjoudj
Halima Kerdjoudj is Professor in Cell Biology at Reims Champagne Ardenne University.She studies the immunomodulatory effect of biomaterials on mesenchymal stem cells and she is also developing perinatal tissuebased materials for bone tissue engineering.She received her PhD from Nancy University in 2007.During here PhD, she worked on layer-by-layer assembly of polyelectrolytes and vascular tissue engineering.In 2008, she worked as a post-doc with Prof. Alexander Seifalian at UCL (London) on progenitor cell differentiation and muscle tissue engineering.She is co-author of 83 publications and 2 patents.
the rst paper of Gero Decher using polyelectrolytes, 1 a tremendous interest has been shown with thousands of annual publications with a wide spectrum of applications in healthcare.A variety of (macro)molecules or nano/micromaterials (synthetic or natural polymers, nanostructures, proteins, or enzymes) have been used to develop LbL nanolms over time.Several reviews report on the physical chemistry and applications of synthetic and natural polymer-based LbL lms. 2,3Most of these works focus on the physical-chemistry aspects of LbL lms and their use in specic biomedical applications.
Proteins play a crucial role in several biological mechanisms, such as DNA replication, metabolic reactions, molecule transport, cellular adhesion, etc.The use of proteins in LbL assembly is limited, despite the growing potential, due to their structural complexities.Besides electrostatic interactions, hydrogen bonding and hydrophobic interactions play a crucial role in protein-based LbL.Frequent literature is available on enzymebased lms for biosensing applications 4,5 and only minor or scattered patches of highlights exist on non-enzyme proteins in general reviews.Regarding the physical chemistry of proteinbased lms, we recommend the review from Dupon-Gillain and co-workers, which gives a guide on the choice of polyelectrolytes and the building conditions that lead to their successful growth. 2LbL lms based solely on proteins are rare due to specic interactions limiting the possible combinations, such as bronectin with gelatin (Gel) or elastin (ELP), 6 or to their ampholyte nature limiting the construction pH as for gelatin. 7It can be noticed that type I collagen (COL)/bronectin (Fn) LbL lms were reported to show unsuccessful build-up despite using three different buffers and incorporation of an anchoring layer. 8COL, [9][10][11][12][13][14] Gel, 6,7,[15][16][17][18][19][20] Fn, 6,21,22 brinogen, 21 ELP, 6,23 laminin, 24 lysozyme (Ly), 25,26 casein, 26 and bovine albumin (BSA) 24,27,28 were mostly associated with synthetic polymers, polysaccharides, or tannic acid (TA, polyphenol).
In this review aer a general introduction to the LbL technique, we will mainly focus on the LbL lms developed using proteins, as at least one component, and addressing different biomedical applications: as antibacterial coatings, to promote mammalian cell adhesion, blood contacting surfaces, drug and gene delivery, bone tissue engineering, and neural tissue engineering.We are not considering the biosensing application or electrochemistry aspects using specic proteins such as enzymes.

Principle of layer-by-layer films
The LbL assembly was introduced in 1992 by Gero Decher using electrostatic interactions between oppositely charged polyelectrolytes, polyanion, and polycation, to obtain polyelectrolyte multilayer lms (Fig. 1a). 1,29Poly(styrene sulfonate)/ poly(allylamine hydrochloride) (PSS/PAH) LbL lms were deposited by the dipping method.Aer a silanization step to obtain a positively charged surface, the substrate was dipped in the polyanion (PSS) aqueous solution.Aer the rinsing step to remove weakly bound molecules, the surface became negatively charged by overcompensation of charges.With the adsorption of the polycation (PAH) layer, a positively charged surface was obtained by reversing the charge.This process can be repeated to obtain the desired lm thickness.Later, the process was done on raw substrate, usually negatively charged.PSS/PAH LbL lms Fig. 1 Layer-by-layer (LbL) method and different processes of buildup.(a) Schematic representation of the alternated deposition of polyanions and polycations, each deposition is followed by a rinsing step.(b) The build-up of LbL films can be achieved by various processes like dipping, spin coating, spray coating, and brushing.

Fouzia Boulmedais
Fouzia Boulmedais is CNRS senior researcher at Institut Charles Sadron (Strasbourg) where she is developing layer-bylayer lms as biomaterial coatings, particularly with antibacterial properties and electrodeposited polymeric lms for biosensing.She graduated from University Louis Pasteur (Strasbourg) where she received her PhD in chemistry and physical chemistry in 2003.In 2004, she worked as a post-doc with prof.Marcus Textor at ETH Zurich (Switzerland) and with Prof. Gleb Sukhorukhov at Max Planck Institute (Golm, Allemagne) on electrodissolution of polyelectrolytes multilayers.She is co-author of 130 publications and 4 patents.
The growth of PSS/PAH lms is linear, i.e., the thickness and the mass increment of the adsorbed pair of layers is constant whatever the number of adsorption cycles.The adsorbed polyelectrolytes interact only with the top outer layer of the lm forming stratied lms. 32Different parameters such as the pH, temperature, ionic strength, type of salt, and the characteristics of each polyelectrolyte strongly inuence the build-up of the lms.Thus, depending on the conditions of the build-up, the polyelectrolytes can adopt different conformations inuencing the growth of the LbL lms, i.e., the mass adsorbed and their thickness.At low ionic strength, they present a "at" or rigid rod-like conformation since the polyelectrolyte charges repel each other resulting in a thin deposited thickness.At high ionic strength, polyelectrolytes adopt a "loopy" conformation by charge screening resulting in a large deposited thickness.Strong polyelectrolytes are fully charged regardless of the pH, but the weak polyelectrolytes, mostly containing amine or carboxylic groups, are pH sensitive possessing a pK a .Below the pK a , weak polyanions are protonated with low density of charges and a at conformation.Above the pK a , the density of charges per chain increases leading to a loopy conformation.
Other types of LbL build-up were reported later.The superlinear growth (faster growth than linear) of LbL lms was attributed to an increase of the lm roughness along the whole buildup process. 33,34Exponentially growing lms were rst observed by Hubbel and co-workers. 35The lm thickness and mass increase exponentially with the number of adsorption cycles.Picart and co-workers explained the growth by the diffusion of at least one of the polyelectrolytes into the entire lm during each deposition step leading to a reservoir of polymer chains able to diffuse out and complex with the oppositely charged polymer chains at the next deposition step.7][38] Aer several deposition steps, exponentially growing lms enter a linear growth phase. 39,40his could be due to a restructuring of the lm that gradually prohibits the diffusion of polyelectrolytes throughout the entire lm and limits it to a constant lm thickness leading to linear growth.The growth regime of LbL lms is related to the interaction strength between the two polyelectrolytes.The build-up process of linearly growing lms is based on polyelectrolyte complexation both enthalpically and entropically driven.On the other hand, the complexation is entirely entropically driven in the case of exponentially growing lms. 41Protein-based LbL lms usually have linear growth and a few have exponential growth, especially when associated with a polysaccharide. 2

Other interactions
The hydrophobic interactions play a signicant role in the adsorption of proteins favouring their LbL build-up. 42,438][49] Apart from electrostatic interactions, hydrogen bonding is one of the most studied driving forces because it allows the insertion of uncharged molecules that can operate as hydrogen bonding donors and acceptors. 50Built at a specic pH, where polyelectrolytes are not charged, H-bonded LbL lms are highly sensitive to post-build-up treatment, such as pH and temperature but not the ionic strength.For example, poly(acrylic acid)/poly(4-vinyl pyridine) LbL lms disassemble above pH 6.9 due to ionization of carboxylic groups of poly(acrylic acid). 51Thanks to the pH responsiveness, H-bonded LbL lms or capsules with controlled degradation are extensively used for drug delivery systems. 52Thanks to specic lectincarbohydrate interaction, LbL microcapsules were fabricated from Concanavalin A, a plant lectin, and glycogen, a polysaccharide. 49Protein LbL lms composed of avidin and biotinlabelled antibody were also prepared using the high specic binding constant (K a : z10 15 M −1 ) between avidin and biotin.
5][56] Haemoglobinbased microcapsules were obtained by alternated deposition with a cross-linker, glutaraldehyde.The build-up was based on the Schiff base reaction between the amino sites of haemoglobin and aldehyde groups of the cross-linker. 53 More chemically and mechanically stable, covalent cross-linked lms are probably the most used lms in various applications like cell-surface interactions, drug delivery, surface patterning, electrooptical devices, catalytic substrates, antifriction etc. 57

Deposition processes
LbL lms have been developed using various methods namely, dipping, 1 spin-coating, 58 spray-coating, 59 and more recently the brushing 60 method (Fig. 1b).The dip coating method can be used on various substrates of practically any possible shape, with economical consumption of products i.e., the same solutions can be used for several deposition steps.It offers a wide selection of materials, from synthetic to biological molecules, which can be deposited, and ne control on the lm structure, thickness, and functionality.However, each deposition step requires prolonged time, generally between 5 to 20 min for each deposition or rinsing.
The problem with long deposition time was addressed by using spin-coating to develop LbL lms on planar surfaces.This method involves dispensing a droplet of polyelectrolyte solution in the centre of the surface and rotating the surface at a controlled speed.The rotating surface exerts a centrifugal force on the polyelectrolyte solution causing it to spread radially outwards on the surface.Under this motion, the excess material is ejected off the surface, leaving behind a uniformly deposited layer.The required time to deposit one layer is typically around 1 min which is extremely fast compared to the dip-coating.Generally, there is no rinsing step required in this method which allows a faster lm deposition.The rotation speed, viscosity or concentration, and polyelectrolyte type impact the lm's roughness and thickness.A high concentration of polyelectrolytes and low rotation speed led to thicker LbL lms. 61hough, spin-coating allows fast deposition time but is limited to a planar surface with reasonable size.
The spraying method was rst reported by Schlenoff et al. to develop PSS and poly(diallyl dimethyl ammonium chloride) LbL lms. 59Polyelectrolyte solutions and rinsing water were sprayed on the substrate held vertically to allow solution drainage under gravity.Like spin-coating, this is also a fast method that allows the coating of larger surface areas, but this process consumes a high amount of solution.
Recently, an attractive and simple method to develop LbL lms has been proposed by brushing.Unlike the dip-coating, this method is tremendously fast requiring only a few seconds of deposition time for one layer, and does not require specic and expensive material.The brushing process allowed to design of chitosan/alginate multilayer lms for drug delivery, 62 and collagen/tannic acid for myoblast differentiation into myotubes. 63The solution concentrations and the brush type were reported to impact the lm thickness and nanotopography.
In 2001, the rst protein-based LbL lm was reported using COL, a brillar protein mostly found in extracellular matrix, to promote cell adhesion on biomaterial surfaces. 64Since 2006, the LbL assembly of extracellular matrix proteins gained huge interest in developing multifunctional biomaterials despite various difficulties associated with their use. 65In the following, we will present the major biomedical issues and how the LbL lms addressed them (Scheme 1).

