Cell membrane and extracellular vesicle membrane‐coated nanoparticles: An envisaged approach for the management of skin conditions

Abstract

Cell membrane‐coated biomimetic nanosystems have been recognized as promising drug delivery vehicles in recent years for the management of diverse skin conditions. Nanoparticles (NPs) coated with biomembranes, derived from either cell membranes or subcellular structures (e.g., extracellular vesicles), offer an opportunity to combine the biological interfacial characteristics of the coating alongside with the internal core component at the nanoscale. The biomimetic coating enhances the biocompatibility of NPs and their interaction with the skin, improving skin affinity, contact, and retention. This coating also enables the controlled release of drugs and provides skin‐targeting capabilities, which collectively improve the effectiveness, safety, and stability of topical and transdermal formulations. In this context, the current review delves into the recent progress in using biomimetic NPs for skin therapeutics. Specifically, it examines the various types of coatings, including their origins, heterogeneous functions, and surface molecular repertoires, in great detail. Additionally, this review presents the methods of preparing and characterizing biomimetic‐coated NPs. Furthermore, the potential of bioinspired NPs in treating a range of skin‐related conditions has been meticulously explored. Last, the toxicological aspects of these NPs have been thoroughly examined to provide a thorough summary of the evolution of biomimetic‐coated nanosystems for skin applications.

Abbreviations

• ADSC
• adipose-derived stem cell
• AMC
• adipose mesenchymal cell
• ATP
• adenosine triphosphate
• bFGF-R
• basic fibroblast growth factor receptor
• CuS
• copper sulfide
• CXCCR
• C-X-C motif chemokine receptor
• DLS
• dynamic light scattering
• ECM
• extracellular matrix
• EGF-R
• epidermal growth factor receptor
• EV
• extracellular vesicle
• EXO
• exosome
• GF
• growth factor
• HSA
• human serum albumin
• HGF-R
• hepatocyte growth factor receptor
• HSP
• heat shock protein
• ICG
• indocyanine green
• IL-R
• interleukin receptor
• iPSC
• induce pluripotent stem cell
• ME
• microfluidic electroporation
• MHC
• major histocompatibility complex
• miRNA
• micro RNA
• MMP
• metalloproteinase
• mRNA
• messenger RNA
• MS
• microfluidic sonication
• MSC
• mesenchymal stem cell
• MV
• microvesicle
• ncRNA
• noncoding RNA
• NIR
• near-infrared spectroscopy
• NP
• nanoparticle
• PAMP
• pathogens-associated molecular patterns
• PDGF-R
• platelet-derived growth factor receptor
• PDT
• photodynamic therapy
• PFTBA
• perfluorortributylamine
• PLGA
• poly-lactic-co-glycolic acid
• PTT
• photothermal therapy
• RBC
• red blood cell
• TEM
• transmission electron microscopy
• TGFβ-R
• transforming growth factor-beta receptor
• TLR
• Toll-like receptor
• TNF-R
• tumor necrosis receptor
• TNF-α
• tumor necrosis factor alfa
• VCAM-1
• vascular adhesion molecule 1

INTRODUCTION

Skin is the most substantial tissue in a person's body, accounting for around 16% of body weight.¹ It acts as a natural physic hurdle against several physical, chemical, and biological aggressors, such as ultraviolet radiation, abrasions, and microorganisms.² In addition, the skin performs essential functions in maintaining internal homeostasis by constantly participating in numerous physiological processes, including regulating hydro-mineral balance, thermoregulation, hormone synthesis, and activation, and the production of vitamin D, neuropeptides, and cytokines.^3,4 The skin is an elaborate anatomical structure made up of three primary strata: the epidermis, dermis, and hypodermis.⁵ The basal layer, spinous layer, granular layer, lucid layer, and horny layer are the five layers that make up the epidermis, arranged from innermost to outermost.¹ The physical barrier offered by the skin is primarily attributed to the epidermis, which is attributable to the presence of the corneal layer and the tight junctions between the keratinocytes.¹ The keratinocytes in the horny layer lack nuclei and are arranged in a pattern similar to bricks, held together by a lipid-rich extracellular matrix (ECM) that prevents dehydration and protects against toxins and bacteria. Furthermore, it is widely recognized that skin cell composition changes as individuals age.⁶

Skin diseases cover a broad spectrum of disorders, ranging from minor conditions like acne and dermatitis to severe illnesses like skin cancer and psoriasis.^7,8 The global burden of these conditions is significant, causing severe health, social, and economic impacts,⁹ and besides they do not represent a serious life threat, they affect the quality of life and mental health of the patients.^10,11 Even after resolution, these disorders can cause significant long-term effects that impact the patient's mental and physical state, leading to higher costs for national healthcare systems worldwide.⁹ For instance, patients with scabies, a human skin infestation by Sarcoptes scabiei var. hominis, can frequently develop a secondary bacterial skin infection by Streptococcus pyogenes. This condition can lead to glomerulonephritis and acute rheumatic fever, which, when occurring in children, is associated with severe permanent consequences such as chronic kidney disease and rheumatic heart disease.¹²

Conventional therapies for skin diseases involve drug administration, either topically or systemically. However, these methods often have limitations such as restricted effectiveness, adverse effects, and the possibility of drug resistance.¹³ Although topical administration provides many advantages, including being a noninvasive and simple administration route and offering a localized treatment, the efficacy of topical treatments for skin diseases is often constrained by the limited drug penetration of the skin barrier and the defective targeting to the site of action. These are the major drawbacks since the precise and direct delivery of drugs to the target has significant relevance for the therapeutic outcome.^14,15 Other than that, conventional topical therapies have low efficacy, low bioavailability at the target site and require a higher drug dose.¹⁴ Moreover, systemic treatments can cause unwanted adverse effects as a result of the unspecific distribution of therapeutic agents throughout the body, as well as reduced patient compliance.^16,17 Therefore, there is a need for developing targeted and functional strategies that can effectively lower the burden that skin conditions represent globally.⁹

In recent years, novel approaches based on nanotechnology have emerged as potential solutions to the limitations of conventional treatments for skin diseases. These approaches hold promise for overcoming some of the challenges associated with drug's capacity to pierce the skin barrier and the nonspecific distribution of drugs in systemic treatments.^18–21

Within the pharmaceutical and medicinal fields, nanotechnology is a rapidly developing area, owing to the unique features of nanoparticles (NPs), as small stature (from 1 to 1000 nm), which enables a larger contact area with body tissues. NPs exhibit intriguing physical, chemical, electrical, and optical properties that enable their broad-spectrum implementation.²² Recently, therapeutic applications based on nanotechnology has extended beyond treatment to encompass the diagnosis, prophylaxis, and treatment of diverse clinical conditions.²³

Concerning skin conditions, the clinical application of NPs, either as an intrinsic therapeutic system or as a nano-based drug delivery system, represents an excellent approach,^24–26 guaranteeing the delivery of active ingredients to the target site at a suitable dose in a controlled and sustained way.⁵

Over the years, to endow NPs with superior permeability, prolonged action, improved loading capacity, enhanced biocompatibility, low immunogenicity, and high specificity, biomimetic coatings are being develop. By now, they englobe cell membranes and extracellular vesicles (EVs).^27,28 This approach addresses some of the major challenges associated to the low efficacy and safety of NPs, as reduced skin penetration and permeation, lack of stability as a result of the quick degradation or agglomeration of NPs, burst drug release even outside of the targeted area, which lead to unwanted side effects and toxicity, immune system clearance, low biodistribution, and potential long-term safety issues.^29–32

In this sense cell membrane coatings emerge as an outstanding approach, paving the way to the development of improved drug delivery systems. The potentialities of cell membrane coatings arise from the carbohydrates (2–10%), commonly found as glycoconjugates with proteins or lipids, due to their active role in cell signaling and recognition, facilitating specific targeting and acting as mediators of cellular responses. Therefore, they are responsible for the modulation of NP delivery, providing stability, biocompatibility, and targeting to NPs.³³ Moreover, the presence of proteins in cell membranes, over half of all cellular constituent, further contributes to the cellular recognition, cell-to-cell contact, and the transport of various substances,³⁴ conferring specificity of the drug delivery system and preventing their recognition as “non-self.” For example, the transmembrane protein CD47 is expressed in several and distinct cells, such as red blood cells (RBCs), and through its interactivity with a receptor on the macrophage's surface, it can inhibit phagocytosis. This extends the circulation time of NPs and enhances their bioavailability.

Cell membrane coatings replicate the intricate characteristics of native cell membrane surfaces,²⁷ offering several advantages over using individual cellular components, including multiple functional effects without the need for complex synthesis methods.^35,36 The use of hybrid cell membranes, incorporating multiple functions from different cell types, has emerged as an integrated biomimetic system that addresses the possible functional limitations of a single-cell type.^37,38 Moreover, EVs, comprising exosomes (EXOs) and microvesicles (MVs), have gained significant attention in the development of bioinspired NPs (as shown in Figure 1). Altogether, the progress in the field of bioinspired NPs holds great potential for medical applications, enabling the identification, prevention, management, and a solution for a range of human disorders, such as skin-related conditions.

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The article offers a summary of recent advancements in developing cell membrane-coated NPs for therapeutic uses in the skin. The review begins with a detailed discussion of biological vectors’ origins, diverse functions, and molecular makeup, specifically cell membranes and EVs. Subsequently, the production and characterization techniques for biomimetic-coated NPs are introduced. The review then comprehensively explores the current applications of these bioinspired NPs in skin therapeutics, including wound healing, bacterial infections, skin cancer treatment, acne, and psoriasis treatment. Finally, the review discusses new perspectives and potential safety concerns associated with using biomimetic NPs in skin applications.

BIOMIMETIC MEMBRANE-COATED NANOSYSTEMS AS NEW THERAPY APPROACHES FOR THE MANAGEMENT OF SKIN CONDITIONS

The biomimetic coating of NPs has received considerable attention in the pharmaceutical field for the design of multifunctional drug delivery systems, including novel topical formulations. The growing interest in cell membrane coatings arises from their numerous benefits compared with conventional therapies and uncoated synthetic NPs. These benefits include lowered nonspecific internalization, enhanced residence time, homotypic targeting, improved circulation half-life, and immune modulation. These bioinspired NPs can be applied as depot systems intended to produce a local effect and enhance treatment efficacy and as drug delivery systems that significantly increase safety and therapeutic efficacy.³⁹ Likewise, EVs, such as EXOs, are also being studied for their application in the coating of NPs loaded with therapeutic agents for skin condition management.^3,40

Bioinspired nanotechnology utilizes cell membranes and EVs to cloak drug-loaded NPs, which can have an organic or inorganic core,⁴¹ for targeted delivery, either directly or indirectly. This approach helps to evade the immune system's detection and ultimately prevents early elimination from the blood.⁴² Thus, biomimetic-coated NPs unite the physical-chemical characteristics of NPs with the bionatural properties of membranes.^28,35,38,39 To perform this technique, the cell membranes are first isolated without damage. Then the NPs are coated with the membranes.²⁷ So far, several types of cells have been used, including leukocytes, RBCs, stem cells, platelets, cancer cells, and bacterial cells.^43,44 The incorporation of cell membranes into artificial NPs mainly involves three phases: membrane extraction, production of synthetic core, and coating (fusion process). Using the same source cells, the first step is to isolate the cell membranes without damaging them.²⁷ Membrane vesicle extraction from source cells is often a consistent process that includes cell isolation, cell lysis, centrifugation, fractionation, and purification. Biomimetic NPs can then be produced using various methods such as sonication, coextrusion, and electroporation. It is crucial to remember that in order to maintain the bioactivity of cell membranes, the entire procedure needs to be carried out as softly as possible and under low temperatures.^27,45 Biomimetic NPs have potential applications in various fields, including diagnosis, photothermal therapy (PTT), drug delivery, and vaccination.⁴⁶ However, despite the promising results that have been obtained,⁴² further research is still necessary.³⁸

EVs-derived NPs

EVs are small, membrane-bound vesicles that facilitate cell-to-cell contact during both normal and pathological processes. They also transport cargo produced by cells. EVs are present in various body fluids such as breast milk, urine, saliva, and blood.⁴⁷ The term EVs encompass three subtypes: EXOs, MVs, or ectosomes, and apoptotic bodies according to their biogenesis.⁴⁸ Even though the molecular makeup and biological functions of the three EV subtypes are similar, they present differences in terms of content, particle size, and origin.²² Concerning the particle size, the values in the literature are not consensual. For instance, the size of EXOs can range from 30 to 100 nm,^1,22 MVs from 50 to 1000 nm, and apoptotic bodies from 50 nm to 5 µm.^41,49,50 Their relatively large size limits the application of apoptotic bodies, so the layout of bioinspired, specific drug delivery nanostructures mainly focuses on using EXOs and MVs.⁵¹

It is important to note that the content and functions that a vesicle exerts in the body depend on the type of cell that gave rise to it and the type of vesicle.^1,49 In the case of EXOs, these vesicles are derived from multivesicular bodies and are formed when the plasma membrane fuses with the endoplasmic reticulum. On the other hand, apoptotic bodies result from cell fragmentation after apoptosis,⁵² while MVs or ectosomes result from plasma membrane rupture.²² EVs are usually composed of proteins, lipids, and nucleic acids, more concertedly RNA (mainly microRNA (miRNA), but also messenger RNA (mRNA) and noncoding RNA (ncRNA)). Some proteins are constitutive, while others depend on the cell of origin. The lipids that make up the membrane of vesicles are originated from the plasma membrane of the cell of origin. Besides the membrane constituents, specific markers (such as CD63, CD81, and CD9), heat shock proteins (HSPs) (e.g. Hsp60, Hsp70, and Hsp90), and proteins that promote cell interaction (such as Alix/PDCD6IP) can also be found (Table 1) (Figure 2).⁵³

TABLE 1 An outline of the primary markers on the exosome membrane and their purposes.

