Polylysine for skin regeneration: A review of recent advances and future perspectives

Abstract There have been several attempts to find promising biomaterials for skin regeneration, among which polylysine (a homopolypeptide) has shown benefits in the regeneration and treatment of skin disorders. This class of biomaterials has shown exceptional abilities due to their macromolecular structure. Polylysine‐based biomaterials can be used as tissue engineering scaffolds for skin regeneration, and as drug carriers or even gene delivery vectors for the treatment of skin diseases. In addition, polylysine can play a preservative role in extending the lifetime of skin tissue by minimizing the appearance of photodamaged skin. Research on polylysine is growing today, opening new scenarios that expand the potential of these biomaterials from traditional treatments to a new era of tissue regeneration. This review aims to address the basic concepts, recent trends, and prospects of polylysine‐based biomaterials for skin regeneration. Undoubtedly, this class of biomaterials needs further evaluations and explorations, and many critical questions have yet to be answered.

needs further evaluations and explorations, and many critical questions have yet to be answered.

K E Y W O R D S
cationic polymer, polyelectrolyte, poly-L-lysine, regenerative medicine, tissue engineering

| INTRODUCTION
The ultimate goal of tissue engineering is to develop materials that can mimic the natural properties of tissue, either as a substitute for tissue repair or to encourage regeneration. Tissues damage can occur due to various causes, such as accidents and disease. The materials utilized for tissue regeneration should mimic the natural behavior of tissue and possess suitable mechanical properties and architectural structure. In this regard, several sophisticated molecules and materials have been designed to meet these requirements. The design of scaffolds for tissue engineering is a tradeoff between the complexity of the molecules and the simplicity of the application, and requires profound knowledge about the relationship between material science and biology. [1][2][3] Polymers have attracted significant attention in the realm of regenerative medicine, because of their unique properties and chemical versatility. These macromolecules can be synthesized as various chemical and physical structures resulting in wide range of physicochemical properties. For example, polymers can be designed to meet the requirements for mimicking soft tissue (e.g., skin) and for hard tissue (e.g., bone). 4,5 In this regard, many natural and synthetic polymers, such as chitosan, 6,7 gelatin, 8,9 and agarose 10,11 have been investigated for tissue regeneration because of their beneficial properties.
These polymers can be categorized based on their charge, as neutral, cationic, or anionic polymers. For example, most of the polysaccharides are anionic polymers due to the presence of pendant anionic functional groups (e.g., hydroxyl and carboxyl) on their main chains.
On the other hand, cationic polymers are positively charged polyelectrolytes, carrying positive charges either on their backbone or on the side chains, and have attracted much interest among the polymer families. Cationic polymers have been more often used in biomedical applications compared to anionic polymers, because of their interaction with negatively charged biomolecules, peptides, proteins, and nucleic acids. Polycationic polymers can interact with negatively charged cell membranes and enhance cellular activities. [12][13][14][15][16] Polycationic polymers such as poly-L-lysine (PLL), polyethyleneimine (PEI), and poly(amidoamine) (PAMAM) dendrimers have been widely utilized to carry cargos (e.g., drugs, genes, and so on) across the cell membrane. [17][18][19] They can be used to form polyelectrolyte complexes (PECs) by combining with anionic species such as polyanions, nucleic acid chains, and some drugs. 20 Besides, they can form layer-by-layer assemblies. 21 Moreover, some polycationic polymers exhibit intrinsic bioactivity, with antibacterial, antioxidant, and antitumor properties. 22,23 Polylysine is a cationic polymer, which has attracted significant attention in tissue engineering because of its biodegradability and biocompatibility. However, because of its poor mechanical properties, it is not often used alone, and it should be copolymerized or blended with other polymers to improve its mechanical properties. Controlled polymerization, grafting, and modification techniques have been used to prepare PLL-based materials with the desired architecture, properties, and multi-functionality as biomimicking scaffolds for tissue engineering applications. 22 Due to the unmet need for skin tissue regeneration and considering the adjustable and unique properties of the polylysine, it is necessary to review the polylysine performance in skin tissue engineering.