Antibacterial LbL films
Microbial contamination and infection pose severe health and economic threats, mainly because of the increasing resistance of bacteria toward conventional antibiotic treatments.The antibacterial surface design has attracted great attention over the past few decades.In this context, LbL lms have been widely developed to achieve mainly three properties: (i) antiadhesive lms to prevent bacteria attachment, (ii) contact-killing lms to inactivate bacteria upon contact, and (iii) release-killing LbL lms to leach out antimicrobial agents (Scheme 2).A comprehensive description of these coatings using organic and inorganic molecules, polyelectrolytes, antimicrobial peptides, nanoparticles, etc. is reviewed elsewhere. 66

Adhesion-resistant lms
To prevent the early attachment of bacteria and further biolm formation, adhesion-resistant LbL lms are usually highly hydrophilic lms 67,68 or possess specic stiffnesses to prevent bacterial adhesion. 69,70To enhance their hydrophilicity, synthetic polyelectrolytes have to be modied by poly(ethylene glycol) 67,68 or phosphoryl choline 71 before deposition to build adhesion-resistant LbL.In contrast, hyaluronic acid (HA)-based LbL lms present an intrinsic high hydrophilicity preventing the adhesion of bacteria.Both synthetic and natural polyelectrolytes-based LbL present specic lm rigidity able to present bacterial adhesion. 69,72The hydrophilicity 73 and rigidity 69,72 of LbL lms can be tuned by the adjustment of physical-chemical parameters of the lm build-up, such as pH and/or ionic strength.
Regarding protein-based LbL, antiadhesive lms were obtained using COL and HA. 54Aer cross-linking of the lm using glutaraldehyde, the attachment of Escherichia coli (E.coli) was decreased by 40% on HA-terminating LbL lm compared to uncoated tissue culture plastic substrate (Fig. 2a).No effect on the bacteria adhesion was observed on COL-terminating lms.Highlighted by the effect of the ending layer, it was suggested that the HA inhibited the attachment of negatively charged E. coli due to the high lm hydration and electrostatic repulsion between HA and the bacteria cell wall.Adhesion-resistant lms prevent the rst step in biolm formation but fail to kill them, requiring the use of an antibacterial agent.

Contact-killing lms
Antibacterial polymers are positively charged leading to the disruption of the bacteria wall integrity, leakage of the intercellular constituents, and cell death. 74][86] Those lms are known to avoid any resistance of the bacteria.Other contact-killing strategies were developed using proteins, particularly antibacterial enzymes, or peptides.Lysozyme (Ly) is an antimicrobial enzyme produced by animals able to hydrolyse 1,4-beta-linkages between N-acetylmuramic acid and N-acetyl-Dglucosamine residues in peptidoglycan, which is the major component of the Gram-positive bacterial wall, hence higher antibacterial activity against Staphylococcus aureus (S. aureus) strain.Silk broin and nylon-6 electrospun nanobrous mats were functionalized by 10 bilayers of COL/Ly LbL to obtain over 98% reduction of viable S. aureus and 87% for E. coli, independently of the ending layer (Fig. 2b). 87Interestingly, for 5 bilayers, Ly-ending lms showed higher antibacterial activity compared to COL-ending ones (Fig. 2b).Quorum-sensing degrading enzymes, such as acylase or amylase, were immobilized with synthetic polyelectrolytes to suppress the biolm formation of Pseudomonas aeruginosa (P.aeruginosa) and S.
aureus.An antimicrobial peptide, LL-37, was physiosorbed or chemically immobilized on cross-linked COL/HA LbL to obtain contact-killing properties toward E. coli. 54The concentration of the immobilized peptide played an important role in increasing the contact-killing efficiency reaching 85-90% against E. coli aer 24 h of contact with the highest concentrations (Fig. 2a).The action of contact-killing LbL lms is efficient with time but limited to the vicinity of the functionalized surface.

Release-killing lms
Avoiding the use of excess amounts of antibiotics in the systemic circulation, the release-killing lms are capable of neutralizing bacterial loads in their surrounding environment.This strategy is pertinent to prevent bacterial adhesion and proliferation during the post-implantation period lasting 6-12 h up to a few days.It has been demonstrated that this period is crucial for the bio(integration) of the biomaterial. 88LbL lms releasing antimicrobial agents were developed usually via (i) the degradation/dissolution of the LbL lm or (ii) diffusion from the lms.
Antibiotics were loaded in LbL lms and the amount was tuned by varying the assembly pH, the incubation time, and the number of deposited layers, and also by using heat treatment. 89he degradation of synthetic LbL lms, containing antibiotics, was obtained using the hydrolytic degradation of one of the synthesized polyelectrolytes, 90 by pH changes, 91 or by an electric potential. 92In the last two cases, the charge of one of the components was neutralized leading to the dissolution and release of the antibiotics.
Due to solubility issues, COL-based LbL lms were usually built at acidic pH leading to their total or partial dissolution at physiological pH due to changes in the overall charge.This property was used to obtain the release of an antimicrobial peptide (Tet213).The antimicrobial peptide was chemically graed on type-IV collagen and assembled into LbL lm with HA.The antibacterial property of the LbL lms was associated with the sustained release of the peptide in a physiological medium (pH 7.4) following the degradation of LbL. 9358.5% and 56.4% of Porphyromonas gingivalis (P.gingivalis) and S. aureus inhibition of the proliferation were obtained respectively aer 24 h of contact with 10 layers of COL-peptide/HA as well as signicant prevention of early biolm formation.CHI-ending (COL/CHI) 10 LbL lms functionalized on silk broin/ polycaprolactone nanobrous mat showed excellent killing efficacy of around 88% and 80% against S. aureus and E. coli, respectively.Around 30-35% killing efficacy comes from one CHI layer with an increase of efficiency with the CHI number of layers. 94The activity can be associated with CHI release in the surrounding bacteria probably due to the degradation of COL/ CHI LbL lm.The same LbL lm was deposited on polycaprolactone/nylon 6 mats to reach 95% inhibition of E. coli and S. aureus proliferation. 95annic acid (TA), a polyphenol known for its antibacterial properties, was associated with COL to obtain a release-killing antibacterial effect towards S. aureus. 96Our group showed the effect of the buffer on the build-up of COL/TA LbL lms leading to a dramatic effect on their antibacterial properties.On the contrary to COL/TA acetate lms, the granular topography of COL/TA citrate lms led to a local release of TA preventing S. aureus proliferation for 24 h (Fig. 3a).Nisin, an antimicrobial peptide extensively used in the food industry, was immobilized with poly(acrylic acid) to obtain a release-killing effect thanks to the dissolution of the lm.
The diffusion of the antibacterial agent from LbL lms was rst obtained by immobilizing silver nanoparticles (AgNPs) allowing the release of Ag + ions.Indeed, AgNP's antibacterial properties come from its dissociation in silver ions, which bind to the microbial wall, diffuse into the cell, and interact with proteins/enzymes/DNA. 97 AgNPs were mostly immobilized in synthetic polyelectrolyte-based LbL by direct incorporation 98 or reduction of Ag + -loaded lms leading to an efficient decrease of the bacteria proliferation. 98,99Avoiding the use of AgNPs, the diffusion of silver ions was obtained from liposomes containing AgNO 3 salt embedded in PLL/HA lms and using the temperature as a trigger.Exponentially growing lms based on polypeptides were exploited to obtain a release-killing effect.][83] Reduced extracellular pH (tissue acidosis) is common in the case of tissue injury, inammation, or infection.Thus, the pHtriggered release could be pertinent at the implant-tissue interface.Minocycline hydrochloride (MH), an antibiotic with anti-inammatory and antibacterial properties, was incorporated into Gel/dextran sulphate (DS) LbL lms thanks to the ability of MH and the polymers to form chelates with Ca 2+ ions (Fig. 3b). 100 (DS + Ca 2+ /MH + Ca 2+ /Gel + Ca 2+ ) 8 LbL lms released the antibiotic for 13 days at pH 6.This release may be due to the weaker chelation between Ca 2+ ions and DS at lower pH.The coatings prevented bacterial proliferation and biolm formation of seven pathogenic strains as well as clinical isolate bacteria.The lms showed no cytotoxicity towards NIH3T3 mouse broblasts.MH released inhibited the production of nitric oxide (NO) from lipopolysaccharide-treated RAW264.7 murine macrophages, thus showing anti-inammatory potential.
The use of proteins allows to design of gas-release lms.Cellobiose dehydrogenase was embedded in zwitterionic polycation/PSS LbL.In the presence of cellobiose, this enzyme produced H 2 O 2 leading to an antibiolm effect, i.e. a reduction of 53% of S. aureus biolm in comparison to uncoated PDMS catheter. 102Gel/TA LbL lms were designed to gra, on the secondary amines of Gel, N-diazeniumdiolates, and NOgenerating moieties (Fig. 3c). 101The developed Gel/TA LbL lms released NO gas reducing S. aureus planktonic growth by 35% with no cytotoxicity towards human dermal broblasts.The amount of NO release was controlled by optimizing the lm thickness, i.e., the number of bilayers.In addition, thanks to the rough porous structure and high surface area of the LbL lm, NO release showed a burst release pattern initially (good for the antibacterial property) followed later by a sustained release (suitable for cell signalling).