Name	Function	References
Integrins	Mediate tropism and attachment to a specific cell type Cell targeting and adhesion	54
Lipid anchors	Allow binding/adhesion to certain cells	55
Surface proteoglycans	Internalizing receptors facilitate exosome entry into cells	54
MVB biogenesis	Facilitate/promote the activation of exosomes	56
Membrane transporter	Allow transport of molecules into the exosomes	56
Antigen presentation (MHC I and II)	Antigen presentation Activation of the immune system	57
Immunomodulatory	Immune system activation, triggering the liberation of certain proinflammatory cytokines from macrophages, improving the release of tumor necrosis factors (TNFs), boosting the activity of natural killer (NK) cells, encouraging the maturation of dendritic cells (DC), delivering major histocompatibility complex (MHC) peptide molecules, and antigen presentation.	55
Tetraspanins (CD81, CD9, CD63)	Facilitate exosome uptake by other cells Cell targeting and adhesion	54
Heat shock proteins (HSP70 e HSP90)	Present in exosomes that carry out the antigen presentation task Aid in the MHC class I molecules’ antigenic peptide loading process	57
Rab GTPases and flotilina-1	Important for membrane fusion	55

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According to the evidence, EVs have been shown to regulate various physiological processes, such as tissue repair, by intervening in angiogenesis, apoptosis (either by activation or inhibition), coagulation, inflammation, and differentiation. Furthermore, EVs can modulate the immune system, as they can act at the level of antigen presentation, although they may also be associated with specific pathologies. Moreover, EVs can readily evade the immune system due to their low immunogenicity and cytotoxicity and can remain stable for prolonged periods.⁵⁸ Hence, they are valid for targeted drug delivery to particular bodily locations, either through receptors on their membrane or through the camouflage of NPs with relevant clinical action.^59,60 Furthermore, these vesicles can exert anti-inflammatory effects (if derived from cancer cells) or proinflammatory effects (if derived from mesenchymal stem cells [MSCs], T cells, or dendritic cells) depending on the cell type that secretes them.¹ When NPs are coated with EV membranes, their detection by immune system can be hampered, enabling better selection of the intended site, as the surface of these vesicles contains proteins that interact with recipient cells, triggering various mechanisms.⁶¹ It should be noted that NPs’ core can be organic or inorganic in nature, depending on the properties and characteristics desired for a particular situation.⁴¹

At the skin level, particularly in wound healing, MSC-derived EVs are common because of their ability to modulate the immune system and promote tissue regeneration. The regenerative capacity is attributed to their ability to stimulate cell proliferation and new blood vessel formation.⁴⁶ EVs derived from MSCs act as mediators of cell communication, as they are associated with many physiological processes, such as the maintenance/recovery of homeostasis, modulation of the immune system, and angiogenesis. According to the literature, the advantages of EVs, whether derived from MSCs or other kinds of cells, are due to their paracrine action, which leads to the conclusion that these vesicles obtain these additional benefits of MSCs. MSC-derived EVs have been studied for various applications in different organs, including the skin, heart, kidneys, liver, and others.⁶² In short, these EVs have also been studied due to their promising application for noninvasive diagnostic and prognostic purposes.⁶³

Among EVs, EXOs are the most widely used EVs due to their diagnostic and therapeutic potential, ease of handling, high stability, inherent ability to target a specific cell/ tissue, multicargo loading and small size (30–200 nm).^64,65

EXOs are spheroid structures bounded by a lipid bilayer that do not self-replicate. They originate from endosomes and usually vary in size from 30 to 100 nm. Given their unique characteristics, EXOs are suitable for both diagnostic and therapeutic reasons.^1,66 Notably, EXOs express many of the same characteristics as the cells that produced them and contain essential molecules within their structure.⁶⁷ EXOs are released by various cells, such as fibroblasts, adipocytes, neurons, and tumor cells. They are located in various biological fluids, including breast milk, blood, urine, saliva, cerebrospinal fluid, and amniotic fluid.^50,57 Like other EVs, EXOs have a membrane rich in lipids, transmembrane proteins, receptors, and functional markers.

EXOs have become an important clinical tool because of their drug-delivery capabilities, particularly in immunotherapy for cancer treatment. Moreover, they are valid for gene delivery, as they are rich in miRNA and mRNA.⁶⁸ Emerging evidence suggests that EXOs can be applied therapeutically for wound healing treatment. Research has revealed that stem cell-derived EXOs promote cell migration, proliferation, and differentiation, as well as and re-epithelialization and angiogenesis, through activating specific signaling pathways (AKT, STAT3 ERK, Wnt/β-catenin). Additionally, EXOs promote skin rejuvenation, indicating possible applications in the cosmetic field.^2,69 The use of EXOs or their membranes to coat NPs for regenerative medicine and tissue engineering is advantageous as a result of their biological properties, including low immunogenicity, biocompatibility, low toxicity, and the ability to exchange molecules with cells.^2,57

Several recent investigations have shown that EXOs derived from MSCs or their membranes quicken the healing of wounds and reduce the development of scars. These EXOs are derived from different kinds of cells, including adipose mesenchymal cells, amniotic epithelial cells, umbilical cord endothelial progenitor cells, and induced pluripotent stem cells (iPSCs).^1,70 These vesicles can attach themselves to target cells using adhesion molecules, including integrins present on the surface of most cells, which enables them to have this effect. Recently, several gels incorporating either EXOs or NPs coated with EXO membranes have been designed for use in skin lesions and be highly effective in wound healing. By coating NPs with EXO membranes, we can fuse the NPs' characteristics with the biological properties of EXOs, which can be further enhanced by adding drugs with specific properties depending on the problem at hand.⁴¹

Research has demonstrated that MSC-derived EXOs express surface markers CD9, CD63, and CD81. Knock-out mouse models for the CD9 marker have been shown to be inefficient in re-epithelialization and wound healing. This is likely due to extended activation of c-Jun NH2-terminal kinase in keratinocytes, leading to extremely elevated concentrations of metalloproteinase-9 (MMP-9) and abnormal destruction of collagen IV. Consequently, the use of MSC membranes may be useful in aiding wound healing in these knock-out mouse models. The surface marker CD63 has demonstrated to increase cell survival and keratinocyte migration by stimulating MMP-1, and overall, it accelerates angiogenesis. CD81 stimulates cell migration in a generalized way. Despite the various studies, the mechanism and specific molecules involved in wound repair are still unclear.³

In this regard, there is evidence that NPs coated with exosomal membranes offer numerous benefits, such as prolonged residence time in the bloodstream, better biocompatibility, and improved targeted delivery. In individuals with impaired healing, these NPs can enhance wound healing thanks to the membrane markers mentioned above.⁴⁰

Cell membranes-derived NPs

Fibroblast cell membrane-coated NPs

Fibroblasts are cells that regulate tissue homeostasis and synthesize the ECM. Thus, these cells encode varied information from various connective tissues, which have crucial roles at the level of different organs, preventing various organ and tissue injuries from arising. Fibroblasts are also responsible for producing or inducing the production of mediators, such as cytokines and growth factors (GFs), among others. It should also be noted that these cells give rise to other types of mesenchymal cells, such as osteoblasts or adipocytes, and they also intervene in the formation of new tissue, either in organ formation or in injury repair. Fibroblasts form a rigid, adhesive, yet adaptable (elastic) structure in the skin that supports the keratinocytes and skin appendages.⁷¹ After the connective tissue is formed, the cells that give rise to the skin fibroblasts differentiate into different fibroblast types with different molecular markers.

According to studies in neonatal mice, papillary fibroblasts from the skin of these animals express transmembrane factors such as CD26/Dpp4, Lrig1, integrin Itga8, and the transcriptional repressor Prdm1/Blimp1. The cells that give rise to reticular fibroblasts, on the other hand, possess transcription factors Pparge Ebf2, transmembrane protein Pdpn, and the multifunctional signaling factor Sca1/Atxn1. As fibroblasts mature, the markers they express change.⁷¹ In most organs, fibroblasts produce type I collagen and distinctive markers, namely platelet-derived growth factor receptor (PDGF-R), and PDGF-Rα. Other markers that are also present include vimentin, CD90 (Thy-1), the transcription factor TCF21, and fibroblast-specific protein-1 (FSP-1/S100A4).⁷²

Currently, new discoveries have emerged in the field of oncology, showing that activated fibroblasts can increase angiogenesis, leading to the occurrence of metastasis and uncontrolled proliferation of cancer cells. Researchers have used the membranes of fibroblasts to coat NPs to direct antitumor therapeutics more effectively to target cells.³⁸ The study focused on using fibroblast-associated nanosystems for application in cancer, specifically in the phototheranostics of the enhanced multimodal cancer tumor microenvironment. Coated NPs were prepared using coextrusion, utilizing semiconductor NPs and cellular membranes derived from activated fibroblasts.⁷³ Tests were then conducted to determine whether this coating had any negative influence on the NIR fluorescence, photothermal, and photodynamic properties of the NPs. It was discovered that the coating did not alter these characteristics. Due to homologous binding, it was found that these NPs had a preferential targeting to fibroblasts inside the microenvironment of the tumor, leading to accumulation in the tumor tissues. In this way, these NPs acted as valuable agents for the detection and treatment of cancer.⁷³

MSC membrane-coated NPs

MSCs are multipotent stem cells isolated from different tissues, such as bone marrow, umbilical cord, amniotic fluid, adipose tissue, placenta, liver, and lung.^62,74 MSCs express specific surface markers, namely CD90, CD105, and CD73, and do not present others, such as CD14, CD34, and CD45. The unique properties of MSCs result from the expression of receptors on their membrane, including the ability to accelerate wound healing due to interactions between cells at the damaged site and interactions between ECM proteins and other biomolecules.⁷⁵ These receptors include chemokine and cytokine receptors, ECM receptors, receptors responsible for cell–cell interactions, and GF receptors, which regulate different signaling pathways associated with protein synthesis. Therefore, understanding the function of all these receptors is essential to develop and improve the use of MSC membranes. Chemokines are recognized to have a significant impact on adhesion and migration to the damaged or tumor site. The chemokine receptors found on the membrane of MSCs are C-X-C motif chemokine receptor (CXCCR) CXCR1, CXCR2, CXCR4, CXCR5, CXCR6, CCR1, CCR2, CCR4, CCR6, CCR7, CCR9, CCR10, and CX3CR1. The chemokine receptors are coupled to the G protein.⁷⁶

The second class of receptors most expressed on the membrane of MSCs are the GF receptors, whose primary action is to promote the migration and differentiation of MSCs. These include the epidermal growth factor receptor (EGF-R), the hepatocyte growth factor receptor (HGF-R), the primary fibroblast growth factor receptor 1 (bFGF-R), the PDGF-R, and the transforming growth factor-beta receptor (TGFβ-R). Cytokine receptors, on the other hand, direct MSCs to the site of damage. The most prominent cytokine receptors found on these cells are the IL-R (interleukin receptor), including IL-1R, IL-3R, IL-4R, IL-6R, IL-7R, IFNγR, and tumor necrosis factor receptor (TNF-R). Finally, ECM receptors are notable for their action in cellular localization and in adhesion, differentiation, and maintenance of cells. These include the integrins (α1, α2, α3, α5, α6, αV, β1, β3, and β5) (Table 2).⁷⁶

TABLE 2 A summary of the principal MSC membrane markers and their role.

Type of receptor	Functions	Types	References
Chemokine receptor	Encourages migration and adherence to the tumor or damaged tissues.	C-X-C motif chemokine receptor (CXCR1) CXCR2, CXCR4, CXCR5, CXCR6 C-C chemokine receptor type 1 (CCR1), CCR2, CCR4, CCR6, CCR7, CCR9, CCR10	76
Growth Factor Receptors	Promotes migration and differentiation of mesenchymal stem cells.	Epidermal growth factor receptor (EGF-R). Hepatocyte growth factor receptor (HGF-R). Basic fibroblast growth factor receptor 1 (bFGF-R). Platelet-derived growth factor receptor (PDGF-R). Transforming growth factor-beta receptor (TGFβ-R)	76
Cytokine receptors	Direct mesenchymal stem cells to the site where damage has occurred.	Interleukin 1 receptor (IL-1R), IL-3R, IL-4R, IL-6R, IL-7R, IFNγR, and TNFR	76
Extracellular matrix receptors	Act at the level of cellular localization. However, they are also relevant in the adhesion, differentiation, and maintenance of cells.	Integrin (α1, α2, α3, α5, α6, αV, β1, β3 and β5)	76

Numerous recent studies have demonstrated that cell membranes confer NPs the ability to be endocytosed by host cells, further enabling their transport into the cell.⁴⁷ To better understand the action of these membranes, gelatin nanogels coated with an MSC membrane layer were created from bone marrow and functionalized with a tumor-specific antigen to target tumors. In the study, the nanogels exhibited optimal targeting in vitro and more significant in vivo tumor penetration and retention, likely due to the coating preventing their removal by the reticuloendothelial system and the membrane ligand's presence aiming for the tumor and inflamed tissues.¹⁷ Despite these advances, the exact mechanism that promotes this targeting is still unclear, although it is thought to occur similarly to the immune system's chemotaxis to the location of damage.¹⁶ This coating study also demonstrated that loading MSC-coated nanogels with doxorubicin enhanced the efficiency of this antitumor therapy, resulting in better results.⁷⁷

Another study used poly-lactic-co-glycolic acid (PLGA) NPs coated with umbilical cord MSC plasma membrane to target the delivery of chemotherapy to tumors. This coating provided a considerable cell absorption efficiency of the NPs, resulting in the death of tumor cells due to targeting and subsequent accumulation in the tumor environment.^78,79 However, despite all these advantages, NPs coated with the MSC membrane still face a challenge due to the limited availability of such cells produced in the placenta, umbilical cord, and bone marrow. As a result, MSCs have a considerably higher cost than other cell types that can be used to coat NPs.¹⁶ It is now well established that stem cells have different roles at the biological level, particularly in tissue regeneration.⁴⁸ Recent research has demonstrated that NPs covered by stem cell membranes have optimal activity against tumors.⁸⁰