| THE CHEMISTRY OF POLYLYSINE
Lysine (an essential amino acid for humans) is the primary building block of polylysine, and is available in two chiral forms: L-lysine and D-lysine. Polylysine can be synthesized through condensation polymerization or by fermentation, which results in α-polylysine or ε-polylysine, respectively. ε-Polylysine allows the linking of ε-amino and α-carboxylic acid moieties and is utilized as a food preservative due to its nontoxicity and antibacterial properties. The conformation of poly-L-lysine (PLL) depends on pH, temperature, ionic strength, and solvent type. 24,25 PLL in aqueous solution exhibits the P II conformation at low temperature and neutral pH, while by raising the pH the conformation changes to an α-helical conformation, and by raising the temperature it changes to a β-sheet structure. It is thought that the steric interactions between the PLL side chains (uncharged), the hydrogen bonds of the solvent or PLL, and the proton donor and acceptor groups in the solvent, stabilize the P II conformation. 24 The different forms of lysine and their different polymerization structures are depicted in Figure 1.
Amino acid N-carboxyanhydrides (NCAs), which are also known as leuchs anhydrides are well-known reactive amino acid derivatives. 26 Ring-opening polymerization of NCAs is a useful method for polypeptide synthesis because of its good yield. 27,28 PLL has been synthesized using the conversion of and ε-primary amine-protected L-lysine derivative into the cyclic N ε -(benzyloxycarbonyl)-L-lysine N-carboxyanhydride monomer. NCA was polymerized via ringopening polymerization starting with a primary amine initiator which can operate in two ways: (1) attacking the C5 of NCA as a nucleophile (amine mechanism); and (2) deprotonating the N 3 of NCA as a base (active monomer mechanism). Moreover, living polymerization using metal complexes has been used for NCA polymerization ( Figure 2). 22,29 3 | INTERACTIONS BETWEEN CELLS AND POLYLYSINE SUBSTRATES

| Simple polylysine substrates
The interactions between cells and their neighboring environment play a key role in governing cellular activity, such as during the processes of morphogenesis, embryogenesis, homeostasis, thrombosis, inflammation, and wound healing. 30,31 The extracellular matrix (ECM) binding-site affects the shape of the cell and its motility. The first step in cellsubstrate interaction is the binding of integrin receptors to ECM molecules, followed by the aggregation of receptors at the contact point. [32][33][34] This is called the focal adhesion complex. Intracellular signaling cascades are triggered, resulting in the binding of the actin cytoskeleton to the ECM mediated by integrins. 35,36 Surface treatment of surfaces using polycationic polymers, such as polylysine, has been used to improve cellsubstrate interactions and facilitate cell adhesion. 37 The use of polyamine acids enhances cellular attachment to the substrate. Cellular functions are affected by this attachment and by ECM components. Some cells are able to produce ECM components, while other cells require an adjuvant to attach to the surface (especially in serum-free media). Polylysine (because of its positively charged nature) can easily interact with the negatively charged surface on many types of cells. Therefore, cells can rapidly attach to polylysine substrates. Interestingly, some cells (e.g., sea urchin eggs) that normally have difficulty to attach to solid surfaces, can adhere to the polylysine surface, as well as mammalian cells. 37 Polylysine chains with higher molecular weights exhibit a higher tendency to bind to cell membranes. Moreover, flexible polycation chains exhibit higher cellbinding compared to rigid chains. 38 It has been observed that culturing mesenchymal stem cells (MSCs) on PLL-coated substrates could preserve stemness and prevent senescence of these cells. 39 PLLcoated plates enhanced the growth of MSCs, and up-regulated some genes that contribute to cell-adhesion, cell differentiation, proliferation, and signaling. 39 Cell-proliferation and osteogenic differentiation were improved by porous PLL-coated poly(lactide-co-glycolide) (PLGA)/hydroxyapatite (HA) scaffolds. 40 Quirk et al. used coating with polylysine to attach the cells to the PLA surface. 41 The optimum concentration of polylysine for cellular attachment was reported to be approximately 0.05-0.5 μg/cm 2 while higher concentrations may result in toxicity. 42 Sakai et al. used