LbL films modulate mammalian cell adhesion
A primary function of biomaterials is to repair, conserve, or promote a tissue function or an entire organ.Much attention has been devoted to designing cell-biomaterial interfaces, where cells meet the biomaterials.Hence, the surface properties become important to modulate inammatory cell activation along with mammalian cell adhesion, proliferation, and subsequently differentiation to ensure successful biointegration.Various strategies were used based on LbL lms such as tuning their mechanical properties by ionic or covalent cross-linking and the surface chemistry by introducing positive charges or cell recognition molecules (Scheme 3).LbL lms based on polyelectrolytes to modulate cell-surface interactions have been extensively reviewed elsewhere. 103,104ynthetic polyelectrolytes can have intrinsic properties, such as PSS, favouring hepatocyte adhesion 30 progenitor endothelial cells maturation 31 or the differentiation of myoblasts into myotubes. 105Although the underlying mechanism is not understood, we can hypothesis that PSS property could be due to its heparin-like structure.PSS can interact with broblast growth factor-2 (FGF2), which plays a role in a range of biological functions such as wound healing, angiogenesis, and bone regeneration.However, to enhance the cell adhesion of primary cells, most synthetic lms have to be functionalized by adhesive moieties, such as RGD (arginine-glycine-aspartic) peptide 106 through the modication of one of the polyelectrolytes.RGD sequence makes up an anchoring place for both a and b integrin sub-units, which enhances the adhesion and proliferation of many kinds of eukaryotic cells.The presence of RGD on top of the lms improved the short-term adhesion of primary osteoblasts.Mannose is a common component of lectin, present at the surface of cell membranes, and playing a role in the recognition within the immune system.Mannose-graed lms allowed primary chondrocyte adhesion and proliferation while preventing chondrosarcoma cell growth. 107COL, Gel, Fn and ELP have been mostly used to promote cell adhesion by recognition of pro-adhesive moieties through RGD sequence, naturally present in these proteins.Table 1 summarizes protein-based LbL lms developed to favour cell adhesion.

Collagen-based lms
Nicholas A. Kotov and co-workers reported the rst COL-based LbL lm with PSS to improve the adhesion of C2C12 mouse myoblast on COL-terminating LbL lms. 64Since then, COL has predominantly been used with various counterparts such as ECM components, polysaccharides, and inorganic micro-to nano-particles in LbL assembly.In general, the lms have a brillar topography due to the tropocollagen triple helix   structure.Using HA as a partner was logical as the polysaccharide is one of the main components of the ECM.Fibrillar COL/HA LbL lms showed terminating-layer dependent chondrosarcoma cell adhesion property.Cells seeded on COL-ended lms were able to synthesize ECM components. 108No cellular matrix was observed on HA-ending lms which was attributed to the high water content of HA and the repulsion between the lm and the HA produced by the cells at their surface.In this work, the instability of COL/HA lms in physiological conditions at room temperature was not addressed, which was put in evidence later. 9T. Fujie et al. developed freestanding COL/HA LbL lms, called nanosheet, using a supporting lm method, i.e., the deposition of a sacricial poly(vinyl alcohol) layer on the substrate before the LbL build-up. 109They showed that the incubation of the nanosheet in physiological conditions at 37 °C led to COL brinogenesis and HA release, which enhanced both the mechanical stiffness of the surface and adhesive elongation of broblasts in contrast to the native nanosheet.The authors suggested that the increased stiffness due to long COL brils favoured the formation of the focal adhesion complex and the removal of HA-related repulsive interactions towards the cells improved ligand-receptor binding between COL and integrins.COL/HA LbL lms were used to improve the adhesion of pre-osteoblasts and human gingival broblasts on titanium disks 110 and osteoblasts on PLLA substrates. 111Native COL/HA LbL lms were used in vivo to cope with the foreign body response 112 and cross-linked ones to promote osseointegration. 113When implanted in the back of a rat model, HAended COL/HA LbL lms decreased the thickness of brosis by 29-57% compared to uncoated PDMS with only a few macrophage aggregates observed close to implant-tissue interfaces. 114o avoid the antiadhesive effect of HA, COL was assembled into LbL lms using chondroitin sulphate (CS).COL-ending COL/CS lms showed satisfactory spreading of murine embryonic broblasts with higher adhesion, cell density, and area than COL/HA lms (Fig. 4a). 115This was attributed to the presence of a higher amount of COL bres and a higher contact area in COL/CS lms than in COL/HA lms (>20 mg cm −2 vs. 10 mg cm −2 , respectively).The cell attachment was improved using oxidized CS (oCS) which led to cross-linked and more rigid COLbased LbL lms.
Alginate (Alg), a polysaccharide extracted from algae, was associated with COL in LbL lms and subsequently cross-linked by carbodiimide chemistry, 121 glutaraldehyde or genipin 55, 56 to stabilize the lm in physiological conditions for human periodontal ligament cells, 121 endothelial cells, 55 astrocytes and human gingival broblasts culture. 56In particular, the two latter cells were aligned on COL-oriented bres obtained by mechanical stretching of the COL/ALG functionalized PDMS substrates.
COL is an amphoteric macromolecule and thus was also associated with polycations such as CHI 120 and PLL.Deposited on poly(caprolactone)/cellulose acetate electrospun nano-brous mat, COL/CHI LbL lms improved the migration of human dermal broblasts in vitro and promoted skin regeneration in vivo. 120Thanks to the increasing number of COL layers, higher binding site densities were exposed for cells to attach on the surface with a positive effect in neovascularization.
Native COL was also associated with chemically modied COL prepared as deamidated, succinylated, maleylated, and citraconylated derivatives to coat polyacrylonitrile and poly(DLlactide-co-glycolide) bres 116 or onto hepatocyte cell layers in a microuidic setup. 125The resulting COL LbL lms showed good attachment and spreading of murine broblasts.Other properties have been added to COL-based lms thanks to the partner.M. K. Saums et al. associated COL with lumican, a small leucine-rich proteoglycan involved in the modulation of cell proliferation and differentiation.A better attachment and differentiation of hepatic stellate cells was obtained on COL/ lumican than on COL/poly(glutamic acid) lms.The brous network of COL and the presence of lumican favoured cell differentiation into myobroblastic phenotype. 122OL was also used to coat gold nanoparticles (NPs) improving the broblast's adhesion in comparison to COL/PLL thanks to the lm rigidity.117 By using photosensitizer-coupled PLL combined with COL-coated NPs, the cells were selectively detached from the LbL-coated substrate by irradiating with a laser.Reactive oxygen species were produced by the photosensitizer leading to cell death and detachment.118 COLbased lms were used to improve the cytocompatibility of quantum dots (QDs) NPs-based LbL.119 Used for diagnosis and therapeutical applications but highly toxic, semiconductor QDsbased LbL were coated by COL/polyacrylic acid LbL to alleviate their cytotoxic nature towards C2C12 myoblasts thanks to the deceleration of QDs decomposition.119 Recently, our group reported the use of commercially available cheap nylon paintbrushes to build linearly growing COL/TA LbL lms.The brushing method allowed the deposition of aligned COL layers, thanks to the shearing effect of the brushing process. Theorientation of COL and release of TA from the LbL lm led to the alignment and differentiation of human myoblasts into myotubes opening the route of the development of 3D human muscle bres in vitro (Fig. 4b).124 Gelatin-based lms Gel having an isoelectric point of 5, the assembly pH of Gel/PSS LbL lms affected chondrocyte viability.130 For assembly at pH 5, the highest chondrocyte viability was observed for only 2 bilayers reaching a plateau for higher bilayers.For assemblies at pH 3 and 7, the chondrocyte viability was observed starting from 4 bilayers.At pH 3 and 7, Gel molecules are highly positively or negatively charged, respectively, which restricts their adsorption in the LbL assembly.At pH 5, a high Gel content was adsorbed with few layers leading to good cytocompatibility.Assembled at pH 6, Gel/CHI LbL lms were unstable in physiological pH (7.4) failing to be used for broblast culture contrary to Gel/poly(diallyldimethylammonium chloride) (PDADMA). 126The pH change induced the deprotonation of the amino groups of CHI leading to a loss of electrostatic interaction between Gel and CHI.Polyethylene terephthalate (PET) engineered ligament gras, coated with Gel/HA LbL lms, signicantly suppressed the chronic inammatory response with the formation of new blood vessels, in the rabbit and porcine model for anterior cruciate ligament reconstruction (Fig. 5a).133 Fibronectin-and laminin-based lms Fn is a glycoprotein known to support cell adhesion, migration, proliferation, and differentiation via transmembrane integrin interactions.The protein has been deposited on the top of synthetic polyelectrolyte-based lms to promote a faster attachment and further growth of smooth muscle cells in comparison to the native lms. 129Developed by lithography and li-off, Gel and Fn-ended LbL patterned surfaces were compared as adhesive proteins with rat aorta smooth muscle cells.128 Cells seeded on the synthetic lms can migrate toward Fn-ended lms leading to good localization of cells on the patterned surface contrary to Gel-ended lms.
Having an isoelectric point between 5.5 and 6, Fn was associated with PLL to build LbL lms which build-up saturated aer 10 bilayers. 134It could be due to the interfacial aggregation of Fn leading to the suppression of the available charges on the terminal layer required for successive LbL growth.It was found that Fn can also be associated with PSS at pH 5.8 with an efficient build-up with overnight incubation of the protein. 128It was not clear if the adsorption of Fn was electrostatic or due to some other attractive force.E. Brynda et al. showed that type IV collagen and Gel-based lms associated with DS or laminin, a glycoprotein from the ECM, led to better murine embryonic stem cell attachment in comparison to BSA-based LbL lms. 127astin-based lms ELP, a brous ECM protein composed of single tropoelastin subunits, presents repeating amino acids sequence VPGVP (V: valine, P: proline, G: glycine).The cell-ELP interactions are attributed to elastin receptors, G protein-coupled receptors, and integrins. 135Elastin-like polypeptides have amino acid sequences derived from tropoelastin consisting of repeating units of a pentapeptide VPGXG, where X can be any amino acid except for proline.Obtained by recombinant DNA techniques, they are produced by bacteria cells and are called also elastinlike recombinamers (ELR).They exhibit a reversible phasetransition in aqueous solution and are soluble below the transition temperature (T t ) and phase-separate into coacervates above T t, which is mostly 37 °C.Thermoresponsive LbL lms were prepared using ionic ELP containing lysine units (polycation) and glutamic acid units (polyanion).Interestingly, long exposure (72 h) of the lms with a salt solution at 37 °C (higher than T t ) caused the lm to shrink, thus improving its stability. 131lickable ELR LbL lms were developed to favour endothelialisation of stents withstanding high shear stress ow, limiting platelet adhesion and blood coagulation.The LbL lms led to a conuent layer of endothelial progenitor cells aer 1 day of culture, thanks to the presence of the RGD sequence (Fig. 6). 136LR-based LbL lms were built using poly(ethylene imine)-ELP and polyacrylic acid-ELP, obtained by chemical coupling, to favour broblasts' focal adhesion point. 132