RBC membrane-coated NPs

RBCs, commonly referred to as erythrocytes, are the most prevalent blood cells that transport and deliver oxygen to the body.⁸¹ They typically have a diameter between 7 and 8 µm.³⁸ These cells are eliminated from the body by the immune system 100–120 days after their synthesis.^38,42 Therefore, RBC-coated NPs express surface antigens that give them a “self” identity (CD47), which allows them to circulate for a more extended period since macrophages do not recognize and eliminate them. Furthermore, the semi-permeability of RBC membrane-coated NPs, makes them biodegradable and less toxic, allowing for the controlled and gradual release of some cargo that the NP carries.³⁸ Due to these reasons, RBCs were the first cells from which the membrane was removed to coat synthetic NPs.^27,82 RBC membrane-coated NPs have been shown to promote better therapeutic efficacy. Another critical factor is that the glycans that comprise the RBC membrane have a targeting action to specific sites and a stabilizing action.²⁸ In this regard, it is established that NPs covered by RBC membrane can be used either in diagnostics (imaging) or personalized therapeutics (drug delivery to a specific target).⁸³

However, the process of coating NPs with RBC membrane presents some difficulties due to the chemical and protein complexity of the cell membrane. In this sense, conventional strategies cannot be applied, so top-down techniques are used to transfer the entire RBC membrane to the synthetic NPs. Two steps need to be performed to coat an NP with an RBC membrane: first, the membrane is isolated from the cell, and then it is fused with the NP. In the case of RBCs, after being obtained from blood, their intracellular elements should be removed by hypotonic agents. Then, the empty RBCs are washed, and their membrane is extruded into porous membranes, creating hollow vesicles that finally fuse with various synthetic NP cores. RBC membrane-coated NPs are helpful in treating some tumors and severe infections caused by bacteria, for instance, by Staphylococcus aureus and other bacteria.²⁸

Macrophage cell membrane-coated NPs

One subtype of white blood cell called a macrophage is capable of identifying, engulfing, and digesting foreign objects and biological trash.⁸⁴ They engage in an essential role by maintaining tissue homeostasis and being recruited to damaged tissue sites.^85,86 Studies have shown that macrophages are critical in skin regeneration, as they have the ability to phagocytize cellular debris, foreign organisms, and cells undergoing apoptosis at the site of injury. Additionally, these cells release chemokines, MMP, and inflammatory mediators that trigger the injury response. Macrophages perform several functions, such as phagocytosis of foreign organisms and cellular debris, presenting antigens by associating them with the MHC and synthesizing various cytokines such as IL-1, IL-6, and TNF-α.⁸⁷

There are two different phenotypes of macrophages, which exhibit different functions depending on the microenvironment.⁸⁸ IFN-γ and TNF-α are examples of Th1 cytokines that induce the M1 macrophage phenotype, but this polarization can also occur through the recognition of bacterial lipopolysaccharides. M1 macrophages release proinflammatory cytokines such as TNF-α, IL-1α, IL-1β, IL-6, IL-12, IL-23, and cyclooxygenase-2. Thus, this phenotype promotes the removal of foreign organisms with antimicrobial and antitumor effects. On the other hand, M2 macrophages prevent the occurrence of chronic inflammatory responses due to their anti-inflammatory activity. Therefore, Th2 cytokines such as IL-4, IL-13, and IL-10 and release IL-10 and TGF-β induce the M2 macrophage phenotype. In this way, these cells phagocytize cellular debris or cells undergoing apoptosis, enhancing tissue regeneration and wound healing. It should be noted that M2 macrophages are divided into four subfamilies (M2a, M2b, M2c, and M2d) depending on the activation stimulus. In the presence of extensive inflammation, macrophages first exhibit the M1 phenotype, releasing TNF-α, IL-1β, IL-12, and IL-23. Then, M2 macrophages emerge and release IL-10 and TGF-β to halt inflammation and restore homeostasis. However, the M1 phase has the potential to harm tissue if it persists. As a result, M2 macrophages release high concentrations of TGF-β and IL-10 to reduce inflammation and support angiogenesis, tissue remodeling, repair, and homeostasis.⁸⁶

Recently, macrophages have been acknowledged as increasingly significant cells since they perform several essential functions, such as identifying and initiating a response to certain stimuli, injuries, infections, or pathologies. Therefore, typically, macrophages serve as the initial line of defense against infection as they detect foreign microbes by expressing many receptors, among which Toll-like receptors (TLRs), such as TLR4, are prominent. These receptors establish interactions with pathogen-associated molecular patterns (PAMPs) from viruses, bacteria, mycobacteria, fungi, and parasites.⁸⁶ These receptors activate when macrophages detect microorganisms or foreign entities.⁸⁷

NPs coated with macrophage membranes acquire the proteins on the macrophage membrane surface, providing them with the ideal properties for targeting and imaging in the field of oncology, specifically in the PTT area. This is because macrophage membrane enables NPs to attach themselves to cancerous cells through exchanges between the α4 integrin present in the macrophage membrane and vascular adhesion molecule-1 (VCAM-1) found in tumor cells (Table 3).⁴² Scientific evidence has shown that coating NPs and other nanosystems with pretreated macrophage membranes enhance their efficiency in targeting bacteria. Furthermore, these nanosystems can transport drugs with antibacterial properties and increase their effectiveness. Importantly, these nanosystems can adhere better to bacterial surfaces and, therefore, are retained at the site of infection, regardless of whether they are administered locally or systemically. As expected, the nanosystems are biocompatible and exhibit longer blood circulation times.^43,89,90

TABLE 3 Overview of the main macrophage markers and their functions.

Type of receptor	Subtypes of receptor	Function	References
Antigen presentation	MHC I and II	Antigen presentation Activation of the immune system	57
TLR	TLR1, TLR2, TLR3, TL4, TLR6	Detection of foreign pathogens	43
CD63	_	Anti-inflammatory activity by augmenting anti-inflammatory cytokine release Cell targeting and adhesion	54, 91, 92
CD206	_	Endocytic receptor promotes phagocytosis of microorganisms and foreign substances	91
Integrin	Integrin-α4	Mediate tropism and attachment to a specific cell type Cell targeting and adhesion Act at the level of cellular localization. However, they are also relevant in cell adhesion, differentiation, and maintenance	54, 76
VCAM-1	_	Promotes cell adhesion	42

Epidermal stem cell membrane-coated NPs

The function of epidermal stem cells (EPSCs) is essential in promoting skin homeostasis and wound healing. These cells are present in the interfollicular epidermis and in skin appendages like sweat glands and hair follicles. The EPSCs found in the interfollicular epidermis are unipotent, while those in the hair follicles are multipotent. The former is rich in integrins β1 and α6, as well as leucine-rich repeats and immunoglobulin 1-like domains (LRIG1), whereas the latter express differentiation cluster markers 34 (CD34) and keratin 15 (K15).³⁵ Nevertheless, all EPSCs, regardless of location and composition, aid in preserving homeostasis and encouraging wound healing.^35,93 These cells can regenerate all types of epithelial cells.⁹⁴ The skin is one of the organs with the highest regenerative capacity, as it contains various types of stem cells responsible for maintaining homeostasis and repairing skin damage. EPSCs are particularly abundant among these stem cells and can be easily obtained without significant ethical concerns. They are similar to adipose stem cells, which have been thoroughly investigated for clinical applications in tissue regeneration.²

For the aforementioned reasons, EPSCs have been examined as potential solutions to the limitations of traditional therapies. These cells have been demonstrated to speed wound healing and treat severe wounds. Effective treatment methods are needed for severe injuries, and EPSCs, or parts of them, are promising as they are easy to isolate and have a significant skin-regenerating action. Recent studies have demonstrated that modifying silver NPs with these cells increased wound healing and further stimulated the physiological activity of EPSCs. EPSCs possess essential characteristics, such as being multipotent and capable of promoting cell differentiation in the epidermis. Therefore, using these cells has shown promise in tissue regeneration.² Studies focused on understanding the significance of EPSCs in wound healing have concluded that the use of these cells promoted re-epithelialization and the restructuring of the skin barrier. However, long-term in vitro tests have suggested that EPSCs may not have a prolonged reparative action on adult skin, and their potential diminishes with age.^95–97

Cancer cell membrane-coated NPs

Cancer cells bind to each other, promoting tumor growth through homotypic bonds.⁴² Thus, when NPs are coated with cancer cell membranes (CCMs), internalization and tumor targeting are boosted. This layer enables NPs’ application in anticancer vaccination, drug delivery, and PTT, as these NPs can undergo homologous adhesion to cancer cells.^38,42 The coating can also provide high stability to the NPs. CCMs are optimal for use in the oncology field, as they are robust, simple to cultivate on a large scale, and self-recognizable homologous cells (homotypic targeting), which give NPs coated with these membranes the aptitude to target tumors and metastatic nodules.^44,45

Various methods are performed to isolate the cancer-derived cell membrane from the remaining cellular constituents, including hypotonic lysis, mechanical perturbations, and differential centrifugation. The membranes are then physically extruded through a 400 nm porous membrane to produce vesicles. The core and membrane are coextruded to coat the NP utilizing a 200 nm diameter porous membrane. This way, these coated NPs acquire the antigens and immune adjuvants on their surface, making them easier to access cancer cells and stimulating anticancer responses. The coating increases the bonds established between the NPs and the cells and also enhances the ability of these NPs to identify and reach cancer metastases.⁴² When the core of the NP is magnetic, it can be used in diagnostic applications, specifically in magnetic resonance imaging scans, as it will have a greater ability to concentrate on the tumor due to the coating with cancer-derived cell membranes. Moreover, carrying cytostatic drugs increases the effectiveness and efficiency of antitumor therapy.⁹⁸ For these reasons, cancer cell-coated NPs may serve as a drug delivery structure as they can target the tumor site or in PTT, making this intervention more efficacious.^38,82

Hybrid membrane-coated NPs

Several scientists have been focusing on creating hybrid membranes for coating NPs in the recent years. Hybrid membranes blend the best characteristics of both cell types, making cell specificity even more significant, and NPs coated with this type of membrane acquire optimal properties.³⁸ Based on all the studies discussed so far, it is evident that coating NPs with cell membranes using different types of cells (such as RBCs, platelets, cancer cells, leukocytes, and stem cells) can provide nanocarriers with surface functionalities of the source cells, resulting in better results in skin applications. However, using hybrid membranes that combine different cell membranes obtained from different cell types to camouflage NPs can also be desirable. It can improve the biocompatibility of NPs and maximize therapeutic targeting by exploiting the synergistic effect between distinct intrinsic membrane properties of the different source cells. As a result, most hybrid membrane-camouflaged NPs have become a cross-feature of both single membrane-coated NPs, producing a multipurpose, integrated, focused nanodelivery approach.^38,42.

For instance, hybrid membranes are generally formed by fusing RBC membranes with platelet membranes.⁸² Thus, RBC membranes can lengthen blood circulation time, while platelet membranes introduce a ligand that facilitates target localization. A study was conducted with a 1:1 mass ratio of RBC and platelet membranes, which coated PLGA NPs via the sonication method. These hybrid membrane-coated NPs were similar to those coated with RBC or platelet membranes alone at the morphological level, but at the functional level, these NPs exhibited properties of both membranes.⁹⁹

PREPARATION AND CHARACTERIZATION OF BIOMIMETIC-COATED NANOSYSTEMS

Preparation methods

The coating of NPs with cell membranes and EVs occurs essentially in three phases. First, the cell membranes or EVs are isolated to form vesicles. Then, NPs are synthesized, and finally, the cell membrane vesicles or EVs are fused with the NPs. This last phase determines the success or failure of the production of NPs coated with cell membranes.^37,82

Techniques for isolating EVs

Differential ultracentrifugation is the most popular technique for isolating EVs; despite its unselectively in terms of size and density.¹ To overcome this, other techniques have emerged, such as ultracentrifugation, consecutive centrifugation, ultrafiltration, immunoaffinity precipitation, or size-exclusion chromatography. The yield and number of vesicles isolated varies depending on the method used.⁵³ Filtration is a faster method compared with differential ultracentrifugation. However, the isolated product has low purity.⁵⁷ Size-exclusion chromatography isolates EVs according to molecular weight or size and is a high throughput and high purity technique.⁵⁷ Thus, depending on the method used, the cost, yield, time, ease of execution, material required, and final purity vary. Furthermore, depending on the theoretical principle of separation, the technique will be more or less specific.⁶¹ To try to overcome the disadvantages of the different methods, it has been decided to combine two or more methods to optimize the isolation of EVs.⁶²

Techniques for isolating cell membranes

Cell membranes consist of a lipid bilayer separating inner and outer contents and containing various proteins with different functions. The membrane performs a broad variety of physiological actions, such as the influx and efflux of substances and the recognition of other cells. Large volumes of cells are typically necessary to isolate the cell membrane, and the procedure aims to remove internal cellular components while preserving the essential membrane constituents.⁴⁵ To achieve this, intracellular contents are eliminated through successive hypotonic lysis or freeze/thaw cycles. Subsequently, by using differential centrifugation, soluble proteins are eliminated, and the membrane forms nanovesicles via an extrusion process.⁴² Upon membrane purification, DNases and RNases are used to eliminate intranuclear components.²⁵ The exact method of membrane isolation depends on the type of cell.¹⁰⁰ For example, this process is straightforward in the case of anucleated cells such as RBCs and platelets. However, in the case of eukaryotic cells such as stem cells, tumor cells, and leukocytes, membrane isolation is more complex, requiring hypotonic lysis, mechanical rupture, and discontinuous sucrose gradient centrifugation to eliminate the nucleus and cytoplasm. Finally, in these cases, the cells are washed with isotonic buffers, sonicated, and extruded to form the resulting nanovesicle cell membrane.³⁸ During extrusion, nonuniform vesicles are sequentially extruded until uniformly sized vesicles are obtained.³⁷ Among the cell membrane isolation methods, successive freeze/thaw repetitions are the most suitable, as it does not affect surface antigens.¹⁰¹ To prevent proteins from losing their conformation and functionality, it is crucial that the membrane extraction/isolation process is as gentle as possible, ideally performed at 4°C and with protease inhibitors.²⁷ The process of removing cell membranes aims to enhance the biointerfacing capabilities of NPs, and several processes have been developed for extraction and coating. The following sections will discuss different methods used to isolate cell membranes, including sonication, electroporation, hypotonic lysis buffer, and repeated freezing and thawing.