| Patterned polylysine substrates
Besides physical and chemical cues which affect the cell behavior, other geometrical cues or anisotropic physical features (e.g., parallel microgrooves and collagen fibers) also affect the cell orientation on both two-(2D and three-dimensional (3D) substrates, which is known as the contact guidance phenomenon. 44,45 Contact guidance affects the shape, adhesion, and migration of cells. Indeed, cell migration, which is a key factor in many biological processes, is affected by both ECM guidance cues (e.g., a chemical species gradient or stiffness) and by contact guidance. Contact guidance results in cancer cell movement across the neighboring ECM and then to metastasis, by a process known as intravasation. 46,47 Furthermore, this phenomenon plays a key role in the wound healing process, where platelets and fibroblasts can induce a local contraction in the wound site, resulting in ECM fibers becoming radially oriented. This local fiber orientation guides the cell migration. 48 Appropriate signaling between cells, which is mediated via ECM proteins, significantly affects the redistribution of molecules that allow cell adhesion in dot or focal patterns. Cells are able to attach to the patterned polylysine substrate, while actin stress fibers are not created between neighboring dots. Irregular morphology was observed for cultured cells in which filopodia-like extensions were abundant; besides no clustering was observed for focal adhesion molecules. 49  Patterned co-cultures can be created using three different techniques. Photolithography is a robust method to create patterned surfaces composed of different materials, that allow selective adhesion of various cells onto the predetermined area. For example, when coculturing hepatocytes and fibroblasts on micropatterned substrates, hepatocytes selectively adhered to the collagen islands which optimized the interaction between the different cells. 50,51 The second approach involves the delivery of cells to the specific areas on the substrate. For instance, patterning of various types of cells onto specific regions was achieved using a microfluidic device. 52,53 Furthermore, cell attachment to specific regions of the substrate can be excluded using elastomeric membranes. After removing the membrane, the remaining surface can be used to attach another different cell type. 54 However, these strategies suffer from some limitations.
The construction of multicellular systems may be limited using photolithography, as each step requires the selective attachment of a single cell type. Moreover, geometrical constraints induced by laminar flow and separation between two different cell types because of channel interspacing are limitations associated with the microfluidic method. 55 Stimuli-responsive surfaces can be used as a general strategy to overcome these limitations; cell repelling surfaces can be switched to cell adhesive surfaces via the application of a specific stimulus. 56,57 For example, the degree of hydrophilicity of electroactive or thermosensitive substrates can be tuned to enhance or diminish cell adhesion. However, high-cost materials and sophisticated synthetic procedures are required to prepare such switchable surfaces, and this F I G U R E 2 Synthesis of poly-L-lysine (PLL). PLL was synthesized using the conversion of the ε-primary amine-protected L-lysine into the cyclic N ε -(benzyloxycarbonyl)-L-lysine N-carboxyanhydride monomer. NCA via ring-opening polymerization was polymerized starting by primary amine initiator which can operate in two ways by (A) attacking the C5 of NCA as a nucleophile (amine mechanism) and (B) deprotonate the N 3 of NCA as a base (active monomer mechanism) may limit their application. Accordingly, there is an urgent need to develop improved surface engineering strategies to fabricate patterned cell co-cultures. Layer-by-layer assembly (L-b-L) of polyelectrolytes can be potentially used to fabricate such switchable surfaces, especially thin films. 58 Khademhosseini et al. co-cultured either hepatocytes or embryonic stem cells with fibroblasts on a micropatterned hyaluronic acid (HA)/polylysine surface. This method was also used for patterned and controlled cell co-culture. 59 HA is a biocompatible and biodegradable polysaccharide that has been widely studied for  64 This group studied stem cell distribution, RhoA activation, and cell differentiation. Cells growing on a glass substrate proliferated more than cells on a soft substrate. Consequently, the finding that during a long incubation period of 21 days, stem cells plated on a charged, rigid, glass substrate became differentiated, even with no chemical induction signals. 60 Better knowledge about the cell-substrate interactions allows us to understand aspects of cellular activity, such as adhesion and migration and helps us to design a biomimetic surface for the best in-vivo function and to achieve proper tissue regeneration.