Designing hemocompatible surfaces
Blood circulation ensures the supply of all tissues with oxygen, and nutrients, and the removal of metabolites.It is ensured by

LbL lms can prevent platelet adhesion
To suppress blood activation processes, highly hydrophilic lms provide a stealth effect on the surface, which should prevent the interaction with cells and proteins in the blood.Synthetic LbL lms were used to decrease the protein adsorption leading to the suppression of platelet adhesion.Usually, heparin-like, 137 zwitterionic, 138 phosphorylcholine, 139,140 or poly(ethylene glycol) based LbL 141 were used for this purpose requiring chemical synthesis.Protein-based LbL lms were used to functionalize diverse types of surfaces using bovin serum albumin (BSA), a plasma protein with anti-thrombogenic properties, to reduce non-specic platelet adhesion.Jian Ji et al.
assembled BSA/PEI LbL lms on poly(vinyl chloride) (PVC) 142 and 316 L stainless steel (SS) 143 surfaces.Built at around physiological pH, the lms were stable for up to 45 days in physiological medium under static conditions with less than 10% BSA released.On 4 BSA/PEI bilayers-coated PVC surfaces, platelet adhesion was negligible.Whereas on 316L SS, due to granular topography, the homogeneous surface coverage was obtained aer 8 bilayers which reduced the platelet adhesion by 90%.Similarly, sulphated polysaccharide heparin (HEP), a commercially known anticoagulant drug, was incorporated into LbL lms with BSA, [144][145][146] streptavidin, 147 Fn, 148,149 and COL. 150,151SA/HEP LbL lms were used to functionalize PVC, 144 polystyrene (PS) surfaces 145 and poly(ether sulfone) (PES) foils. 146uilt at pH 3.9 on PVC sheets, BSA/HEP lms were unstable in PBS due to the reversal charge of BSA (pI 4.9) at physiological pH.Aer cross-linking by glutaraldehyde, HEP-ending BSA/HEP lms suppressed platelet aggregation and decreased platelet adhesion in comparison to untreated PVC substrate.Higher anticoagulant efficiency of uncross-linked lms was attributed to the release of HEP. 144On PS substrates, BSA/HEP lms built at pH 4 and cross-linked by glutaraldehyde showed a reduced platelet adhesion with the increase in bilayers.This result was similar with only BSA cross-linked LbL lms.Dried and reswollen (BSA/HEP) 3 coatings showed a moderate platelet attachment.HEP once incorporated into LbL lms loses its thrombin inhibition by less than 10%, perhaps due to HEP interactions within LbL assembly.Thrombin inhibition efficacy was also improved by increasing the number of bilayers. 145BSA/ HEP multilayers with two kinds of HEP (standard, and high anticoagulant fraction) were built at pH 4 on PES foils.Interestingly, the type of HEP solely affected the biological properties of LbL lms.Platelet adhesion was roughly comparable on BSA/ HEP and BSA control coatings.However, BSA/HEP reduced the coagulation activation especially when using a high anticoagulant fraction of HEP (Fig. 7a).Such coatings could be used in blood purication systems. 146eparinylated multilayers were constructed using streptavidin/biotin bio-specic interaction on titanium.The lms showed reduced platelet adhesion and enhanced clotting time. 147Fn is known to interact with leukocytes.Fn/PLL LbL lms were deposited on hydrophobic polyurethane to study the protein conformation and the response of monocytes.Fn adsorbed in unfolded state in Fn/PLL LbL, due to the negatively charged cell binding domain (RGD domain) and sub-domains with the C-terminal of Fn interacting with positively charged PLL.This leads to the low availability of Fn cell binding domain suppressing monocyte activation (Fig. 7b). 149These lms alleviated the pro-inammatory response of monocytes in contact with degradable polar hydrophobic polyurethane for 3 days.
Titanium was modied by COL/HEP using electrostatic LbL assembly.The coated surfaces show decreased platelet adhesion and activation, and an increment in the clotting time. 150,151xidized dopamine (oDOP) used as precursor layer on titanium allowed obtaining COL/HEP LbL lms with higher thickness, prolonged thromboplastin activation time, lower haemolysis ratios, and platelet coverage. 152COL/HEP coatings were compared with DOP/HEP.Though DOP/HEP showed a better

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Chemical Science adhesion of the coating and stability of the lm, COL/HEP is better at maintaining anti-coagulant efficiency. 153Yet, the lm's stability over a prolonged time remains a challenge.

LbL lms can promote endothelial cell adhesion
Endothelial cells (ECs) form the inner layer of blood and lymphatic vessels.ECs possess anti-thrombogenic potential by maintaining a physiological barrier between outer host tissue and circulating blood.An incomplete endothelialisation can cause other damages like restenosis and neointimal hyperplasia.8][159][160] Deposited in the inner part of arteries, PSS/PAH lms showed good in vivo retention of the luminal surface. 161PAH/PSS LbL lm improves also the EPC's maturation into functional and mature ECs. 31his could be related to the heparin-like properties of PSS.
Our group investigated the effect of chemical cross-linking on the endothelialisation of COL/ALG LbL lms.On the contrary to glutaraldehyde, genipin, a natural plant-derived agent, cross-linked coatings showed rapid attachment of human vascular ECs within 1 h, leading to a conuent layer in 5 days (Fig. 8a). 55Combining the benets of COL biocompatibility and HEP anticoagulant capacity, COL/HEP coatings on intravascular stents showed antithrombic properties with good adhesion and proliferation towards human umbilical vein endothelial cells (HUVECs). 154In vivo assessments were performed to better elucidate the vascularization potential of multilayer lms.Better angiogenesis was obtained with COL/ HEP coated porous hydroxyapatite scaffolds with enhanced mechanical properties using the chicken chorioallantois membrane model. 155COL IV/HEP modied cardiovascular titanium stent surfaces exhibited new angiogenesis aer 15 days in dog femoral artery model. 156Another perspective within the vascularization approach is to develop multifunctional coatings with selectivity towards ECs to support early and fast endothelialisation.For this, COL/HEP LbL lms were immobilized REDV peptide, recognized by ECs.Hydrophobic, Teon® (ePTFE) lms were coated with COL/HEP lms and showed weak platelet activation and adhesion, prolonged coagulation time, and reduced haemolysis.REDV-containing lms showed enhanced early cell attachment, proliferation (cell density aer 72 h), and cell activity. 162The delivery of growth factor to the host site can potentially enhance endothelialisation and angiogenesis.However, their controlled delivery is challenging due to the extremely small half-life of growth factors (less than 1

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Perspective hour) and susceptibility to degradation in a physiological environment or under the action of enzymes. 153Their embedding into LbL lms could overcome this issue. 163Prolonged release (above 35 days) of b-FGF from COL/HEP LbL lms showed enhanced angiogenesis of subcutaneous tissue of rat model, i.e. high blood vessels' density and diameter. 153Antibodies can improve EC specicity by their cell-surface antigen interactions.LbL assembly provides a platform for antibody immobilization to selectively capture EC from the circulating blood for vascular tissue engineering.CD34 is an antigen that specically presents EPCs.Electrostatically immobilized anti-CD34 into COL/HEP LbL lms on Ti surface formed compact EC lining just aer 2 days, 164 compared to 3 days reported for COL/HEP lms without the antibody. 165Stable under static and ow conditions, COL/HEP LbL lms ended by anti-CD34 antibody enhanced in vitro early attachment (1 h) of EPCs and signicantly reduced neo-intimal formation in rabbit femoral artery model in vivo (Fig. 8b). 166Commercially available ePTFE gras were functionalized with COL/HEP LbL loaded with anti-CD 133.CD133 is more specic than CD34 as a surface marker for EPCs.Antibodies functionalized gras showed enhanced EPCs adhesion and in situ rapid early endothelialisation in a porcine carotid artery transplantation model.However, further investigations with smaller diameters (<6 mm) of the gras and host-relevant antibodies are essential. 167ug, cytokine, growth factor, and gene delivery Sustained and controlled release of drug formulations is essential for higher drug efficiency and lower risks of toxicity.Towards this, the LbL technology offers enormous possibilities and is a far better replacement for conventional drug encapsulation techniques. 168For instance, the LbL lms allow the nanometric control over the order, location, and concentration or loading of various cargo layers like polymer, drug, growth factor, cytokine, or gene. 169Park et al. reviewed the various methods used for preparing LbL lms with drugs. 170LbL-assembled lms were also used to develop hollow micro-nano capsule payload carriers. 171bL capsules offer unique advantages over conventional carriers (e.g.liposomes).The versatility of the LbL method allows for control of the inner cavity of the capsules as well as their surface allowing multi-functionalisation for therapy, diagnosis, or both (e.g., theranostic).A recent review summarizes the recent development of this type of capsule. 172In this context, protein-based LbL lms were coated onto polystyrene, calcium carbonate, or melamine formaldehyde nano-micro particles to be further dissolved to obtain hollow capsules.The main advantages of using proteins, instead of synthetic polyelectrolytes, are to develop microcapsules with the possibility of obtaining the (i) dissolution in a physiological medium due to a pH change and/or (ii) degradation by enzymes for the drug release (Scheme 5).They are also used to give specic properties to the lms or capsules such as controlling mammalian cell fate by using growth factors, plasmids, or recognition by using antibodies.A summary of proteinbased LbL drug and gene delivery systems is given in Table 2.