Sonication

Sonication is one method for isolating cell membranes, which uses sound energy to move cells, typically using ultrasonic waves with a frequency of 20–50 kHz. The frequency used varies, resulting in cycles of compression and rarefaction. During rarefaction, tiny empty bubbles form due to the ultrasonic waves. After that, the mixture is extruded by forcing it through porous polycarbonate membranes.²⁵ The energy source connected to the sonicator converts energy into mechanical energy, which is then transferred to a metal probe placed on the sample (cells), resulting in cavitation and cell lysis. However, this technique can generate high temperatures of up to 5000 K and pressures of 2000 atm. This process has some disadvantages, including high heating of the sample (cells), which can lead to protein denaturation, variation in yield, and free radical formation. However, the potential disadvantage of protein denaturation can be mitigated using an ice-cold lysis buffer.¹⁰⁰

Electroporation

Electroporation is a method that promotes cell lysis by exposing cells to strong electrical fields, which cause the cell membrane to become porous and release intracellular constituents. However, electroporation can also cause structural damage and denaturation of membrane proteins, so extreme electroporation conditions should be avoided to prevent irreversible and severe loss of innate membrane potential.⁴⁵

Hypotonic lysis buffer

Isolation of the cell membrane can be achieved through a combination of hypotonic treatments, mechanical disruption, and high-speed differential centrifugation.²⁵ The most common process for isolating tumor cell membranes is mechanical rupture using a homogenizer after osmosis-based lysis in a hypotonic solution. Like other cells with a nucleus, tumor cells require gentler lysis and higher-speed centrifugation than anucleated cells. Thus, the specifics of membrane isolation by osmosis depend on the cell type.⁴⁵ A solution with a lower solute concentration than another solution on the opposite side of the membrane separating them is called a hypotonic solution. If the cytoplasm is more hypotonic than the extracellular environment, there is an outflow of water molecules into the external environment, which leads to cellular plasmolysis. Conversely, when the cytoplasm is less hypotonic than the extracellular medium, water molecules move into the cell, leading to cell turgescence or lysis. Therefore, the buffer solution used to cause cell lysis must be hypotonic compared with the cell's internal environment and must contain buffering salts and ions to regulate pH and osmolarity. Additionally, the buffer solution may contain detergents, such as Triton X-100 or sodium dodecyl sulfate, to cause the membrane to rupture. It is important to mention that the buffer solution's composition is based on the cell type and its origin, as well as the structure we want to isolate.¹⁰⁰

Repeated freezing and thawing

Repeated freezing and thawing are one of the most widely used techniques for isolating cell membranes since they maintain integrity, including the structure of the lipid membrane and the antigens and other biomarkers existing on the cell's surface membrane. In this process, it involves repeatedly freezing and thawing the cells until cell membranes are detached. Remaining cellular constituents through several centrifugation steps under different conditions, resulting in pure membranes.^25,76 In this method, cells are frozen at −80°C (in an ethanol bath or dry ice) and then thawed at 37°C or room temperature. These steps are repeated alternately for several cycles. These repetitions can damage the membrane due to ice crystals, which promote swelling of the cells, followed by their contraction, leading to the elimination of the cytoplasm and clumping of the membranes. It is important to note that this method is best suited for anucleated cells, such as RBCs or platelets.^45,100 As a rule, two or three freeze/thaw cycles are performed, but the minimum number of cycles should be used to avoid decreasing the activity of some enzymes or proteins.

Selection of NP core

Nanomaterials have several benefits over traditional drug delivery systems, which make these nanostructures interesting for different clinical applications, both for treatment and diagnosis (Table 4). Depending on their physicochemical properties, size, hydrophobicity/hydrophilicity, and morphology, NPs can have different applications.^18–21,102 For example, the nanometer particle dimensions and high specific surface area make them increasingly interesting for dermal applications.^78,79 The penetration of different types of NPs into the corneal layer depends on their shape, particle size, core constitution, and surface constitution. In addition, NPs can carry drugs that increase the safety and efficacy of treatment.⁴ Functionalized NPs have demonstrated strong efficacy in wound healing. Specifically, metallic inorganic NPs, such as copper, superparamagnetic iron oxide (Fe₃O₄), silver, and gold, have been widely applied because of their inherent antibacterial activity in the healing of wounds and their great capacity to penetrate the skin cell membrane.⁴⁹

TABLE 4 Summary of the advantages of nanoparticles and limitations of traditional drug delivery methods.

	Characteristics	References
Advantages of nanoparticles	Designed with controlled size and shape Superior skin permeation Superior retention of the active ingredient at the skin level Prevent the degradation of the active components Allow controlled release of the active ingredient Surface modified for vectored release of active ingredients Flexible nanostructure design for therapeutic, diagnostic, and theranostic purposes Decreases particle size, increasing surface area and the retention time of the active ingredient at the location of action	4
Limitations of traditional drug delivery methods	Low bioavailability at specific sites Non favorable biodistribution Minimal buildup at the intended location Off-target impacts	41

According to the intended use, a wide variety of NPs are suitable as a core to produce biomimetic NPs. Regardless of the core material, the main requirement is for the NPs’ zeta potential to be negative. This characteristic will allow for the appropriate guidance of cell membranes near the NPs, due to the electrostatic repulsion between negatively charged extracellular membrane components and the NP surface.¹⁰³ Another factor to consider when selecting the NP core is size because it should be similar in size to the cell for interaction with the biological system to mimic cellular activity most efficiently.¹⁰⁴ The NP core can be organic or inorganic, have different shapes and dimensions, and consequently, different actions.¹⁰⁵ Organic NPs are those whose core is composed of lipids and/or polymers. These are most often synthesized using the emulsification and/or precipitation method. The most applied one within this class of NPs is PLGA, which has advantages such as being biodegradable, nontoxic, and biocompatible. The other class of NPs is called inorganic NPs, and there is a vast diversity, often camouflaged with different cell membrane vesicles. Another critical issue is that these cores are cheap and relatively simple to produce. In addition, they have optical, electrical, and magnetic properties that vary depending on the size and morphology of the NPs.⁴² Recently, there have been several materials that constitute the core of NPs and are coated by cell membranes, most notably PLGA,^27,40,42,106 silicon dioxide (SiO₂), silica, gold,^41,107,108 magnetic iron oxide,^4,27,42 silver,^3,109 and albumin.^27,42,110 The type of core NPs is selected according to the required properties.⁴¹ The cores of NPs can be natural (liposomes, serum albumin, viruses, among others) or synthetic polymers. Natural ones result from biological and chemical processes that occur naturally. Polymer derivatives usually consist of PLGA, polylactic acid, chitosan, gelatin, and so on.²⁷ Overall, the composition of the NP is crucial depending on the application since this is determinant in the release and effectiveness of the cargo it carries at the desired location. It should be noted that this target is realized a lot thanks to the cell membrane coating.⁴⁵

Polymeric-based NPs

Poly-lactic-co-glycolic acid. Because of its ability to encapsulate a wide range of compounds, United States Food and Drug Administration approval, biodegradability, and biocompatibility, PLGA is one of the most often utilized NP cores. PLGA is a biodegradable material that can be of natural or synthetic origin and is broken down into biocompatible by-products in vivo through enzymatic or nonenzymatic means. Therefore, this acid is α-carbon asymmetric and can have various enantiomeric forms. Additionally, it is soluble in a variety of commonly used solvents, including acetone and ethyl acetate, although hydrolysis of the ester linkages in water degrades it.^106,111 In addition, PLGA NPs present favorable optical characteristics, which are very useful in imaging and PTT. However, they have some drawbacks, namely that they are not selective for specific cell types, like cancer cells, and are rapidly eliminated by the body. These disadvantages can be solved by putting a cell membrane on the NP. In this regard, some studies have shown that coatings of this type of NPs with an RBC membrane are more effective in delivering antitumor agents, especially for solid tumors.^40,42 There is also scientific evidence that PLGA cores extend the spectrum of activity of drugs and simultaneously reduce their hematological toxicity.²⁷

Metal-based NPs

Silver NPs. Silver is one of the most widely used metals in the production of NPs and nanosystems, mainly because of its antibacterial and healing properties.¹¹² For these reasons, these NPs are suitable components for incorporating into various topical products, such as a wound or burn bandages.¹¹³ Chemical, physical, or biological methods can produce these NPs. The biological route is the simplest, least costly, and most environmentally friendly.¹¹⁴ This process can involve the use of microorganisms, such as bacteria, algae and fungi, or plant extracts.¹⁰⁹ A study has shown the advantages of applying silver NPs to the healing of wounds, especially burns, and has also demonstrated high benefits when these silver NPs are coated with EXO membranes. In the latter case, complete re-epithelialization was observed. The aforementioned study also revealed that treatment with silver NPs, either coated with the EXO membrane or uncoated, resulted in faster burn healing than other known local topical treatments.³

Magnetic iron oxide NPs. Iron oxide NPs possess magnetization characteristics in an external magnetic field, which makes them applicable to PTT.¹¹⁵ These types of NPs can alter macrophages’ adhesion and polarization phenotypes under a frequency-dependent magnetic field. The capacity of magnetic iron oxide NPs to stimulate the polarization of M1 macrophages (proinflammatory) into M2 macrophages (anti-inflammatory) is crucial in wound tissue regeneration as it affects the inflammation phase. For this reason, iron oxide NPs are used to transport drugs such as antibiotics, anti-inflammatory drugs, antioxidants, GFs, and nucleic acids used explicitly in wound care to enhance wound therapy.⁴ In recent years, research has indicated that magnetic iron oxide NPs coated with RBC membrane by microfluidic electroporation (ME) acquire a longer circulation time in the body, are less eliminated, and can be applied in PTT. Additionally, they are able to transform NIR rays into the visible range and can be used in fluorescence imaging with advantageous optical characteristics, like minimal toxicity, narrow emission peaks, and optimal photo-stability.^42,116

Gold NPs. The therapeutic interest in gold NPs has increased due to their unique characteristics and biocompatibility. These NPs have the most significant ability to transport drugs, are the most inert, have the lowest toxicity, and are, therefore, the easiest to circulate in the body without causing any side effects.¹¹⁷ An optimal feature of gold NPs is that they can be functionalized since they quickly form strong bonds with sulfur, which allows them to bind and adhere to different target molecules such as nucleic acids, proteins, antibodies, peptides, and carbohydrates. It should be noted that these gold-sulfur bonds are not weak, which ensures good stability during transport. Another critical factor is the ease with which gold NPs with different sizes and morphologies can be obtained, usually by reducing Au³⁺ to Au, which leads to nucleation. In this reduction process, reaction conditions such as pH, time, and temperature, among others, vary depending on the drug it will carry and the therapeutic purpose of the NPs.^118,119 It is now known that gold NPs can heat-producing optical energy optical energy into heat and thus kill cancer cells by hyperthermia, enhance drug delivery, or induce gene expression without damaging healthy tissues.⁴¹ Additionally, applying this type of NPs has an efficient antibacterial effect, prevents the development of drug resistance and can destroy multidrug-resistant strains. For the reasons mentioned above, gold NPs are very useful in PTT applications thanks to their stability, biocompatibility, and high efficiency of light-to-thermal energy conversion.¹⁰⁷ In addition to all that has already been mentioned, gold NPs can also be used in disease diagnosis.¹⁰⁸

Other NPs

Human serum albumin-based NPs

Human serum albumin (HSA) is a protein with several domains, synthesized in the liver with a molecular weight of 66.5 kDa, and a concentration between 35 and 50 g/L. This protein has the ability to bind to drugs, making it optimal for therapeutic applications since it promotes the alteration of drug distribution and elimination, enhancing their therapeutic activity.¹¹⁰ According to research, HSA, the most abundant serum protein in plasma, is one of the most commonly used materials to form NPs with several applications in the clinical and pharmaceutical fields. Additionally, these HSA NPs can also be cloaked with RBC membranes, establishing a biomimetic platform that not only enhances drug delivery but also increases the length of stay in the body and improves the results of PTT.^27,42

Techniques for coating NPs with cell membranes

After removing the cell's internal constituents, the plasma membrane forms a vesicle that coats the NPs, forming biomimetic NPs. Essentially, the production of biomimetic NPs involves two steps: the production of the NP core and its coating with the plasma membrane of different cells. This coating can be accomplished with different methods such as physical extrusion, sonication, and electroporation.⁴⁰ To create a stable structure, all of these techniques make use of the electrostatic interactions that exist between the NP core and the cell membrane.⁴⁵ When the NP core has a positive charge, there is an electrostatic attraction with the negatively charged cell membrane, which spontaneously forms the coating on the NP. In this method, the driving force resulting from the electrostatic attraction promotes the fusion of the membranes with the NP. Since this coating occurs spontaneously and is poorly controlled due to electrostatic and hydrophobic interactions, the coating may not be complete and efficient, and there is a substantial probability of inducing a multilamellar coating (Table 5).³⁷ According to current scientific knowledge, extrusion and sonication are the most widely employed techniques. It is also known that the relationship between the potential of the NP core and the cell membranes that will coat it is one of the most relevant factors that affect the manufacture and action of biomimetic NPs. Therefore, this parameter should be given more importance. In summary, when coating NPs with cell membranes, it is necessary to consider many factors, such as the membrane/NP ratio, surface charge, and NP diameter.²⁵

TABLE 5 Overview of each coating strategy, advantages, and disadvantages.