| POLYLYSINE ANTIBACTERIAL ACTIVITY
4.1 | Gram-negative bacteria such as Escherichia coli ε-Poly-L-lysine (ε-PL) exhibits the most pronounced antibacterial activity within the polylysine family, and inhibits the proliferation of most F I G U R E 3 Cell spreading on polylysine. B16 cells were cultured on patterned substrata of fibronectin (red; A) or polylysine (red; B,C) and labeled for actin (green; A,B) and paxillin (green; C). Cells can adhere to polylysine dots, however, the actin cytoskeleton is not reorganized (B) and paxillin does not accumulate over the dots (C). Scale bars: 10 μm. Reprinted with permission from Reference [49] microorganisms (Table 1). Generally, three mechanistic models including, barrel-stave, toroidal, and the carpet mechanism have been proposed to explain the antibacterial activity of polycationic polymers like PLL. The barrel-stave mechanism involves a rapid interaction with the hydrophobic core of the membrane lipid bilayer, while the other mechanisms rely on an interaction with the membrane surface and the headgroups. The residues with a hydrophobic nature not only can replace the headgroups of phospholipid units through a mechanism based on toroidal pore modeling, but can also induce pore lumen formation by altering the positive membrane curvature. The carpet-like mechanism is based on a theory in which membranes are permeabilized in a nonspecific manner, involving only a contact between the positively charged peptide and the negatively charged head-groups of the phospholipid. 71,72 The three models discussed above may not be sufficient to explain all the possible interactions within the membrane, so other models should be taken into consideration. 72 Scientists have also tried to explain the antimicrobial activity by other models, such as the gripand-dip, as well as the disordered toroidal pore model. 73,74 Moreover, many believe that some interactions could be mediated by a process analogous to electroporation, called the sinking raft model. 75 It should be noted that in addition to the peptide structure and headgroup constitution of the membrane, the composition of existing acyl chains could also affect the peptide interaction with the membranes. 76 Hyldgaard et al. clarified the mechanism of the antibacterial properties of ε-polylysine against E. coli as Gram-negative and Listeria innocua as Gram-positive model bacteria. It was found that some components of the E. coli lipopolysaccharide layer, such as the divalent cations, along with heptose (Types I and II)-phosphate could play a role in the binding process with polylysine. Polylysine destroyed the E. coli lipopolysaccharide layer and altered its morphology, while L. innocua showed only minimal morphological changes. The membranes of both the bacteria were attacked by polylysine, as shown by cytoplasmic membrane permeabilization. The carpet-like model could also explain how polylysine could disrupt the membrane stability. This involves the polylysine binding to negatively charged phospholipids and releasing existing divalent cations, and consequently changing the membrane curvature into negative folding, thereby producing structures similar to vesicles or micelles ( Figure 6). 69 The concentration of divalent cations in the ambient medium will competitively inhibit the binding of ε-PL to the bacterial membranes.
Moreover, an increase in the pH will decrease the positive charge on the ε-PL, and subsequently, reduce the electrostatic interaction with the cells, F I G U R E 4 Polylysine and stem cell differentiation. (A) Confocal laser scanning microscopy images of adherent hADSC on flat (F) PEM. The platforms were cross-linked using EDC (50, 10, and 2 mg mL À1 ). Afterward, the cells were cultured and incubated overnight (actin cytoskeleton (red), nucleus (blue), and vinculin in focal adhesions (green)). S, M, and L refer to small, medium, and large, respectively. PLL/HA and 50 mg mL À1 EDC cross-linked nanostructured platforms were viewed. Nanostructured platforms were treated with PLL/HA and cross-linked with 50 mg mL À1 EDC. (B) hADSC chondrogenic differentiation. (C) hADSC osteogenic differentiation. Reproduced with permission from Reference [21] and lessen the bactericidal activity. ε-PL shows selective antimicrobial activity against a wide range of microorganisms, without any pronounced cytotoxicity towards eukaryotic cells, because the phospholipid head groups are different in eukaryotic and prokaryotic membranes.
In addition to membrane disruption, the antibacterial activity of   without affecting the cytoplasm, it is thought to be unlikely to produce any antimicrobial resistance even after repeated application. 80

| Mycelial fungal cells such as Penicillium digitatum
Other have reports revealed that eukaryotic fungal cells (such as P. digitatum) could be inhibited by ε-PL. 81 ε-PL affected mycelial production, and the spore germination rate and germ tube duration were significantly inhibited. Moreover, ε-PL could also affect the cell wall morphology in eukaryotic fungal cells, and disrupt their cell membrane. 82 These findings, therefore, showed that mycelial inhibition, spore development, and damage to the cell wall and membrane were all caused by ε-PL and were linked to the death of eukaryotic fungal cells.