Release by diffusion/erosion
Once implanted into the human body, immune cells, mainly monocytes and macrophages, are recruited to the implant surfaces within hours post-surgery and initiate the cascades of early inammatory responses.Although initial inammation is required for tissue healing, adverse immune reactions in the implanted biomaterials take place, leading to brous encapsulation.Immunosuppressive drugs, such as sirolimus, inhibit or decrease the intensity of the immune response.Cross-linked LbL COL coatings were developed to be used as a drug reservoir for the sustained release of sirolimus. 173Approved by the Food and Drug Administration (FDA), the drug reduces neointimal thickening in models of vascular injury.COL/ sirolimus LbL lms were built by spray coating and further cross-linked using genipin.From cross-linked COL lms, sirolimus was eluted for up to 28 days depending on the number of deposited bilayers.One of the key components of the inammatory response is macrophage polarization.The cells can differentiate into pro-inammatory M1 macrophages, associated with classic signs of inammation, or anti-inammatory and pro-healing M2 macrophages, alleviating inammation and generating a favourable immune microenvironment for tissue healing.A prolonged inammation due to the accumulation of M1 macrophages gives rise to the formation of brous encapsulation, a barrier to implant integration.Cytokines are small cellsignaling proteins secreted by immune cells to help control the inammation of the body.Thus, LbL lms were developed to release Interleukin-4 (IL-4, a M2 polarizing cytokine).In contrast to the uncoated substrates where the monocytes produced high levels of pro-inammatory cytokines, the released IL-4 from PLL/HA-Aldehyde LbL drives the monocyte differentiation into M2 macrophages over M1 macrophages. 201he release of IL-4 from CHI/dermatan sulphate LbL, built on polypropylene mesh, was tuneable based on the number of coating bilayers.IL-4 was detected up to 14, 22, and 30 days for coatings of 20, 40 and 60 bilayers.In vitro, macrophage culture assays showed that implants coated with IL-4 promote the polarization towards M2 macrophages, although the concentration of released IL-4 (2.25 ng mL −1 ) was lower than the positive control (20 ng mL −1 ).Aer 7 days of implantation in mice subcutaneous pocket, the coated polypropylene mesh revealed within the rst 50 mm the presence of the greatest

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Chemical Science amount of M2 macrophages than M1 macrophages versus uncoated mesh, suggesting that the effects of IL-4 released from the LbL coating are limited locally.At 90 days postimplantation, IL-4 loaded mesh had a reduced capsule area and thickness compared to the prominent and dense capsules surrounding uncoated mesh. 202ransforming growth factor-beta 1 (TGF-b1), platelet-derived growth factor bb (PDGF-bb), and insulin growth factor 1 (IGF-1) involved in tissue morphogenesis were loaded with the highest loading in combination with HEP or DS.The release of the growth factors was obtained by diffusion and erosion in a controlled manner with the bioactivity maintained for up to 14 days (Fig. 9a). 200The activity of growth factors was preserved and allowed increasing broblast proliferation as well as enhancing myobroblast differentiation as put in evidence by a-SMA labelling.
Protamine sulphate (PRM) is a natural arginine-rich protein involved in the condensation of DNA.It is also FDA-approved for the treatment of heparin overdose or excessive bleeding.The LbL-assembled lms of protamine and plasmid DNA encoding hepatocyte growth factor were used to promote the growth of HUVECs and hinder the growth of artery smooth muscle cells in a co-culture. 197Both cells were transfected upon contact with the plasmid-based LbL lm, enhancing the competitiveness of HUVECs over smooth muscle cells (Fig. 9b). 197Indeed in comparison to glass and sh sperm DNA (control), the cell density of HUVECs was higher than the cell density of smooth muscle cells aer 3 days of culture.Graphene oxide (GO) nanosheets were coated with PRM/Alg LbL lm, which not only improved the dispersibility and stability under physiological conditions but also reduced protein adsorption.

Chemical Science Perspective
The LbL-coated GO nanosheets loaded with doxorubicin (Dox) showed better cellular uptake and cytotoxicity against MCF-7 cells. 192In comparison with bare Ibuprofen crystals, the uncoated microcapsules showed faster release of Ibuprofen in gastric uid and slower release in intestinal uid.The PRM/PSS coating prevented the initial burst release and resulted in sustained release of the drug in both uids. 195he LbL method was used for the direct functionalization of drug-based crystals to serve as a diffusion barrier to control the release of the drug payload.For example, Ibuprofen crystals were coated with HSA/l-a-dimyristoyl phosphatidic acid LbL lm for controlled drug release in a solution of pH 7.4.The release rate was controlled by increasing the number of layers and the crystal size. 188Furosemide dye microcrystals were coated with Gel/PSS LbL lm (thickness around 40-115 nm for 2-6 bilayers) that reduced the release rate of furosemide by 50-300 times in aqueous solutions (Fig. 9c).The media at pH 7.4 showed 6-8 times faster release than the one at pH 1.4. 178design of the experiment with aims to enable scale-up production and sustained dissolution (from 120 min to 270 min). 177Gel/PSS LbLcoated Naproxen microcrystals, a non-steroidal anti-inammatory drug, showed a delayed release of the core payload in PBS pH 7.4 (approximately 50% lower dissolution rate). 176lease at physiological pH Polyphenols, plant-derived secondary metabolites, possess potentially broad bio-functionalities like antibacterial, antioxidant, and anticancer properties.However, low bioavailability and half-life limit their use as free compounds both in vitro and in vivo.Hence, a targeted delivery approach with controlled release is necessary.Lvov and co-workers developed Gel/ epigallocatechin gallate (EGCG)-based hollow microcapsules with antioxidant properties boosted by increasing the number of bilayers from 1 to 10 (Fig. 10a).Only the permeability of the capsules was tested using dextran of different molecular weights. 174Moreover, they developed cross-linked type A Gel nanoparticles (NPs), of around 200 nm diameter using the desolvation method, coated with LbL lms, e.g., carboxymethyl cellulose/type A Gel (CMC/Gel) n .EGCG; a chemopreventive polyphenol, was successfully loaded at pH 4 and subsequently released at pH 7.5 from the nanoparticles to block the HGFintracellular signalling in the breast cancer (MBA-MD-231) cells.The LbL-coated Gel NPs showed pH-triggered controlled release of EGCG, unlike the burst release within the rst 15 min from the uncoated Gel NPs. 199 multi-drug carrier, based on BSA/CHI LbL, was designed to deliver both hydrophobic (pyrene and curcumin) and hydrophilic DOX drugs.Microcapsules were built at acidic pH using a mixture of BSA/hydrophobic drug and CHI solutions.The hydrophilic drug was incorporated in a second step by soaking the lm.The release of both types of drug was obtained in physiological pH.182 BSA nanoparticles/CHI LbL were designed to load and deliver doxorubicin (Dox) under a pH trigger.184 A proteolytic enzyme, a-chymotrypsin, was loaded and subsequently released from PRM-based LbL capsules under acidic and above neutral pH, respectively.191 Using the hydrolytic nature of a synthetic polymer, the LbL strategy was rst reported by Hammond and workers for dual drug delivery in the murine ear skin model in vivo.They described the use of a cationic

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Chemical Science polymer, poly(amino ester) named poly-1, with a protein antigen, ovalbumin (ova), and/or immunostimulatory CpG (cytosine-phosphate-diester-guanine-rich) DNA oligonucleotide adjuvant molecules for LbL assembly.Applied as a skin patch, such dual delivery LbL lms released ovalbumin and the CpG in a rapid and sustained manner by hydrolytic degradation (Fig. 10b). 189In vivo experiments showed the penetration of ovalbumin and CpG and colocalization with Langherans cells (cells from the immune system).