Coating strategy	Principle	Advantages	Disadvantages	References
Extrusion/coextrusion	Mechanical force exerted during extrusion forces the nanoparticles to penetrate the cell membrane's phospholipid layer.	Robust, in the sense that it creates uniform nanoparticles, both in terms of size, zeta potential, membrane thickness. Reproducible method	The process is very time consuming.	37, 45
Microfluidic sonication	Using this technique, a microfluidic device is submerged in the nanoparticles and cell membranes inside an ultrasonic bath, which generates an intensive compressive pressure that promotes coating.	Versatile Effective Continuous Fast	Can cause irreversible pores in the membrane	40
Sonication	The core of the nanoparticle and the cell membrane are exposed to the ultrasonic energy force, and the cavitation bubbles that form due to the ultrasonic waves partially destroy the structure of the membrane and subsequently help reconstitute it around the nanoparticles.	Low material waste Maintenance of nanoparticle and membrane properties Allows the use of different membranes simultaneously Fast High throughput	Can lead to a nonuniform coating and consequently an uneven size Can cause irreversible pores in the membrane	37, 82, 100, 120
Microfluidic electroporation	This procedure causes several pores in the cell membrane and thus the nanoparticles penetrate through them, thus becoming coated by the cell membrane.	Efficient Reliable High performance Good parallelism	High cost High complexity Can cause irreversible pores in the membrane	37, 38, 82, 100
Spontaneous formation by electrostatic attractions	This method is based on electrostatic and hydrophobic interactions of the negatively charged cell membrane with the positively charged nanoparticle.	Spontaneous reaction	Incomplete coating Multilamellar coating Polydisperse particles	37

Coextrusion/Extrusion

Extrusion is a technique in which a material is forced through a mesh to reduce its cross-section. This method can be performed either hot or cold. If the procedure is hot, it involves high temperatures that facilitate the passage of the material through the extrusion mesh. In this case, extrusion is an anhydrous process that occurs in fewer steps, increasing bioavailability and yield. In the case of cold extrusion, the process occurs at room temperature.¹²¹ When this technique is used, the cell membrane and NPs core are successively extruded through a porous polycarbonate membrane. The mechanical forces exerted during this process are responsible for coating the NPs.⁴⁰ This is a very effective and practical method, but challenging to apply on a large scale. In this technique, the NP core is incubated and subsequently extruded together with the cell membrane. However, this is a very time-consuming process, which becomes a major disadvantage.³⁷ However, the extrusion method is quite robust because it creates uniform NPs in terms of particle size, zeta potential, and membrane thickness, among other factors, and is, therefore, a reproducible method.⁴⁵ In this case, the two components, NP core and cell membrane, undergo successive coextrusions through a 200 nm porous polycarbonate membrane, resulting in automatic adsorption and subsequent fusion of cell membrane vesicles with the NP surface. Coating occurs because the mechanical force exerted during extrusion forces the NPs through the phospholipid layer of the cell membrane.¹²² This method allows coating with multiple functional layers using fewer steps, making it advantageous.¹⁰⁰ Also, the size of the coated NPs can be adjusted depending on the pore diameter of the extrusion membrane. One of the strengths of this strategy is that it ensures that the membrane retains its bioactivity even after coating.³⁷

Sonication

Sonication is one of the most widely used techniques for coating NPs with cell membranes. In this method, both NP's core and cell membrane are exposed to ultrasonic energy, which creates cavitation bubbles that partially destroy the structure of the membrane. Subsequently, the bubbles help reconstitute the membrane around the NPs.¹²³ This method has several advantages, including low material waste and the preservation of the properties of NPs.⁸² Furthermore, it allows for the simultaneous use of different membranes, resulting in the fusion of multiple membranes. As a result, the biomimetic NPs acquire the functionality and properties of different cells.^100,124

Sonication is a quick method for coating NPs but can result in a nonuniform coating and varying size.^42,125 To minimize these differences, various parameters such as power, frequency, and sonication duration ought to be investigated in order to reduce cell membrane damage and maximize fusion efficiency.^25,37 Compared with extrusion, sonication has a higher yield and lower material waste.¹²⁰ To optimize NP coating, two methods that complement each other, such as sonication (acoustic force) and extrusion (shear force), can be applied simultaneously.²⁵

Microfluidic sonication

Microfluidic sonication (MS) is a recently emerged technique that involves immersing a microfluidic device with NPs and cell membranes inside an ultrasonic bath. In the first section of this device, there are three inlets and a single straight microchannel. In the second part, there is one inlet, a single microchannel with a double spiral, and one outlet.¹²¹ The mixture of NPs with cell membranes is injected into the device, and thanks to sonication, which generates an intensive compressive pressure (frequency of 80 kHz and power of 100 W), the coating of the core of the NPs with the cell membrane occurs almost instantaneously (<30 ms). This technique has a very high coating efficiency (up to 93%).⁴⁰ In a comparative study, in which dynamic light scattering (DLS) was used to calculate the diameter and polydispersity index, the MS method provided favorable results, with the average diameter and polydispersity index values of 177.4 nm and 0.193, respectively. Without using MS, the size and polydispersity index values were 237.6 and 0.474 nm, respectively. MS is a more versatile, effective, and continuous method than other, more conventional approaches for producing biomimetic NPs. Its distinct advantages include the ability to perform multiple reactions on the same chip, rapid coating formation around NPs, and efficient coating even with multiple cell membranes.⁴⁰

Microfluidic electroporation

Electroporation is a procedure similar to sonication, which creates several pores in the cell membrane to allow NPs to penetrate and become coated with the cell membrane.³⁸ ME is one of the techniques used to obtain biomimetic NPs, and it does not rely on mechanical forces.⁴² In more detail, the NPs and membranes are introduced by two inlets into the microfluidic chip. In the Y-shaped channel, fusion occurs, and in the S-shaped channel, the compounds mix entirely. As this mixture flows and passes, the electrical impulses that are produced cause temporary holes in the membranes through the electroporation zone, allowing the NPs to enter the interior of the membranes. Finally, the coated NPs are collected.³⁷

In this technique, the main factors to consider are the voltage and duration of the electrical pulses, which encourage membrane pore formation. However, these pores may be reversible or irreversible, and the flow rate should also be considered.^37,82 If these parameters are well controlled and defined, we can obtain entirely and uniformly coated NPs with adequate stability.⁴⁵ The primary drawback of this technique is its expensiveness and complexity. However, it offers significant advantages, such as excellent parallelism, fast throughput, and quantitative determination.³⁷ It is crucial to note that this method maintains cell membranes' integrity and minimizes membrane protein loss.²⁷

Isolation and purification of cell membrane-coated NPs

Thereupon the formation of cell membrane-coated NPs, they should be purified in order to remove the excess of synthetic NPs and cell membrane fragments. Despite the importance of this step, very few investigation has been conducted in this area. Until now, centrifugation between 1000 and 6000×g seems to be the most used method to remove the excess of cell membranes.^126–129

Despite this, centrifugation is not the most accurate method to purify these nanoplatforms. Therefore, more effective methods should be developed for this purpose.

Characterization of cell membrane-coated NPs

Verification of surface molecular repertoire

NP coatings with cell membranes are biomimetic nanosystems with specific physical, chemical, and biological characteristics, which should be evaluated to determine if the NP coating succeeded.³⁸ Techniques such as transmission electron microscopy (TEM) or scanning electron microscopy are able to supply data mainly on the morphology of the coated NPs.⁴² It is crucial to remember that the cell membrane coating is thin, typically around 10 nm thick, and the most commonly used method to evaluate this parameter is DLS.^38,42,82 To confirm successful coating, zeta potential measurements, and surface charge values must also be evaluated.^42,82 Zeta potential is a measurement that indicates the surface electric potential of NPs, and it shows differences before and after coating, typically increasing by ≈10 mV after coating with the cell membrane.³⁸ Usually, the zeta potential varies within a range of ±100 mV, and this parameter provides information on stability. NPs with a zeta potential greater than +30 mV or less than −30 mV are highly stable, whereas NPs with a zeta potential less than +25 mV or greater than −25 mV are less stable since they are more likely to form interparticle bonds and undergo an aggregation process.¹⁰⁰

On the other hand, evaluating the integrity of the membrane is also relevant. For this purpose, an analysis of the protein content is conducted. This can be achieved using polyacrylamide gel electrophoresis succeeded by staining or Western blotting technique. These analyzes aim to study the existence of protein markers, antigens, and adhesion proteins in the membrane coating the NPs.^42,82 In short, to determine whether the coating occurred efficiently, we can rely on particle size, surface charge (zeta potential), and protein composition.³⁸ However, only the physicochemical properties can confirm the coating's effectiveness, whereas the biological characteristics of the membrane assess the biomimetic function. Therefore, verifying the integrity and bioactivity of the membrane is crucial for an efficient coating to have taken place.^25,38,45,82

ROLE OF BIOMIMETIC-COATED NANOSYSTEMS IN SKIN APPLICATIONS

NPs coated with biomimetic cell membranes have been used in different areas of medicine, as the different cell membranes and different cores of the NPs allow for this diversity of applications, namely in drug delivery, PTT, and anticancer therapy (radiotherapy).⁴²

Wound healing

Wounds are cellular injuries that occur in given tissues due to physiological or pathological conditions or physical or thermal damage.^130,131 Skin wounds can be divided into various categories based on the depth of the injury.¹³¹ Wounds can become complicated in several situations, particularly in the aging population or with specific pathologies such as diabetes, obesity, sensory neuropathies, autoimmune diseases, or cardiovascular diseases.⁴⁹ In these cases, a complete repair of the injured tissue does not occur, leading to continued inflammation and altered angiogenesis.⁴ Thus, promoting wound healing is a matter that must be accomplished, given that even today, chronic wounds are an economic and health problem with an increasing trend that leads to the death of many people due to poor healing.¹³² The process of wound healing is dynamic and intricate, involving several molecular and cellular mechanisms, which include the migration and proliferation of specific cells at the damaged site.^74,133 One process that most promote wound healing is re-epithelialization, which occurs through the proliferation and displacement of basal keratinocytes.¹³² Another component that promotes skin healing is tight junctions, as they promote cell proliferation, migration, and differentiation. Moreover, they regulate immune system response and homeostasis.⁴ It is important to remember that healing is extremely dependent on the individual's immune system, which helps in the barrier against pathogen entry, mobilizes cells to the site, and promotes remodeling of the ECM. In this sense, some biomaterials can be used to promote the action of the immune system.⁶

Despite all that is known about the pathogenesis of chronic wounds, it is still very complex, and conventional therapy seems less and less appropriate. Uncontrolled inflammation and the imbalance of matrix MMPs and their inhibitors prevent the proper formation of ECM. These successive failed cycles lead to the depletion of EPSCs that help regenerate the epidermis and wound healing.² Thus, with the growing concern to treat severe wounds, especially chronic wounds and avoid the problems that arise from them, new biomaterials have been developed to improve healing.¹³³

Regarding wound healing, there are numerous conventional treatments, including dressings, autologous grafts, hyperbaric oxygen therapy, and so on, but they have limitations.^74,133 Nowadays, the most common treatments are skin flap replacement and autologous tissue transplants skin flap transplantation and autologous grafts; nevertheless, they are not very efficient since there is a shortage of suitable tissue. Furthermore, they possess a high cost, donor site morbidity, immunogenicity, and antigenicity. Thus, bioactive molecules provide better prospects.¹³⁰ In this sense, NPs, whether coated with cellular membranes or uncoated, have some advantages over conventional dressings. They allow the transport of drugs that enhance wound healing, increase the bioavailability of these drugs, and trigger the activation of cell signaling pathways involved in healing.⁴

Currently, several studies demonstrate the benefits of MSC-derived EVs. In one such study, bone marrow MSC-derived EVs were used, and ex vivo assays showed an increase in fibroblast proliferation and migration and activation of the Akt, ERK, and STAT3 pathways, respectively, which are closely related to wound healing. Other in vitro investigations found that iPSC-derived EVs promoted collagen formation and induced angiogenesis, facilitating healing. Additionally, it has been shown that adipose-derived MSC EVs can accelerate wound healing in mice, as they potentiate the alteration of the phenotype of fibroblasts, particularly in terms of the release of collagen I and III by fibroblasts at an early stage, as this production decreases at a late stage to prevent scarring.⁶²

One factor that can cause skin damage is radiation, which is especially common in people undergoing radiotherapy for health reasons. Studies have revealed that ncRNA regulates the initiation and progression of tumors. Therefore, this study used miR181a, a tumor suppressor, to coat polymeric NPs made of hemoglobin and IF16. The research demonstrated that these NPs were nontoxic to cells, and an in vivo study in mice suggested that they promote wound healing and decrease ROS expression. The study revealed that the amount and characteristics of novel blood vessels formed during wound healing are crucial for promoting the process. The NPs under study promoted increased wound granulation tissue thickness and angiogenesis at the site. These NPs were found to be effective in promoting wound healing caused by radiation damage since they increased the percentage of oxygen present in the damaged site and reduced oxidative stress due to IFI6. Notably, the NPs under study minimize inflammation, encourage angiogenesis and the growth of granulation tissue, and improve the immune microenvironment (Figure 3).¹³⁴