| Yeast cells such as Saccharomyces cervisiae
It was observed that the fungicidal and fungistatic activities of ε-PL were different against yeast species, as shown for S. cerevisiae cells.
As shown in Figure 9, the potential mechanism has been suggested for the antifungal activity of ε-PL, which binds to components of the cell membrane by electrostatic forces. If the concentration ε-PL exceeds a threshold value, ε-PL will rapidly become embedded in the First, cationic ε-PL attaches to negatively charged teichoic acid embedded in the peptidoglycan layers of the S. aureus cell wall. The peptidoglycan structure in the cell wall is destroyed by ε-PL, leading to cell wall fragility. Next, the cell membrane is disturbed by ε-PL, and this disturbance further induces the changes in hydrophobic region and bilayer curvature of cell membrane, causing increase of cell membrane permeability. Finally, ε-PL enters into the cells and interrupts the primary metabolism of S. aureus; thus, killing the cells. Reproduced with kind permission from Reference [79] regeneration. It is possible for acute injuries causing either superficial or deep wounds to repair completely within 21 days, and little to no scar tissue formation is usually observed. In contrast, chronic wounds usually develop when acute wounds fail to heal even after 3 months. Diabetic wounds, which are common in individuals suffering from diabetes, are considered to be chronic. A more complete classification of various wounds is represented in Figure 10. 86 The most important but challenging issue in wound treatment is to recover the tissue function with an acceptable aesthetic outcome in a short period of time. Large wounds especially in unhealthy condition such as diabetes, infectious disease and cancer can be very difficult to recover.
Inadequate wound healing or excessive scar formation can affect many people after surgery, major trauma, or disease. There is still an  Figure 11B). The first thing to happen is the rapid activation of the coagulation cascade to produce a fibrin clot, which the provides the essential matrix architecture for recruiting inflammatory and other cell types into the wound. Platelets immobilized within the fibrin clot secrete localized growth factors and chemokines into the wound vicinity in order to attract other cells. The importance of platelets for effective wound healing has been demonstrated by the therapeutic application of platelet-rich plasma rich in preclinical studies of wound healing, 89 and the incidence of delayed healing in disorders related to platelets. 90 In addition to hemostasis, the early inflammatory responses (both local and systemic) activate wound healing responses ( Figure 11A). Inflammation is more pronounce in deep and more severe injuries ( Figure 11B), It is thought that the chronic inflammatory state of such injuries can fail to resolve, thus, producing chronic wounds. 91 More specifically, recent studies using molecular analysis of severely wounded tissue and F I G U R E 1 1 Molecular and cellular processes for the repair of normal skin. Molecular and cellular pathways for wound healing progression. The first step of wound healing comprises hemostasis and keratinocyte activation and inflammatory cells. The intermediate step includes keratinocytes, fibroblast proliferation, matrix deposition, along with angiogenesis. This spatiotemporal cycle is highly governed through several cell types that release various growth factors, cytokines, and chemokines to obtain wound closure and functional restoration. The chronic wound molecular pathology. Illustrations show the cellular and molecular mechanisms that are damaged in severe injuries. Although the increased inflammatory cells such as macrophages and neutrophils, they do not all work perfectly (validated using misshaped cells). Most fibroblasts mature into senescent ones. For chronic wounds, angiogenesis is decreased, the recruitment and activation of stem cells and the remodeling of ECM relative to the healing of injuries with the normal rate. Histology reflecting features of a diabetic foot ulcer (DFU), venous stasis ulcer (VLU), and bad sore. Such chronic wounds, though different etiology, have specific cellular characteristics depicted in (A): H, hyperproliferative epidermis; F, fibrosis, increased cellular inflammation. 87 (C) Schematic representation of the required properties of a wound dressing material. Reproduced with permission from Reference [88] wound fluid have shown a continuous competitive environment involving both pro-inflammatory and anti-inflammatory signals. 91,92 An increased number of neutrophils and macrophages are major components of the pro-inflammatory cellular infiltrate that contributes to the delayed healing of chronic ulcers. 93 These cells secrete primary pro-inflammatory cytokines, including IL-1β and tumor necrosis factor-α (TNFα), which have been shown to prolong the inflammatory process and thereby delay healing. 92,94 Increased IL-1β and TNFα in chronic wounds have been shown to lead to an increase in metalloproteinase enzymes that cause excessive local degradation of the ECM, and therefore prevent the migration of other cells designed to heal the wound. 95 Recent studies have involved the inflammasome, a multiprotein complex produced by the innate immune system that continuously activates and secretes IL-1β and IL-18 inside chronic wounds. 96,97 Furthermore, the ongoing presence of a relatively high load of bacterial cells within chronic wounds (colonization) also continuously attracts pro-inflammatory cells resulting in delayed wound healing ( Figure 11). 