Release by enzymatic degradation
COL/HA LbL hollow microcapsules showed sustained release of uorescently labelled BSA triggered by collagenase degradation.The release could be controlled by the number of layers, COL cross-linking, and collagenase concentration. 203Microcapsules based on BSA/TA hydrogen-bonded LbL assembly were developed to deliver hydrophilic-uorescently-labelled BSA and hydrophobic 3,4,9,10-tetra-(hectoxy-carbonyl)-perylene, thanks to enzymatic degradation by a-chymotrypsin.The developed capsules were not toxic to murine RAW264.7 macrophages at payload concentrations of around 50 capsules per cell.Such payload carriers may nd applications in intravenous drug delivery with an ability for site-specic release. 186Moreover, BSA/TA microcapsules were reported to be stable in simulated gastric uid but degrade in simulated intestinal uid.Thus, opening possibilities to deliver bioactive compounds and functional foods to the lower gastrointestinal tract.Immunoglobulin G (IgG), a milk protein that can adhere to the human intestinal surface, was incorporated into the LbL lm, while blactoglobulin could not adsorb.IgG incorporation enhanced the adhesion of the capsules to Caco-2 cells by more than 6 times. 185ultiple polyphenols like EGCG, 3,4-O-dicaffeoylquinic acid (3,4-diCQA), and TA were alternatingly assembled with BSA to develop robust microcapsules for delivery of IgG in the gastrointestinal tract.Thanks to the synergistic stability due to the use of multiple polyphenols, the capsules, were stable through stomach digestion, showed higher thermal denaturation temperatures, and improved antioxidant potential. 187ambogic acid, a bioactive species, loaded micelles were coated with PRM/HA LbL lm.The LbL coating enhanced the internalisation of the micelles by human lung adenocarcinoma, where they undergo the removal of HA in a hyaluronidase (HAase)-rich tumour microenvironment and exposed the PRM that activates the "proton sponge" effect.Thus, showing targeted anticancer activity towards human lung adenocarcinoma (A549) tumour xenogras in nude mice in vivo (Fig. 11). 193Localized gene delivery from the LbL lms was studied through an enzymatic trigger.Single-strand DNA (ssDNA) was delivered from a formaldehyde-induced covalently bonded ssDNA/BSA LbL lm via proteinase K degradation.The amount of ssDNA and the protein loading was increased by increasing the number of layers. 183DNA was delivered from PRM/DNA LbL lms under the action of a-chymotrypsin enzymatic degradation. 198

Release by dual stimuli: pH and enzymes
In contrast to the desired sustained drug release proles, in particular cases like cancer treatment and vaccines, a burst release of the drug aer reaching the target site might be more efficient.Dual stimuli-responsive (pH and enzyme) PRM/HEP LbL capsules were loaded with Dox at pH 5 for cancer treatment.Once internalized by the MCF-7 breast cancer cells, the capsules disintegrate inside the cells, leading to a burst release of the Dox and subsequent cell death. 194PRM-carboxymethylcellulose LbL nanocapsules decorated with Fe 3 O 4 magnetic nanoparticles were developed to deliver Dox.The presence of an external magnetic eld enhanced the capsule uptake at the target site in the Balb/c mouse model in vivo with successful Dox delivery under an external magnetic eld. 196

Bone regeneration
Failure to osseointegration, the formation of interfacial brous tissue between bone and implant, is a major reason for implant aseptic loosening.Several types of implant materials have been

Chemical Science Perspective
functionalized using the LbL technique, such as titanium, polyetheretherketone (PEEK), 204 poly(L-lactic acid) (PLLA), 205 stainless steel, 206 poly(3-caprolactone) scaffold 207 and bres, 208 or poly(propylene carbonate). 209A brief introduction to bone repair requirements including differentiation and the types of cells involved can be found elsewhere. 210Subsequently, different LbL lms emerge to address various aspects of bone tissue repair, in particular, to regulate cell behaviour on host tissue/implantable devices' interface.Two types of properties have been addressed: osteoconduction which is the ability of osteoblasts or pre-osteoblasts (bone-forming cells) to form new bone over time in the biomaterials or on its surface, and osteoinduction which is the recruitment and differentiation of mesenchymal stem cells (MSCs) and pre-osteoblasts into osteoblastic lineage (Scheme 6).MSCs are multipotent cells that can be differentiated into various cell types, including osteoblasts, chondrocytes, and adipocytes.MSCs differentiated into preosteoblasts before their differentiation into osteoblasts. 211he differentiation of MSC is commonly obtained thanks to additional growth factors or bioactive calcium phosphates in the cell culture medium or adsorbed in the LbL lms. 212To this end, cell-adhesive proteins have been incorporated into the LbL lms to improve cell adhesion and proliferation by taking advantage of the high adsorbed quantity of proteins, the lm dissolution to release bioactive compounds and/or the bioactivity of the partner LbL component to obtain an additional property.During the bone repair process, M1 macrophages secrete mediators that (i) prevent surrounding cells (i.e., osteoblasts and ECs) from growing and proliferating and (ii) activate the osteoclastic bone resorption.Whereas to restrain the brous encapsulation of biomaterials, M2 macrophages release mediators that promote the proliferation of adjacent cells involved in osteogenesis, angiogenesis, and osseointegration.Thus, a positive regulation on the M1/M2 phenotype switch appears to be key to the implant osseointegration.
Osteoconductive LbL lms COL/HA LbL lms were used to improve the adhesion of preosteoblasts 110 and osteoblasts on PLLA substrates. 111Electrostatically assembled COL/HA LbL lms dissolve in physiological conditions.The dissolution of COL/HA LbL lms in physiological conditions leads to higher osteogenic protein and gene expression aer 14 days, accelerating their differentiation. 213he rate of COL/HA LbL lm's dissolution in PBS was modulated by a disulde cross-linking strategy.Incorporation of RGD in cross-linked COL/HA LbL lms improved murine preosteoblast adhesion with higher bone-specic gene expression levels and matrix mineralization. 214The lms were obtained using a disulphide RGD peptide subsequently cross-linked by the conversion of free sulydryl groups into disulphide linkages.Knowing that COL possesses RGD moieties, the advantage of the presence of RGD on the cross-linker was not discussed in the paper.An in vivo comparison between cross-linked and native COL/HA LbL lms highlighted the importance of lm stability in trabecular bone rabbit models.Cross-linked COL/ HA lms led to better osseointegration with tight de novo bone-implant interfacial contact, reducing the implant loosening, than the native lm. 215For COL/HA lms, the crosslinking strategy resulted in better osteointegration but in the case of COL/Alg the lm crosslinking showed terminating layerdependent property.For example, carbodiimide cross-linked COL/Alg lms show signicantly higher ALP activity aer 12 days of seeding of murine pre-osteoblasts when alginate is the terminating layer.Perhaps, the crosslinking reaction rendered cell-binding domains of COL unavailable for murine preosteoblasts. 216n comparison to uncoated substrates and Fn monolayer, Fn/PLL LbL lms signicantly improved human osteoblast-like and pre-osteoblast 134 adhesion and proliferation. 217,218PRM, used for treating heparin overdose or excessive bleeding

Perspective
Chemical Science disorder, gives rise to hydrophilic coatings on silicon substrates with PSS as a polyanionic counterpart.The increase in layer pair number from 20 to 240 directly impacts the surface roughness and Young's modulus in a hydrated state.Such mechanical properties of the coated surfaces appear to signicantly inuence murine pre-osteoblasts spreading, proliferation, and differentiation with higher ALP activity and mineralization. 219ater contact angle (WCA) deals with the wetting behaviour of a surface and indirectly affects mammalian cell adhesion.In the case of superhydrophilicity, i.e., WCA in the range of 0°to 5°, proteins from cell culture medium that can support cell adhesion are thermodynamically unable to adsorb on the surface, giving rise to antifouling surfaces.Similarly, if a surface is hydrophobic, i.e., WCA above 70°, the cell culture medium proteins adsorb on the surface in a denatured manner that also prevents cell adhesion. 220Therefore, LbL lms are oen used to provide well-controlled hydrophilicity to support cell adhesion and proliferation.Thanks to the hydrophilic character of the proteins and polyelectrolytes used, the LbL lms decreased the water contact angle of relatively hydrophobic surfaces.By increasing the hydrophilicity of poly(propylene carbonate) (PPC), i.e., decreasing the water contact angle from 70°to 58°, Gel/PEI LbL lms enhanced the proliferation of both broblasts and osteoblasts (Fig. 12a). 209Gel/CHI LbL lms were extensively used, due to the osteoconductive properties of CHI and the non-immunogenic character of Gel, as platforms for the controlled release of bioactive agents.Similarly, to COL/HA lms, Gel/CHI LbL lms were built in acidic pH, due to CHI solubility limitation, and became unstable in a physiological medium due to the decrease in electrostatic interactions between partners.Aer cross-linking via carbodiimide chemistry, these lms showed better adhesion, proliferation, and viability of osteoblasts in comparison to the uncoated titanium substrate. 222Unstable in PBS, Gel/CHI LbL lms were exploited for the incorporation and subsequent release of different bioactive agents.The release of Zn ions, below its toxic concentration (2 ppm), improved osteoblasts proliferation and activity up to 7 days, and showed antibacterial activity by reducing S. aureus and E. coli adhesion. 223b-Estradiol, known to upregulate osteoblast maturation and to reduce osteoclast growth, was loaded into mesoporous silica nanoparticles coated by Gel/CHI LbL lms and embedded in the same degradable LbL lms. 224Adhered osteoblasts were able to uptake the drug-loaded nanocarriers, thus displaying higher mineralization than on Gel/CHI lms without the loaded nanoparticles.Titanium nanotube usually obtained by electrochemical anodization were reported to signicantly improve cell spreading and adhesion. 225They can be excellent carriers of bioactive molecules.Icariin, a natural avonoid from Epimedium herbs, potent to treat osteoporosis and inhibit osteoclast differentiation, was delivered from titanium nanotubes coated by the degradable Gel/CHI LbL lms.In physiological conditions, the lms delayed the release of Icariin up to 5 days leading to higher osteoblast proliferation than the uncoated Icariin loaded titanium nanotube. 226Polyaniline-modied CHI (CHI-PANI) was used with Gel in LbL manner to obtain multifunctional antibacterial activity and osteoconductive properties. 227Methacrylamide modied Gel/N-halanine modied poly(N,N 0 -methylene bis(acrylamide)) LbL lms were deposited on BMP-2 loaded titanium nanotubes to obtain a pH triggered release of BMP-2 to favour osteoblasts viability as well as antibacterial properties towards S. aureus and E. coli. 228The antibacterial activity is due to N-halamines presenting an oxidation state of the chloride ions, targeting thiol groups or amino groups of bacterial proteins, leading to growth inhibition or inactivation.Hydroxyapatite (HAP), a mineral that comprises approximately 70% of the bone mass, is naturally found as nanoneedles.Several layers of HAP nanobers were sandwiched in Gel/CHI LbL lms to obtain a lamellar stack hybrid lm (Fig. 12b).These hybrid lms improved osteoblast migration and signicant mineralization for up to 14 days. 221