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Moreover, the literature describes the topical application of functionalized NPs by coating them with cell membranes or incorporating bioactive agents into their surface markers to enhance healing at the damaged site. An example is superparamagnetic iron oxide (Fe₃O₄) NPs, which have drawn more attention as a result of their reaction to an outside magnetic field.⁴ This hypothesis suggests that such NPs can reduce inflammation, increase cell proliferation, and ultimately promote tissue regeneration in damaged areas, thereby demonstrating significant potential in tissue repair (Figure 4).⁴

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Currently, there is increasing interest in using NPs topically to promote wound healing. Biocompatible NPs are employed to deliver drugs to the target and allow for their controlled release, making the treatment more efficient. Furthermore, the core of NPs can mitigate biological responses such as antimicrobial and antifungal action. This property is seen in silver NPs, antioxidant action in gold NPs and cellular regulatory action of specific cells in calcium-based NPs to regulate keratinocyte migration.⁴ It is noteworthy that gold NPs may have several applications, such as the transport of active ingredients, PTT, diagnosis, antioxidant, and antimicrobial activities, and also have more excellent stability than silver NPs.⁴

Bacterial infections

Bacterial infections cause serious public health consequences worldwide, and they also have a significant impact on the economy. In extreme cases, they can evolve into a systemic infection called sepsis, leading to the individual's death. Despite the advances that have been made, some types of infections are still very complicated to fight, and in some cases, they may be impossible to treat.⁴³ One of the causes for the inefficiency of treatment is often antimicrobial resistance's emergence, which can be due to incorrect usage of antibiotics, such as excessive duration of treatment. Therefore, it is increasingly crucial to promote better release of appropriate drugs targeted to the location of infection to promote greater efficiency, less toxicity, and prevent the spread of resistance.⁴³

In this sense, and with the advancement of discoveries in the area of nanotechnology, new therapeutic options have emerged. One of the most exciting and relevant is the combination of synthetic NPs with biomembranes. In this way, the NPs acquire the properties and characteristics of cell membranes.⁴³ The cell membrane of macrophages, which are cells that belong to the immune system, is also helpful in this method and can be used to find and respond appropriately to endogenous and exogenous stimuli. It is important to add that macrophages function as the first line of the body protection against infection because they recognize foreign agents thanks to their receptors, especially TLRs, which have the ability to bind to microbial molecular patterns. In their membrane, macrophages have pathogen recognition receptors that detect distinct PAMPs.⁴³ Thus, a study in the literature was conducted to test the coating action of NPs with photothermal action coated on macrophage membranes pretreated with bacteria, S. aureus (Gram-positive) and Escherichia coli (Gram-negative). Gold and silver NPs were used, and the coating was performed according to the extrusion technique. Notably, after treatment with the bacterial models, the researchers confirmed the presence of receptors linked to pathogens on the membranes of macrophages (Figures 5 and 6). The success and uniformity of the coating were confirmed using TEM, and this method also allowed verifying that the coating's thickness was about 3 nm in the dry state.⁴³ This research was conducted to test the pharmacological possibilities of these NPs in the control/treatment of infections. In this case, a mouse model with subcutaneous infection was used. It was concluded from this experiment that the coated NPs showed more significant accumulation at the site of infection due to bacterial recognition receptors, an extended duration of circulation in the bloodstream thanks to the biocompatible coating with macrophage membranes, and also improved photothermal action due to the longer residence time at the site. This study suggests that NPs coated with macrophage membranes pretreated with bacterial models have enormous advantages in treating local infections.^43,107

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Skin cancer

Among the most common, deadly, and aggressive types of skin cancer is melanoma, which affects melanocytes and has a high risk of metastasis formation.^135,136 Traditional methods for treating cancer include operation for tumor removal the tumor followed by cycles of chemotherapy, chemotherapy alone, or localized radiation therapy. However, chemotherapy is the only viable treatment option if tumors are inoperable or there are metastases.⁴⁵ Due to these therapeutic limitations, there is enormous motivation to seek new strategies, and one possibility that has been studied is immune stimulation to eliminate cancer cells. Some studies are already testing vaccines for prevention and other immunotherapy techniques for treatment, which are more specific and less harmful/toxic than cytotoxic approaches.⁴⁵ The prevalence of this cancer has been steadily increasing in recent decades, which has increasingly worried researchers. Therefore, the need arises to formulate new therapeutic approaches that are more efficient, and the idea of applying NPs coated with cell membranes has emerged.¹³⁷

There was one study in which hollow gold NPs were incorporated with EXOs originating from human placental MSCs. Additionally, further functionalization occurred as they underwent PEGylation and were subsequently coated with melanoma cell membranes (B16F10). This coating was helpful as it improved stability and prevented agglomeration between the NPs.⁴¹ Alternatively, a group of researchers created a nanovaccine against melanoma using polymeric NPs coated with RBC membranes.²⁷ Other studies revealed that HSA NPs coated with RBC membrane enable enhanced use in PTT. A study using an NP coated by the membrane of RBCs containing an infrared dye and perfluorotributylamine (PFTBA) demonstrated that after irradiation with near-infrared spectroscopy (NIR), the NPs achieved a 62% tumor inhibition power in photodynamic therapy (PDT) and a 93% tumor inhibition when the two types of PTT and PDT were associated.³⁸ In another study, bovine serum albumin NPs were coated with 1,2-diaminocyclohexane-platinum (II) and indocyanine green (ICG) with RBC membrane that was modified with specific peptides. It was found that these were able to identify and destroy B16F10 type melanomas and were also able to prevent the development of lung metastases in vivo when combining PDT and PTT.^38,138

Nowadays, one of the methods used in cancer treatment is PTT because its therapeutic action is selective and localized and therefore, less aggressive and harmful to the rest of the organism. Phototherapy includes two techniques: PDT, in which the photosensitizer is excited in a particular wavelength of light, resulting in the creation of oxygen that generates excess heat (local hyperthermia), causing damage and consequent death of cancer cells, and PTT. In this regard, especially in the last decade, different types of NPs coated with cell membranes have improved the effectiveness of phototherapy. In a study, ICG-HSA NPs were used, subsequently coated with the RBC membrane. It was found that this coating resulted in extended duration of circulation in the body and higher oxygen production, which is useful in PDT. The core of the NP was HSA, produced by encapsulation of ICG and PFTBA, while the coating occurred using the extrusion method. Another study used magnetic NPs that were also coated with RBC membrane through electroporation. Like the previous study, these biomimetic NPs improved the results of PTT. Additionally, it was found that coating PLGA NPs with CCMs by the extrusion process showed a homologous orientation directed towards the tumor site, which improves PTT efficiency.^139–141

The biomimetic NPs in the aforementioned studies were shown to be monodisperse with improved photothermal properties and tumor-targeting ability.^139–141 Other investigators designed albumin NPs coated with macrophage membranes, with which they also associated paclitaxel, thus obtaining targeted therapy for melanoma.^142,143 These membranes were isolated by hypotonic lysis, mechanical fragmentation of the membrane, and differential centrifugation to ensure complete emptying of the intracellular contents. Subsequently, the membrane was subjected to an extrusion process to form vesicles. Additionally, albumin NPs were prepared by nanoprecipitation. As a last step, the macrophage membrane vesicles and albumin NPs were coextruded so that the vesicle coated the NP. In this study, albumin NPs coated with paclitaxel-loaded macrophage plasma membrane exhibited higher cytotoxicity and apoptosis in murine melanoma cells (B16F10) than noncoated albumin NPs loaded with paclitaxel. Additionally, the coated NPs showed higher penetration power into B16F10 cells than uncoated NPs. Notably, there was also an extended period of blood circulation and the buildup of these coated and paclitaxel-loaded NPs at the tumor site.¹⁴³

In the literature, a study has shown the potential of using NPs to eliminate lung metastases in murine melanoma (B16F10).¹⁶ Another research study has used NPs of PLGA associated with a TLR-9 agonist, coated with the melanoma cell membrane, to develop an antitumor vaccine that blocked the development of tumors in 86% of the mice tested. When these NPs were associated with antibodies, anti-CTLA4, and anti-PD1, the control of melanoma growth was more significant compared with the use of PLGA NPs that had only the TLR-9 agonist coated with the melanoma cell membrane. As a result, the mice's survival time increased. These studies have demonstrated the promising utility of cancer cell-coated NPs for the chemotherapeutic treatment of B16F10 tumors. This is due to these NPs’ capacity to target and accumulate drugs at the target site and their long residence time in the body.

After years of research, it has been realized that cancer cells exhibit unlimited proliferation, homotypic adhesion, and an enormous ability to evade the immune system. Therefore, a study was conducted in which PLGA NPs were coated with MDA-MB-435 cancer cells-derived membranes using physical extrusion. Experimental studies revealed that cellular uptake was about ten times higher when the NPs were coated with RBC membranes due to the homotypic bonds’ affinity between cancer cells. These bonds exist between cancerous cells, after attachment or clinging, depending on the antigens on their surfaces with homotypic adhesion domains. Using cancer cells to coat NPs is also feasible, given their ability to replicate even in vitro and be acquired in considerable quantities.¹⁴⁴

In addition, hybrid membrane-coated NPs have also been employed in PTT to reduce the growth of tumors. In this regard, Zhang, other collaborators, and Wang et al., used hollow copper sulfide (CuS) NPs containing docetaxel coated with hybrid RBC membranes and melanoma CCMs for use in melanoma treatment. This working group confirmed the excellent coating by evaluating the size and zeta potential. Subsequently, these researchers intravenously administered the hybrid NPs with NIR laser radiation at a wavelength of 1064 nm and observed an increase in tumor temperature to a maximum of 51.2°C, and the tumor growth inhibition rate was 86% in melanoma-bearing mice.^38,144 In another study, melanin NPs coated with hybrid membranes of RBCs and cancer cells were used to optimize the effectiveness of PTT, as it gave them a longer circulation time and a more homotypic orientation.²⁷

Several investigations have utilized CuS NPs, semiconductors with a photothermal effect capable of destroying cancer cells using an NIR laser. This type of NP has also been analyzed for its potential as a drug carrier. In order to avoid phagocytosis and subsequent destruction of these NPs by the body, researchers have attempted to produce biomimetic NPs by coating the core with a hybrid cell membrane composed of RBC membrane and B16F10 CCM. Following coating, there was a slight increase in both zeta potential and mean diameter value as determined by DLS analysis. Additionally, there was also improved colloidal stability, with photoelectric absorption remaining unchanged (1064 nm), and the half-life time was about nine times longer due to the characteristics supplied by the coating of the RBC membrane. With respect to in vivo distribution, there was a higher buildup within the tumor in contrast to the naked CuS NPs, those coated only with the RBC membrane or only with B16F10 CCMs. Concerning photothermal action, these NPs exhibited enhanced efficacy. Cellular uptake was studied in vitro using phosphorescence, and it was concluded that uptake was higher in the NPs coated with the hybrid membrane due to the more specific targeting, mainly due to the CCM. Regarding antitumor action, biomimetic NPs loaded with docetaxel showed cytotoxicity against B16F10 cells. Finally, in this investigation, the hybrid membrane-coated NPs were full of docetaxel, and in melanoma mice, the tumor inhibition rate following NIR laser irradiation was around 100%, which highlights the tremendous potential of these biomimetic NPs for future medical applications (Figure 7).¹⁴⁵

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Another study was conducted in which researchers developed melanoma cell membrane (B16F10)-coated PLGA NPs and revealed that the specific binding and adhesion to tumor cells was better for them when compared with NPs coated with RBC membrane or plain NPs.¹⁴⁶ As previously mentioned, there are several treatments for melanoma. However, they are associated with several negative factors, such as low response rate, high toxicity, side effects, and sometimes resistance to some antineoplastics. Therefore, in this area, a study was conducted in which a multifunctional NP core (specifically a PLGA NP) was used and coated with a hybrid cell membrane resulting from 19LF6 T cells associated with an anti-gp100/HLA-A2 T receptor (TCR), in addition to carrying an antineoplastic drug, Trametinib.¹⁴⁷ This research demonstrated high stability as well as high hemocompatibility and cytocompatibility. When performing cytological and hematological compatibility studies, it was perceived that the NPs had no toxicity to the cell line comparable to isolated PLGA NPs up to a dose of 1000 µg/mL. There were no significant changes in the coagulation and hemolysis assay compared with controls. It was also revealed that the cell membrane coating allowed for a controlled release of the carried drug, and in vitro, the coating with the 19LF6 cell membrane (T cells) provided a considerable increase of these NPs by cells, especially melanoma cells when compared with the use of isolated NPs. This study also concluded that cell binding and uptake depend not only on the membrane coating but also on the TCR receptors associated with it. NPs with a higher concentration of anti-gp100 TCR were demonstrated to be more successful in targeting and enhancing the anticancer action. This study also had an in vivo part in which it was possible to assess the targeting and specificity of these NPs to the tumor site. The results confirmed that T-MNPs (melanoma-binding specific NPs) showed better targeting and accumulation in the tumor than control NPs, that is, nonspecific (D-MNP) and NNPs or bare PLGA NPs. Thus, it was realized that T-MNPs accumulate specifically in the tumor, and therefore accumulation in the liver was reduced compared with that of D-MNPs and bare NPs (Figure 8).¹⁴⁷

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Nowadays, there is a consensus that NPs coated with CCMs show promising potential because of their homologous targeting in cancer treatment, high persistence in the bloodstream, and capacity for immune evasion. In a study, porous silica NPs coated with a biocompatible membrane derived from tumor cells that were in an acidic environment and carrying dacarbazine in conjunction with aPD1 were used to optimized anticancer activity. In vitro studies revealed that these NPs have higher efficacy in causing the death of tumor cells in contrast to the drug alone. In vivo studies revealed that merging chemotherapy with the developed NPs and immunotherapy promoted by aPD1 could efficiently block melanoma development and progression. Additionally, this study has concentrated on evaluating the safety of these NPs, and the results have shown that the NPs have high specificity for the tumor, which consequently decreases systemic toxicity.¹⁴⁸