87 A more complete insight into the mechanisms regulating the inflammatory response is required before better approaches to the healing of chronic wounds can be devised. 87 The ideal wound dressing should provide a suitable milieu for accelerating wound healing, minimizing the scar, increasing cellular activity (i.e., epithelial spreading, angiogenesis, and connective tissue synthesis). Wound dressings should also absorb wound exudate, maintain the proper moisture content and temperature within the wound (which also facilitates blood circulation and epithelial migration), exchange nutrients and oxygen, reduce pain, protect the wound against infection, and be nonadherent to the wound (i.e., easy to remove the wound dressing after healing). Moreover, dressings should encourage leucocyte migration, facilitate the activity of enzymes that can carry out debridement, and must also be sterile, nontoxic, and nonallergic. 84 Table 2 present the guidance for wound management and some synthetic commercial wound dressings available on the market are summarized in Table 3. Moreover, this platform showed good biocompatibility and antibacterial activity against both Gram-positive and -negative bacteria. In-vivo studies showed that the platform possessed an antiinfective properties. 117 Self-healing properties were introduced into the hydrogel by adding plasma amine oxidase to the ε-PL, because of the formation of Schiff base reaction products. 118 As mentioned above, ε-PL attaches to the bacterial surface destroying the cell membrane and causing cell lysis, resulting in cell death. SEM images were used to investigate the antimicrobial mechanism of ε-PL, by visualizing morphological changes occurring during cell-ε-PL interactions. As shown in Figure 12, rodlike (E. coli) and round (S. aureus) cellular morphology was preserved when the bacteria were cultured on a control hydrogel. By contrast, a disrupted and withered surface was observed for bacteria cultured on EHPA hydrogels. 117 S. aureus is a well-known species that contributes to many wound infections. This bacterial species was embedded in EHPA hydrogels, which were then subcutaneously implanted in a mouse model to assess its potential as an antimicrobial hydrogel in clinical application. Sterile saline, noncontaminated EHPA hydrogels, S. aureus bacteria alone, and bacteria loaded-EHPA hydrogels were all injected into groups of mice to evaluate the effects of the hydrogel on infection prevention and treatment. Three days after injection with bacteria alone, the positive control mice were close to death. The animals were divided into two groups; one group was sacrificed and dissected to observe the infected tissue. As shown in Figure 12  staining showed widespread bacterial infection that could be prevented by the EHPA hydrogel (see Figure 12). Furthermore, an inflammatory response was also observed in tissue infected with bacteria alone, with numerous inflammatory cells migrating to the injection site. However, injection of the bacteria-loaded EHPA hydrogel, sterile EHPA hydrogel, or sterile saline resulted in no inflammation ( Figure 12). 117  These polymers also showed good protection of pEGFP from in vitro digestion by DNase I and DNase II. Upon delivery by lipid substituted polymers, the intracellular pEGFP expression was stable for up to 7 days. Additional investigation using flow cytometry showed that modification of PLLs with stearic and myristic acid allowed successful protein expression, and myristic acid showed the lowest toxicity. They found that compared to pristine lipid alone, lipid conjugated PLL led to more efficient gene delivery. 125 Impaired angiogenesis is a significant clinical concern that might explain delayed wound healing, particularly in diabetes patients. One strategy to accelerate wound healing in diabetes patients, used an approach which could enhance angiogenesis. There were two peptides involved: (1) the TG peptide was covalently linked to the fibrin network for sustained release; (2) a polyR peptide was used to enhance the cellular uptake of these nanocondensates. To induce angiogenesis, both modified and unmodified polymers were condensed with plasmid-DNA in vitro. HIF-1α is an oxygen-sensitive transcription factor that contains a domain for oxygen-dependent degradation (ODD), and HIF-1α is naturally degraded under normoxic conditions. The temporary-induced F I G U R E 1 3 Future perspective and desired performance of the polylysine. Reprinted with permission from Reference [131] expression of the angiogenesis-linked genes Acta2, Pecam1, along with HIF-1α and VEGF was observed after PLL-g-PEG polymer-mediated DNA transfer of a HIF-1α À ΔODD plasmid. In addition, the delivery of the modified HIF-1α gene improved wound healing and increased the number of endothelial and smooth muscle cell precursors making more mature blood vessels. 126 Erlach et al. synthesized poly(2-methyl-2-oxazoline)-grafted poly(l-lysine) particles as a nonviral delivery vehicle for therapeutic DNA (TDNA) for improved wound healing. The polyplexes were soluble in serum and after heating to 70 C, and they preserved the condensed DNA from digestion by DNase-I. The efficacy of DNA-PMOXA-g-PLL DNA transfection was strongly dependent upon the number of PMOXA molecules grafted onto the polymer chains; a low grafting density between 7% and 14% and a medium N/P ratio (3.125-6.25) was found to be the best. As an alternative to PLL-g-PEG-DNA polyplexes formed from PLL20-g7PMOXA4 may be promising for the transfection of TDNA. 127