Osteoinductive LbL lms
The choice of LbL partners is crucial for the desired biological response.COL/HEP LbL lms coated on PLLA substrates allowed to reduce human MSCs proliferation whilst stimulating their differentiation and mineralization aer 28 days which provided osteoinductive environments.Here, HEP interacts with telopeptide regions of COL due to interlayer mobility/ diffusion within LbL lm and blocks the cell-binding domains of COL that cause lower cell adhesion. 205A comparative study in which COL was assembled with native or oxidized

Chemical Science Perspective
HA or CS showed that the molecular composition of LbL lms impacted not only the physicochemical properties of the lms but also the adhesion of human adipose-derived MSCs on the lms. 229A favourable microenvironment was obtained with COL/CS lms with a higher COL remodelling by the MSCs.This was attributed to a higher amount of COL deposition in COL/CS lm.PLA lms were functionalized by cross-linked COL/HA embedding silicon-carbonated HAP nanoparticles, leading to better cell attachment, proliferation, and osteogenic activity of human MSCs than on unmodied PLA. 230steoinduction properties can also be obtained by the action of growth factors, proteins that stimulate the growth of specic tissues, on MSCs.Bone Morphogenetic Protein 2 (BMP-2) is a multi-functional growth factor released during bone growth or healing.The embedding of BMP-2 in Gel/CHI LbL allowed better differentiation of MSCs compared to bare alloy substrates (Fig. 13). 231Although, the authors did not report any signicant difference between Gel/CHI, LbL/BPM-2/Fn, and the TCPS group (see mineralization assay), in the physiological medium, the sustained release of Gel and BPM-2 over 14 days induced higher osteoblastic protein expressions in comparison to the uncoated substrate. 231A more pronounced formation of de novo bone was observed in vivo in the rabbit femur model thanks to BMP-2 release.The great advantage of LbL is the possibility to have multiple properties with the same lm.A double functionalization of the porous titanium scaffold was achieved by Gel/CHI LbL lms and ended by Gel/BMP-2 layer and CHI/ antibiotic layer.This led to higher ALP expression (2-fold), matrix mineralization (4-fold) as well as antibacterial activity against S. aureus up to 8 log reduction in planktonic and adherent bacteria in comparison to the uncoated scaffold.In vivo subcutaneous implantation in rats revealed the connective tissue formation with no foreign body response aer 8 weeks. 232ased on the assumption that insulin growth factor (IGF) plays an important role in maintaining bone strength, IGF adsorbed as a last layer on Gel/CHI LbL demonstrated signicantly higher osteogenic differentiation of MSCs and new bone formation in rat with osteoporosis. 233teo-immunomodulatory LbL lms In this context, Zhao et al., empowered an interconnected nanoporous titanium (Ti) network with polydopamine forming a polydopamine anchoring layer then immobilized COL through three reactions: the Schiff base reaction between the NH 2 of COL and quinone of polydopamine, the Michael-type addition reaction between the NH 2 of COL and catechol of polydopamine, and the amide bond (CO-NH) formation reaction between the COOH of COL and NH 2 of polydopamine.The increase in the COL content was achieved by COL/HA LbL.In contrast to the raw Ti network, which favoured M1 macrophage polarization, the COL/HA coating signicantly downregulates the transcription of M1 macrophage genes (i.e., iNOS, CD11C, CD86, and CCR-7) and upregulates the transcription of M2 macrophage genes (i.e., CD163, CD206, IL-10, and arginine-1). 234el/CHI LbL loaded with SEW2871, a macrophage recruitment agent, was built on dopamine-modied Ti.In addition to the increase in RAW264.7 cells in vitro migration, SEW2871 in LbL lm promotes the M2 phenotype.In vivo, better early recruitment of endogenous macrophages at the implants-bone interface was observed in the implanted SEW2871 Gel/CHIcoated Ti versus raw Ti.Suppression of CD86 positive M1 macrophage and a better expression of CD206 positive M2 macrophages aer 7 days of implantation were observed close to SEW2871 Gel/CHI coated Ti in comparison with raw Ti. 235 As promising alternative biomaterials to conventional metals in bone reconstructive surgery, poly(etheretherketone) (PEEK) possesses superior characteristics including excellent mechanical properties, good chemical stability, and biocompatibility, as well as natural radiolucency.However, macrophages persist in the M1 state on the PEEK surface, leading to fusion into multinucleated giant cells, which contribute to the formation of brous encapsulation and inferior osseointegration.O 2 plasmatreated PEEK coated with PAH/poly(acrylic acid) LbL lm built at pH 1.8 and ended by PAH inhibits the early M1 polarization of macrophages, promotes the M2 polarization with prolonged culture time, and suppress the osteoclast formation. 204

Neural interfaces
Protein-based LbL lms can provide physicochemical as well as bio-specic cues for attachment, proliferation, and differentiation of neural cells (Scheme 7).
Laminin (Ln), a tri-peptide glycoprotein, is known to favour neurite outgrowth by integrin-specic interactions.Fn/poly(Dlysine) (PDL) and Ln/PDL were built on silicon rubber, functionalized by (PSS/PEI) 3 precursor layer.Stable in PBS for 30 days, both lms showed comparable neuronal cell attachment, differentiation, and neurite outgrowth with an elongated shape. 236Silicon microelectrode arrays (Si MEAs) can detect

Perspective
Chemical Science neural activity in vivo.However, in terms of impedance, their long-term performance is compromised by brous encapsulation and scar tissue development.Si MEAs were coated with Ln/ PEI, Gel/PEI, and Gel/CHI LbL lms (Fig. 14a).In comparison to Gel/PEI and Gel/CHI LbL lms, Ln/PEI LbL lms coated on Si MEA showed the highest attached neuron density (4 h) and neurite outgrowth with clearly observable growth cones and network connections.The cell spreading was better and neurites were longer and thicker.Gel/CHI showed the least attached neuron density in comparison to Gel/PEI.The authors suggested that the interpenetration of underlying bulky CHI chains renders fewer Gel sites available for neuron attachment, in comparison to linear PEI chains.Nevertheless, the neurite outgrowth was comparable to Gel/PEI aer 24 and 48 h. 237milar results were reported for Gel/CHI LbL lms coated on PLLA electrospun bres.The number and length of dendrites (a differentiated form of neurons responsible for signal transfer) were higher with Gel/CHI multilayers irrespective of the ending layer compared to control monolayers of Gel or CHI.Ln was also assembled with CHI in a LbL manner to coat PLLA electrospun bres.Ln/CHI coatings were stable in PBS at 37 °C with less than 10% loss of Ln aer 20 days.The neuronal cell adhesion, viability, neurite length, and proliferation of dorsal root ganglia neurons improved by increasing the number of Ln/CHI bilayers (Fig. 14b) as well as for neuronal stem cells.The better performance of Ln/CHI lms is associated with the higher amounts of laminin incorporated with each bilayer. 238n the case of COL/HA LbL, neuronal cell density, and neurite length decreased with the increase of layers due probably to higher lm roughness/heterogeneity. 239 The adhesion and outgrowth of hippocampal and cortical neurons showed a preference towards HA/PAH and COL/HA LbL lms, respectively.Cortical neural prostheses are used to record and translate neural signals into movement commands for paralyzed patients.These electrodes oen fail to ensure their role due to inammation and neuronal loss around the implant.To stimulate neurite outgrowth, Gel/neurotrophin nerve growth factor (NGF) based LbL coatings were deposited on the prostheses.In contrast to Hep/NGF and DS/NGF LbL lms, a higher loading and sustained delivery of NGF were obtained for up to 2 weeks under PBS degradation triggering upregulating of neurite outgrowth from PC12 cells. 175rrent status, challenges, and future research directions Since 1992, layer-by-layer (LbL) technology has been used to assemble polyelectrolytes, natural polymers, and proteins for various healthcare applications.Unlike traditional polyelectrolyte LbL lms, there is a lack of in-depth understanding of the structure and dynamics of protein-based LbL lms and their correlation to the intended biological function.Traditional materials characterization techniques based on light, electron, or X-ray are limited by spatial resolution, sensitivity, sample size, and the need for sample preparation.Despite the potential benets, the use of ECM-derived proteins in LbL assembly is limited by their structural complexity.As natural polymers, proteins can vary in molecular weight, leading to batch-to-batch variability and contamination issues, especially when produced in E. coli.When assembled in LbL, proteins can experience denaturation, structural changes, and loss of biological activity due to complexation with the partner material.Optimization of assembly conditions and choice of LbL partner is therefore necessary for controlled adsorption, orientation, and organization of proteins.Attempts have been made to manipulate the complex surface charge properties of proteins, such as the use of protein nanoparticles/crystals and proteinpolymer complexes.Engineered proteins with enhanced stability, specicity, and functionality tuneable properties could overcome some of the limitations associated with native

Chemical Science Perspective
proteins, facilitating their integration into LbL lms.However, protein production and purication can be expensive, tedious, and time-consuming tasks.In addition to the cost and availability of proteins in large quantities, there are still some limitations to overcome for commercial scale-up applications, such as difficult control of lm uniformity (in terms of thickness, roughness, and architecture), extended build-up time (for dip coating), batch-to-batch variability, industrial adaptability of equipment and its automation for lm production, and quality control.In addition, the incorporation of proteins into LbL lms on a commercial scale may raise regulatory concerns regarding safety, potential contamination, and biocompatibility.Therefore, it is essential to maintain a sterile environment (such as a clean room) or choose the right sterilization methods (heat or irradiation can damage proteins).Finally, for practical applications, a robust and stable LbL lm is oen required, which is achieved by chemical crosslinking, which can adversely affect the biological function of proteins.In this regard, maintaining the biological activity of proteins in LbL lms can be challenging.