In another study, DOX and ICG-coloaded CuS NPs (ID-HCuSNP@B16F10) were coated with the melanoma cell membrane and used to treat tumors. These NPs exhibited high accumulation at tumor sites and enhanced anticancer potential due to their homologous characteristics. Additionally, these NPs demonstrated an optimal photothermal effect in melanoma models. In summary, this biomimetic nanosystem possesses a synergistic chemo-photothermal effect, which translates into efficient inhibition of tumor growth seen in animal models.¹⁴⁹ These findings indicate that it is now understood that NPs are very effective drug carriers, especially in the therapy of oncological diseases, as they improve drug solubility and stability in vivo and enhance distribution and targeting. In this way, it is also possible to promote the regulated discharge of the active ingredient and decrease resistance to therapy.¹⁵⁰

Skin burns

Burns are a relatively common skin injury worldwide and can result in significant mortality and morbidity, particularly among the elderly and children. Heat (exposure to hot liquids, open flames, and incandescent bodies), ionizing radiation, electricity, and chemicals are some of the causes.¹⁵¹ It is important to mention that burns lead to the dermal and epidermal layers' cells dying, which can result in complications such as infections. Burned skin is more vulnerable to pathogenic microorganisms because the skin's barrier function is destroyed, so the restoration of the skin is crucial. Severe burns trigger an immune system response and an inflammatory reaction that may result in the failure of several organs. Therefore, rapid recovery and limiting inflammation are crucial for the complete and efficient regeneration of the affected area.¹⁵¹ Burns affect many people and have a significant economic impact, as they are complicated injuries to treat and often require extended hospitalization with high material costs. In addition, these injuries can lead to more severe conditions such as, sepsis, abrupt renal failure, hypovolemic shock, liver or heart failure, and tissue retraction when healing does not occur normally, and the formation of keloids. Significant fluid loss from burns can result in electrolyte imbalance, dehydration, and renal and circulatory failure.⁴⁰

Despite all the possible complications that can arise, the survival rate after burn injuries has increased due to the support given to vital organs such as the heart, lungs, kidneys, and liver, which are often affected. Skin grafts are also used to facilitate the recovery of skin integrity. However, autologous grafts are not the most appropriate for extensive burn injuries as their application is limited to a specific area. For this reason, recent approaches have been designed that target the use of NPs to prevent infection and accelerate healing.^152,153 In a study, third-degree burns were induced in rats to prove the beneficial effect of EXO-coated NPs in treating skin burns. The results of macroscopic and histopathological analyses were studied in the control group (no treatment) and in groups treated with local dressings of silver sulfadiazine, plain gel (G), gel + silver NPs AgNPs (G + AgNPs), gel + EXOs (G+EXO), and gel + AgNPs + EXOs (G + AgNPs + EXO) on day 14 and day 21 after the injury was induced. In this study, fibrin gel was used, and the EXOs were derived from human mesenchymal stromal cells, as they had previously been shown to be beneficial in the healing process of skin burns.³

Immunofluorescence was employed to assess the granulation tissue's maturity, and it was noticed that in terms of the density of the blood vessel network and the immunostaining of smooth muscle actin, the reduction was more significant within the cohorts administered with G + NPS and G + NPS + EXO. It was further found that complete re-epithelialization occurred in the group treated with G + NPS + EXO. Thus, in this study, the groups treated with G + NPS, G + EXO, or G + NPS + EXO showed a more mature granulation tissue with a more organized basement membrane and epidermis and a lower number of inflammatory cells. However, these same groups had total epithelialization/healing with a prominence of the granular and corneal layers.³ It should be added that the animals treated with G + NPS + EXO showed the emergence of the papillary dermis and an initial regeneration involving the epidermal appendages (pilosebaceous unit). To sum up, the findings of this investigation proved that the treatments with G + EXO, G + NPS, or G + NPS + EXO were more efficient in skin recovery/regeneration and were faster and more efficient than the other treatments applied in the study.³

Aging

The signs of skin aging are mainly reflected in sagging and the appearance of wrinkles, which occurs because the dermis and epidermis become thinner and collagen fibers diminish.¹⁵⁴ This can be due to either intrinsic or extrinsic factors.¹⁵⁵ The aging process is intertwined with the continued loss of tissue function and sensitivity. Intrinsic factors result from endogenous mechanisms that occur due to hormonal imbalances, such as estrogen, testosterone, insulin, dehydroepiandrosterone, thyroxine, melatonin, cortisol, and growth hormone, genetic factors, and dysregulated cellular mechanisms such as the mechanism of free radical production and elimination. In the case of women after menopause, with the decrease of estrogens, wrinkles, elasticity loss, disintegration of collagen, and atrophy of the epidermis with consequent visible aging abruptly appear. Extrinsic factors are factors that we can control and modify, and they mostly affect the dermis.¹⁵⁶ These factors include smoking, which breaks down the dermal collagen and elastic fibers, resulting in less elastic skin. Exposure to ultraviolet rays promotes loss of elasticity, formation of wrinkles, and pigments, in addition to lifestyle factors such as alcohol consumption and diet. Oxidative stress, caused mainly by reactive species and oxygen, is also responsible for skin elasticity loss.¹⁵⁵

Recently, the utilization of NPs in aging has evolved into a widely used approach as they can easily penetrate the skin and improve drug solubility, resulting in increased efficacy.¹⁵⁷ Stem cells play a significant and fundamental role in tissue homeostasis at the aging level as they have an enormous capacity to replace damaged cells and replenish tissue functions. Additionally, these cells are undifferentiated and can replace damaged cells. Therefore, preserving the plasticity and differentiation capacity of these cells is crucial as it guarantees their regenerative capacity. However, over time, skin stem cells (SSCs) suffer damage, which makes them unable to reverse the aging of tissues and cells. Another group of cells that also lose their function over time are fibroblasts, which lead to increased cell and tissue damage and decreased ECM synthesis.¹⁵⁶

Following the aforementioned theory, NPs that stimulate the proliferation of SSCs or mimic their action will help modulate the aging process.¹⁵⁶ In some tests performed in vitro, gold NPs showed to have good cellular uptake and proliferation. Moreover, they were able to promote cell migration, which favors damage repair, and they also have an anti-inflammatory action as they capture reactive oxygen species. An important factor is that they have high biocompatibility and can promote the proliferation of keratinocytes and fibroblasts.¹⁵⁶ In summary, using biocompatible polymers or biomimetic NPs may be very useful in the future either alone or to promote drug transport and controlled release.¹⁵⁸

Acne treatment

Acne is a chronic and inflammatory dermatological condition that tends to affect around 85% of individuals in their adolescence phase globally and can last until adulthood.^108,159 This illness may result in physical scarring and significantly impact the psychological and social well-being of young people, making it essential to find appropriate solutions for these situations. The inflammation is typically located in the sebaceous glands associated with the hair follicle.¹⁰⁸ There are several types of acne within this pathology, with the most common types being acne retentional and acne papulo-pustulosa. The more severe types include acne nodular, acne conglobata, and acne fulminans. In addition to the types mentioned above, there are other etiological forms of acne, including neonatal and infantile acne, acne caused by medications, exogenous acne, and acne excoriata. Today, we know that one or more factors cause acne. The four main factors responsible for acne are excessive sebum production (hyperseborrhea) and alteration of the keratinization process (hyperkeratinization), continued inflammation, and bacterial proliferation of the skin saprophytic bacteria, Cutibacterium acnes.

Currently, the existing acne treatments consist of topical ointments containing antibiotics, retinoids, benzoyl peroxide, or azelaic acid. Oral antibiotics, isotretinoin, or zinc gluconate may be used in more complicated cases. In both cases, these treatments are accompanied by a lot of hygiene care with sebum-regulating action. Often, PDT can also be used as a treatment. It should be noted that the only treatment that addresses the four factors that cause acne mentioned above is isotretinoin, but it has some adverse reactions, such as extreme dryness, flaking, and even the creation of resistance to the medication. For these reasons, innovation in acne treatment is necessary.^108,159

Recently, NPs have been utilized in several cases of acne. One material that has aroused much interest in this area is polydopamine (PDA) due to its optimal biocompatibility and ability to improve the structural stability of branched gold and silver NPs and improve the photothermal conversion power of these NPs. Some studies have coated gold and silver NPs with PDA associated with MSC membranes to increase cell uptake, decrease the possible release of harmful metal ions, and improve the photothermal conversion power of these NPs.¹⁰⁸ This study used gold and silver as core materials, as silver has excellent antibacterial action similar to gold. Combining these two materials provides the core with valuable characteristics, such as excellent thermal conductivity, which is beneficial in PTT. The photothermal conversion ability was analyzed by temperature measurements, which showed that the temperature of gold and silver NPs associated with PDA and laser irradiated MSC membranes was higher and dependent on power density. Toxicity was evaluated in human sebaceous cells by analyzing the cell viability of cells that contacted gold and silver NPs associated with PDA and MSC membranes, obtaining a value of 70% of the control (p < .05), indicating that stem cell membrane coating considerably reduces cytotoxicity. In summary, this study has shown that stem cell membranes benefit dermatological applications and that gold and silver NPs associated with PDA and MSC membranes have significant therapeutic potential for acne situations through thermal action on sebaceous gland cells. These systems prevent the spread of sebaceous cells in vitro, induce a decrease in the sebaceous glands’ dimensions, and therefore reduce the production of sebum, which is one of the main causes of acne (Figure 9).¹⁰⁸

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As mentioned earlier, one of the acne treatments is the topical application of retinoids. However, this treatment can promote skin irritation. Thus, researchers have been looking for vesicles or other carriers that can increase the concentration of these retinoids in the skin while reducing their side effects. In this regard, biomembranes have been considered a possible delivery system for these drugs, as they can efficiently and effectively penetrate the stratum corneum.¹⁵⁹ A study was conducted using tretinoin-loaded NPs coated with human umbilical cord MSC (hMSC) membrane to treat acne. These NPs were capable of promoting a controlled drug release, which improved the therapeutic action, reduced skin irritation, and increased the therapeutic efficiency. They showed good skin permeation and increased active ingredient stability. Furthermore, the study revealed that these tretinoin-loaded NPs coated with hMSC membranes could reduce comedones in just 14 days, compared with control groups (including those treated with only tretinoin or stem cell membrane-coated NPs). At the same time, these NPs reduced hyperkeratinization. However, these NPs did not affect the production of IL-8 and TNF-α, which are two inflammatory cytokines associated with acne, indicating that they do not act on the inflammatory aspect of the disease. Another issue on which these NPs had no effect was hyperseborrhea, as sebum production remained the same. This study used rabbits as a model to evaluate the effectiveness of reducing hyperkeratinization. The noneffectiveness in reducing excessive sebum production was evaluated in a golden hamster. Electron microscopy and Fourier transform infrared spectral analyses were used to evaluate improved permeation into the skin. The study also revealed improved skin penetration and permeation as the NPs enhanced endocytosis.¹⁵⁹

Psoriasis treatment

Psoriasis is an autoimmune disease characterized by inflammation resulting from uncontrolled proliferation and differentiation of epidermal keratinocytes. It affects approximately 2–4% of the world's population, significantly impacting patients’ quality of life and mental health. Patients with psoriasis develop erythematous plaques and lamellar scales on their skin, resulting from an immune response involving cytokines such as IL-17, IL-22, IL-23, NF-κB, and TNF-α. These cytokines activate keratinocytes, promoting the inflammatory cytokine release that further intensify the immune system response. Therefore, the keratinocytes and immune cells, especially T lymphocytes, associated with this disease may be valuable targets for treatment.¹⁶⁰ Whitish erythema, scaly plaques, and thickening of the epidermis characterize psoriasis. Currently, the most commonly used drugs for treatment include calcipotriol, tacalcitol, anthralin, tretinoin, glucocorticoids, tacrolimus, pimecrolimus, and camptothecin, among others. However, using these drugs can cause adverse effects, and the abnormally thickened epidermis hinders drug permeation into the skin, reducing their effectiveness.¹⁶¹

Currently, the most commonly used treatment for this condition is PDT or PTT due to their localized action, which minimizes adverse effects. In this study, IR-780 iodide was utilized, which is a dye with good tissue penetration and near-infrared absorption capabilities, which allows it to convert optical energy into thermal energy. This leads to greater production of ROS, consequently controlling the uncontrolled proliferation of keratinocytes. Therefore, in this experimental study, we used azide (N3)-encapsulated IR-780 in a PLGA core, subsequently coated by HEK-293T cell membranes (human embryonic kidney-derived cells). These modifications promote photosensitizer accumulation at sites with psoriasis lesions, thus enhancing PPT or PDT. Experimental activities revealed that these NPs are an effective nanodelivery structure that potentiates PPT or PDT, mainly when applied to skin pathologies. The study showed that the membrane coating of IR-780 conferred superior stability, tumor-targeting, and accumulation into target cells (mainly keratinocytes), improving photothermal and photodynamic cytotoxicity. Furthermore, the study evaluated the in vivo targeting and biodistribution of these nanostructures and concluded that they provide exceptional targeting and accumulation at the target site, which enhances the action of PPT or PDT in vivo. In addition, to better understand the action of these nanostructures, immunohistochemical analysis was performed with IL-6, IL-17, TNF-α, and CD3 antibodies, which showed that these NPs reduced inflammatory cytokine's release and also decreased T cell infiltration, leading to a marked decrease in the inflammatory state associated with psoriasis (Figure 10).¹⁶⁰