| TRANSLATIONAL SUCCESS AND CHALLENGES
Translational research tries to generate meaningful, applicable outcomes that advance human health and translate fundamental science findings more efficiently into practical usages. It is worth mentioning that healthcare is an important subject in translational research, particularly in using advanced materials. Different type of advanced biomaterials have been used as tissue replacements, regenerating scaffolds, drug carriers, and releasing vehicles known as a translational approach. Among these biomaterials, Polylysine can be engineered in different structures to be used in tissue engineering and as a drug/ gene carrier. It is reported that polylysine/CAG-peptide is used for the clinical treatment of cardiovascular diseases. 128 Moreover, polylysine/ chitosan is proposed as a proper substrate for clinical usage as an anti-hemorrhaging hydrogel. 129 However, it is reported that the polylysine show toxicity in some cases, and therefore it should be controlled for the desired application, which is known as a translation challenge. 130 It can be deduced that the successful translational of polylysine is a subtle balance between simplicity and complexity.
Hence, the polylysine-based products should be designed based on final application to achieve the proper results.

| CONCLUDING REMARKS AND FUTURE PERSPECTIVES
Polylysine can be used as a biomaterial to improve wound healing and skin regeneration due to its beneficial features. Several promising results have been obtained based on the preclinical and clinical experiments made in the past years. Herein, we have summarized several instances of polylysine and its composites being used as antimicrobial materials to assist wound healing and act as a gene delivery vector to treat skin diseases. Polylysine could be applied in different formulations: scaffolds, gels, dressings/films, or nanoparticles because of its stimuli-responsive performance, biocompatibility, and FDA approval ( Figure 13).
Suppose everything goes well with polylysine usage in wound management; in that case, it is expected that the polylysine scaffold shows proper antibacterial effect along with proper cell adhesion and proliferation, which causes rapid wound healing. Moreover, as a gene delivery vehicle, polylysine is one of the most preferred carriers for gene delivery which can be tailored to play a suitable role in gene therapy. Despite some promising results with formulations based on polylysine obtained in skin regeneration and wound healing, several challenges remain. For example, preparation methods and characterization techniques should be optimized and standardized. Adjusting the suitable surface properties, design, and pore size required by cells to proliferate and principally to differentiate with the right phenotype is also another challenge. Drug delivery applications should be tailored to biological mechanisms involved in wound healing, and thus, considering the essential role of proper vascularization is another challenge in this realm. Moreover, gene transfer applications should be further investigated to better control the proton sponge effect for lysosomal escape.

DATA AVAILABILITY STATEMENT
Data sharing not applicable to this article as no datasets were generated or analysed during the current study.