Conclusions
The use of proteins as components of LbL lms makes it possible to impart various biological properties to biomaterial surfaces using a simple method, performed at room temperature in water, without chemical modication.The LbL lms could be antibacterial, promote or prevent mammalian cell adhesion/differentiation, or deliver drugs/genes.First, all protein-based lms can be used to deliver (macro)molecules (antibacterial agent, drug, gene, etc.) by enzymatic degradation by a-chemotrypsin or specic enzymes (collagenase).Carrying pro-adhesive moieties (RGD sequence), COL, Gel and Fn-based lms promote broblast, endothelial, and pre-osteoblast adhesion with an inuence of the nal layer.COL-based lms are also osteoinductive, promoting the recruitment and differentiation of stem cells into the osteoblastic lineage.Ln-based lms have been specically used to promote neuronal outgrowth and maturation through integrin-specic interactions.BSA, a plasma protein with anti-thrombogenic properties, was also used to reduce non-specic platelet adhesion.PRMbased lms were mainly used for gene transfection.Antibacterial enzymes were incorporated to achieve antibacterial properties due to their ability to degrade biolm components (proteoglycans) or quorum-sensing molecules.Specic proteins such as immunoglobulins or growth factors have been used for recognition or stem cell differentiation.Gel-based lms have been used to coat drug nanocrystals for sustained release in a physiological medium while avoiding burst release.By a judicious choice of the partners and association, multifunctionality can be obtained.This could be a nice and interesting perspective of such LbL lms.Predicting LbL properties, in particular protein-based lms, remains a challenge.Machine learning methodologies emerge and potentially revolutionize the eld.Recently, Vrana and co-workers used literature data and in-house generated experimental results for coating thickness prediction.These studies could be the rst steps for the prediction of biological properties. 240,241

Fig. 2
Fig. 2 Adhesion-resistant and contact-killing LbL films.(a) Schematic illustration of bacterial tests on native and LL-37 functionalized COL/ HA LbL films.Antiadhesive properties of native LbL (0 mM LL-37) and antibacterial activity of LL-37 functionalized LbL against E. coli (green = live bacteria and red = dead bacteria).This figure has been adapted from ref. 54 with permission from Elsevier, copyright 2016.(b) Scheme of the fabrication process of silk fibroin/nylon-6 nanofibrous mats by electrospinning and the build-up of COL/lysozyme (Ly) LbL films onto the nanofibrous mats, and the antibacterial activity as a function of the number of bilayers against S. aureus and E. coli.This figure has been adapted from ref. 87 with permission from Elsevier, copyright 2020.

Fig. 3
Fig. 3 Release-killing LbL films.(a) Effect of the buffer on the build-up, topography, complexation, and antibacterial property of collagen/ tannic acid (COL/TA) LbL films.This figure has been adapted from ref. 96 with permission from American Chemical Society, copyright 2010.(b) Ca 2+ -binding mediated sustained release of minocycline (MN) from dextran sulfate/gelatin type A (DS/Gel) LbL shows antibacterial activity.This figure has been adapted from ref. 100 with permission from PLOS ONE, copyright 2014.(c) The release of nitric oxide (NO) gas from gelatin/tannic acid (Gel/TA) LbL film shows antibacterial activity towards S. aureus.LbL and NO-LbL stand for Gel/TA and NO-generating moieties functionalized Gel/TA, respectively.This figure has been adapted from ref. 101 with permission from Nature Springer, copyright 2019.

Scheme 3
Scheme 3 Schematic representation of the LbL strategies to promote cell adhesion.103

Fig. 4
Fig. 4 Collagen-based LbL films.(a) Intrinsically crosslinked collagenbased LbL films with oxidized glycosaminoglycans (either hyaluronic acid or chondroitin sulfate) show preservation of COL fibrillar structures and enhanced adhesion, cell size, and polarization of embryonic fibroblasts, thanks to increased film stiffness due to the crosslinking.This figure has been adapted from ref. 115 with permission from American Chemical Society, copyright 2014.(b) Scheme for build-up of oriented collagen/tannic acid (COL/TA) LbL film using a brushing LbL method, and use of the LbL for orientation and differentiation of human myoblasts into myotubes.This figure has been adapted from ref. 124 with permission from American Chemical Society, copyright 2022.

Fig. 5
Fig. 5 Gelatin and fibronectin-based LbL films.Functionalization of polyethylene terephthalate (PET) ligaments with HA/Gel LbL film implanted in vivo in a porcine model showed regeneration of anterior cruciate ligament.This figure has been adapted from ref. 133 with permission from PLoS One, copyright 2012.

Fig. 6
Fig. 6 Use of elastin in LbL films.Clickable elastin-like recombinamers (ELR), modified by either azide or cyclooctyne, LbL film on coronary stent reduces platelet adhesion and promotes endothelial layer formation, thanks to the presence of RGD-sequence on ELR-azide.This figure has been adapted from ref. 136 with permission from Elsevier, copyright 2019.

Fig. 7
Fig. 7 LbL films inhibit platelet adhesion.(a) Bovine serum albumin/ heparin (BSA/HEP) LbL on polyethersulfone foils (PES) reduces the adhesion of platelets and leukocytes upon incubation in slightly heparinized (1.5 IU heparin per mL) human whole blood in vitro.This figure has been adapted from ref. 146 with permission from John Wiley and Sons, copyright 2006.(b) (Fn/PLL) LbL films deposited on different polyurethanes influence the behavior of monocytes.This figure has been adapted from ref. 149 with permission from Elsevier, copyright 2018.

Fig. 8
Fig. 8 LbL films promote endothelial cell adhesion.(a) Human umbilical vein endothelial cells (HUVECs) formed a confluent layer on genipin cross-linked (ALG/COL) LbL after 5 days compared to glutaraldehyde ones.This figure has been adapted from ref. 55 with permission from American Chemical Society, copyright 2012.(b) Collagen/heparin (COL/HEP) LbL ended by CD34 antibody coated on stents showed rapid endothelialisation and no neointimal hyperplasia in rabbit femoral arteries in vivo.This figure has been adapted from ref. 166 with permission from Elsevier, copyright 2010.

Scheme 5
Scheme 5 Schematic illustration of drug/gene release from the LbL films under various stimuli i.e., diffusion or erosion, pH change, and enzymatic degradation.

Fig. 9
Fig. 9 Drug release by diffusion or erosion.(a) Efficient loading and sustained delivery of transforming growth factor beta 1, plateletderived growth factor bb, and insulin growth factor 1 from either growth factor/heparin LbL films.The films resulted in the proliferation, migration, and differentiation of fibroblast cells into myofibroblasts (a-SMA labelling).This figure has been adapted from ref. 200 with permission from the Royal Society of Chemistry, copyright 2020.(b) Protamine sulfate/hepatocyte growth factor-pDNA (PRM/HGF-pDNA) LbL, on top of human umbilical vein endothelial cells (HUVEC) and human umbilical artery smooth muscle cells (SMC), induced the secretion of HGF by the cells and showed competitiveness of ECs over SMCs.This figure has been adapted from ref. 197 with permission from Elsevier, copyright 2013.(c) Direct nano-encapsulation of furosemide microcrystals with Gel/PSS LbL to enable sustained drug release profiles.This figure has been adapted from ref. 178 with permission from Elsevier, copyright 2003.

Fig. 10
Fig. 10 Drug release at physiological pH.(a) Scheme of gelatin A/ epigallocatechin gallate (Gel/EGCG) LbL hollow capsule preparation, and fluorescence/AFM images.This figure has been adapted from ref. 174 with permission from Elsevier, copyright 2009.(b) Hydrolysable polymer associated with ovalbumin and CpG, the release of ovalbumin, an optical image of LbL-coated PDMS substrate, and the scheme of its application onto tape-stripped ear skin and confocal images showing penetration of fluorophore-conjugated ova (red) and CpG (blue) released from LbL films into the skin.This figure has been adapted from ref. 189 with permission from American Chemical Society, copyright 2009.

Fig. 11 Scheme 6
Fig. 11 Drug release by enzymatic degradation of LbL films.Scheme of PRM/HA LbL coated bioactive micelles for targeting tumours, and biodistribution in vivo.This figure has been adapted from ref. 193 with permission from Elsevier, copyright 2018.

Fig. 12
Fig. 12 LbL films supporting osteoconduction.(a) Polyethylene imine/ gelatin (Gel/PEI) LbL increased the proliferation of human osteoblast cells, thanks to the hydrophilic nature of the LbL film.This figure has been adapted from ref. 209 with permission from Elsevier, copyright 2012.(b) Chitosan/gelatin/hydroxyapatite (CHI/Gel/HAP) composite LbL films on titanium alloy, a cross-section of the films by SEM, and mineralisation of osteoblasts after 7 and 14 days of culture.This figure has been adapted from ref. 221 with permission from Elsevier, copyright 2014.

Fig. 13
Fig. 13 LbL films supporting osteoinduction.Gel/CHI/Gel/BMP2 LbL films were built on a titanium alloy rod for implantation in rabbit femur in vivo.Gel and BPM2 were released in a physiological medium.LbLcoated surfaces show better mesenchymal stem cell mineralization, and bone formation (MicroCT and histochemical analysis).This figure has been adapted from ref. 231 with permission from Elsevier, copyright 2012.

Scheme 7
Scheme 7 Schematic illustration of neurogenesis, i.e., differentiation cascade of neural stem cells to form mature neurons, onto LbL films.

Fig. 14
Fig. 14 LbL films promote neural cell adhesion and differentiation.(a) Silicon microelectrode arrays (Si MEAs) coated with various LbL films, with compositions such as PEI/Gel, CHI/Gel, and PEI/laminin (Ln), show better chick cortical neuron differentiation only when using protein-based LbL, i.e., Gel or Ln, among which PEI/Ln was the best composition.This figure has been adapted from ref. 237 with permission from Elsevier, copyright 2005.(b) Chitosan/laminin (CHI/ Ln) LbL film on poly-L-lactide (PLLA) electrospun scaffold, with no preferential orientation of the nanofibers, increased the neurite outgrowth of neurons after 7 days.Increasing the number of bilayers from 1 to 5 enhanced the neurite outgrowth, thanks to a higher amount of Ln incorporated into the LbL film.This figure has been adapted from ref. 238 with permission from John Wiley and Sons, copyright 2013.

Table 1
Protein-based LbL films reported to modulate mammalian cell response a