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Since drug delivery locally in these cases presents an even greater barrier due to excessive thickening of the epidermis, using microneedles can be an alternative that increases the effectiveness of transdermal drug delivery more quickly, even more quickly when compared with nanostructures that already have better permeability than using drugs alone. These microneedles have a different mechanism of action than techniques such as iontophoresis, ultrasound, and electroporation because they induce the formation of microchannels that allow drug delivery painlessly and reversibly. Microneedling is experiencing exponential growth due to its enormous potential in the management of skin conditions such as skin infections, atopic dermatitis, and psoriasis. There are several types of microneedles, and water-soluble ones have been the most studied because they are safer and easier to use. Given their biosafety, ease of use, and dosage accuracy, water-soluble MNs have been a prominent study focus among different types of MNs. In this study, the authors used nonimmunogenic, biodegradable hyaluronic acid coupled with acidic pH-sensitive histidine as copolymers to produce pH-sensitive polymeric micelles that promoted topical delivery of shikonin. Arnebiae radix or Lithospermi radix are the source of the compound shikikonin, which is generated from naphthoquinone. The compound has good biosafety and good action in treating psoriasis since it has enormous anti-inflammatory power and can also suppress IL-23 expression and the uncontrollably high proliferation of human keratinocytes brought on by IL-17A and IL-22. These micelles were coated with the membrane of HaCaT cells (an immortal keratinocyte cell line) to improve the targeting of target cells (keratinocytes) due to their homologous action. Delivery into the epidermis was achieved once the coated micelles were incorporated into water-soluble microneedles produced with karaya gum to actively target the epidermis. Studies conducted with this structure revealed that the polymeric micelles were pH sensitive, releasing more drugs when the medium was more acidified. Furthermore, it was corroborated that these micelles also promote a controlled and diffuse drug release at the intended site. The application of these microneedles containing shikonin polymeric micelles coated with HaCaT cells was shown to promote apoptosis of keratinocytes/HaCaT cells with an excessive proliferative profile promoted by the action of shikonin, causing less cytotoxicity to other normal cells. In summary, when we are facing an inflammatory case, we know that the pH in the tissues presents values below 6.0 and may reach values below 5.0 in some purulent inflammation-affected areas. By using a hyaluronic acid histidine copolymer sensitive to acidic pH, we stimulate the release of shikonin in acidic sites where an inflammatory process is present. Additionally, using these water-soluble polymeric micelles allowed us to resolve the issue of water insolubility of shikonin. This study demonstrated that the microneedles dissolve in the skin and release polymeric micelles with shikonin coated by the membrane of HaCaT cells that target epidermal cells, resulting in a consequent accumulation of these in the epidermis. The study's findings showed that the transdermal passage of shikonin took longer in the coated micelles than in the group in which the micelles were on the microneedles. Furthermore, this study demonstrates that shikonin has an antipsoriatic action as it acts on the IL23 (Figure 11).¹⁶¹

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CELL MEMBRANE VERSUS EV MEMBRANE-COATED NPs FOR SKIN DISEASES

Cell membrane and EV coatings have emerged as an innovative and promising strategy to enhance the potential of NPs for the management and treatment of skin conditions.^27,60 Although they originate from different biological sources, both of these biomimetic coatings result in nanocarriers with excellent biocompatibility, minimal immunogenicity, prolonged and sustained drug release, and targeted drug delivery.^162,163 However, the distinct sources of these coatings have an impact in the final features of the nanosystem, in terms of size, composition, biological function, and surface markers.

EVs are small structures secreted by both healthy and diseased cells composed of a lipid bilayer, antigens, proteins, DNA, mRNA and miRNA derived from the parent cell and whose main function is to establish intercellular communications.⁶⁰ The amount, molecular composition and intrinsic properties of EVs are highly dependent on the selected biological fluid, parent cells and the presence of diseases.¹⁶⁴ For example, MSC-derived EVs contain membrane proteins and markers that are characteristic of MSCs, such as CD29, CD105, CD73, CD90, CD44, and CD117.¹⁶⁵ For cell membrane coatings, the isolation of cells is conducted, such as RBCs, macrophages, and cancer cells, which are large and complex structures compared with EVs and have a vast and varying repertoire of biological functions.^43,45

Regarding the isolation of EVs, it is generally considered a challenging and low yield process that can be carried out with different techniques, each with advantages and disadvantages. Therefore, by now, there hasn't been an agreement on the best method for EV isolation. For example, aggregation and structural changes of EVs can occur when differential ultracentrifugation is the chosen method. Likewise, size-exclusion chromatography may lead to EV loss and a longer isolation process, since EVs must be concentrated before inserting them into purification columns.¹⁶⁶ However, EXOs, the type of EV more commonly explored for therapeutic applications, when isolated, present an optimal nanometer size and, therefore, do not require damaging techniques utilized in cell membrane extraction such as sonication.²⁵

For cell membrane extraction and preparation, similarly to EV isolation, there is no standard procedure. The currently used techniques such as sonication, electroporation, hypotonic lysis buffer, centrifugation, and freezing and thawing are relatively simple and effective in isolating cell membranes.¹⁶⁷ However, during the process, phenomena such as membrane losses, changes in the membrane's homogeneity, and protein denaturation can arise limiting the targeting ability of the biomimetic nanosystem.^45,100 In the case of EVs, although the isolation process is difficult, it typically maintains the whole membrane components, assuring the high targeting ability of the resulting EV-coated nanocarriers. Cell membrane isolation offers a higher yield compared with EV isolation, but a large cell number is still necessary to acquire an optimal amount of membranes.¹⁶⁷

The techniques utilized for coating NPs with cell membranes or EV membranes are mostly the same. Likewise, since both NP-coatings originate highly complex biomimetic nanosystems, the characterization methods used to study their physicochemical properties, such as size and zeta potential, morphology, and the surface molecular repertoire, are identical.^161,168 All things considered, cell membrane-coated NPs possess excellent immune evasion capacity and prolonged circulation, and their isolation and preparation process is simpler and with a higher yield; however, in some cases, their targeting ability may be compromised. Conversely, other than the membrane components of EVs, their cargo could be delivered in combination with the coated NP. Therefore, EVs are difficult to isolate, but their NP-coating leads to biomimetic NPs with excellent targeting ability and increased therapeutic potential.¹⁶⁷

The success of cell membrane and EVs membrane-coated nanosystems for the therapy of skin conditions is dependent on the designed nanosystem and the loaded active ingredients, but also on the source of the chosen membrane. The selection of the biomimetic coating, whether from a cell or from EVs, must be a careful and studied process that takes into consideration the pathophysiology of the disease.⁶⁰ However, there is still a scarcity of studies using biomimetic nanosystems for skin conditions, especially with EV membrane coatings. Thus, depending on the selected and biological condition of the cell source, cell type, and isolation procedure, different outcomes of isolated membranes for nanosystem coatings can be achieved.¹⁶⁷ More studies exploring cell membrane- and EV-coated NP characteristics and skin condition therapeutic potential are necessary, including those comparing the two types of biomimetic systems.

TOXICOLOGICAL ASPECTS/SAFETY CONCERNS

Recent years have seen amazing advancements in the application of nanotechnology across a range of industries. However, it is crucial to analyze nanotechnology's safety in short and long terms for both consumers and the environment. When active ingredients are integrated into NPs or other nanoscale particles, their essential characteristics, such as solubility, biological and chemical reactivity, and efficacy, may change, and their toxicity patterns may also be altered.¹⁶⁹ For example, when the particle size is reduced, the area of surface increases, which can increase reactivity and interaction. This can lead to the production of ROS, which may induce oxidative stress and inflammation. These events can cause cellular damage and even damage to proteins and DNA.^169,170

Based on the scientific information available to date, it is established that NPs have some inherent safety risks since they have a higher probability of causing toxicity due to longer exposure time and larger contact areas. Factors such as shape, charge, surface configuration, chemical composition, and solubility can also contribute to this increased probability of toxicity.¹⁷⁰ It is crucial to consider that NPs’ small dimensions enables them to permeate membranes with more ease and come into contact with cells, tissues, and organs. It is also critical to remember that the kind of NP used will determine the possible toxicity or other side effects. Moreover, the more biocompatible and biodegradable the NPs are, the less likely they are to be toxic.¹⁶⁹

One study has concluded that prolonged contact with silver NPs can cause DNA damage.¹⁶⁹ It is important to note that NPs can cross the placenta, endometrium, yolk sac, or fetus, leading to oxidative stress and irritation, negatively affecting the fetus, and may even trigger prenatal abnormalities, delayed neonatal development, neurotoxicity, placental damage, and newborn reproductive dysfunction.¹⁷⁰ Scientific information also shows that carbon-based nanosystems can potentiate liver toxicity in humans and further promote cellular oxidation.¹⁶⁹ Nanotechnology also has an environmental impact, as it can lodge in tiny spaces and lead to abnormal reactions in various biological systems, either by bioaccumulation, potentiating ROS production, triggering oxidative stress, or even lysosomal dysfunction.¹⁷⁰ Although NPs have been playing a broad and growing role in medicine, especially in the dermatological area, this innovation has also raised concerns about the toxicity these NPs may develop, but there are still no clear conclusions. What is known is that the small size of these particles makes them interact differently and can trigger unexpected effects in the body.¹⁵⁶ Despite the various beneficial interactions that various NPs have, such as the antibacterial and anticancer action of metallic NPs, they can also interact negatively with cells, leading to cytotoxicity. Currently, it is known that NPs that are not very biocompatible can lead to hemolysis. On the other hand, these NPs, especially those smaller than 2 nm, can be more easily excreted by the kidney, which decreases toxicity.¹⁵⁶

Another established factor is that the coating of NPs has a major impact on determining their toxicity or lack thereof. Despite being a topic of discussion for several years, there is still no conclusive evidence regarding the toxicity of NPs, as it is difficult to make precise comparisons due to various factors that can influence and increase their toxicity. As a result, there is still no established standard for accurately assessing the induction of toxicity by NPs.¹⁵⁶ Due to the above, toxicity studies are becoming increasingly important and should be modified.¹⁶⁹ Furthermore, because of their small size, it is challenging to observe the interactions of NPs and their effects on human health and the environment.¹⁷⁰

CONCLUSIONS AND FUTURE PERSPECTIVES

The application of nanotechnology in clinical practice is increasingly proving to be a promising tool, particularly in dermatology. However, only a few studies allow us to understand better the therapeutic ability to use cell membrane- and EV membrane-coated NPs for skin disorders. So far, the area with the most advanced studies on the therapeutic effects of this type of nanosystem is oncology, while other skin pathologies, such as acne, skin wounds, bacterial infections, and psoriasis, have received less attention. In the future, it may be possible to use the entire EXO to take advantage not only of the markers on the surface of the membrane but also of the internal components, such as microRNAs. This could have applications in various skin issues, especially in the treatment of wounds and in delaying aging.⁵⁰ Furthermore, safety and ethical concerns have restricted the use of stem cell treatments for tissue repair, such as wound healing. This way, strategies based on nonliving cells, such as the use of cell membranes and EV membranes for NP coating, are crucial to advance therapy outcomes, considered safer and easier to manage, and their efficacy can be increased with bioengineering techniques Additionally, through the combination of cutting-edge materials with cell or EV membrane coating technology, scientists can resolve different issues regarding NP application onto the skin.¹⁶⁶

While the development of biomimetic NPs using these membrane coatings has made significant advances, there are still difficulties in translating the biomimetic systems to clinical practice, with the investigations reporting their potential use in skin conditions only presenting in vitro tests and in vivo mouse models. For instance, it is necessary to conduct a thorough characterization of the systems, address problems regarding the manufacturing protocol standardization, and clarify their biodistribution, elimination, and targeting abilities.¹⁷¹ In addition, more research is necessary to completely understand their biosafety and potential toxicity.³⁷

This review clearly shows that coating NPs with cell membranes is an innovative and multipurpose strategy that improves their interaction with the body and their circulation time in the bloodstream. The NPs coated with membranes mimic cells, which helps them evade clearance from the immune system, thereby increasing the therapeutic efficacy of drugs.^28,38,42 In the future, cell membrane and EV coatings on NPs are expected to become more and more popular for skin condition therapy. The coating of NPs in engineered EVs that have been modified to enhance the surface expression of target proteins and antigens is one strategy that is expected to see greater use in the future years.⁶⁰ Likewise, cell membrane and EVs’ immune-evasive and activating abilities could be utilized in immunomodulatory NP-based therapies, particularly in personalized therapies using patient-derived sources. This could especially be helpful in skin diseases with an immune dysregulation background, such as atopic dermatitis. However, scalability is crucial as therapies relying on cell or EV membrane isolation from patient samples are constrained by the quantity of membranes possible to be isolated.¹⁷²

Despite all that is already known about biomimetic NPs, many questions still need to be resolved. It is necessary to establish a simple technique to synthesize these NPs as a whole on an industrial scale, ensuring good yield and reproducibility. It could be helpful to establish optimized and standardized protocols for isolation of cell membranes and EVs and for efficiently preparing these biomimetic NPs as a whole on an industrial scale, ensuring good yield and reproducibility since the selected preparation processes have an impact on the membrane protein's integrity and composition. It is also essential to establish reliable methods for characterizing and ensuring the success of all processes, including the coating process. Last, it is important to mention that although these NPs are stable, keeping them free of contaminants and ensuring that membrane proteins do not denaturate is essential to prevent immunological reactions to endogenous elements.

AUTHOR CONTRIBUTION

A. M. S., I. S. O., M. C., P. C. P., A. S., N. K. J., and E. N. Z. contributed to drafting the manuscript. A. M. S. and I. S. O. prepared figures. F. V., B. H. J. G., Y. X., P. M., and A. C. P. S. provided comments, revisions, and reviewed the manuscript. All authors reviewed the manuscript.

ACKNOWLEDGMENTS

A. C.-P. S., F. V., and P. M. acknowledge the financial support from the Fundacão para a Ciência e a Tecnologia I.P. (FCT)—Research and Development Project grant 2022.05270.PTDC.

CONFLICT OF INTEREST STATEMENT

The authors declare no conflict of interest.

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