Examples of four classes of CPPs and delivered cargoes. The list of cargo is not exhaustive and given for illustration. X = 7, 8, or 9 arginine residues.
\r\n\t
\r\n\tThe history of pacing, the newest devices with home monitoring as well as electrophysiologic aspect of cardiac pacing, with an illustration of the standard surgical implantation procedures will be discussed. Major pacing trials will be mentioned to highlight the benefits of the therapy in both sinus node disease and AV block. The rapid evolution of pacing technology will also be touched upon as we will devote a part of the book to pacing algorithms. The book will also discuss cardiac resynchronization therapy, left ventricular pacing leads and ICDs.
The fields of tissue engineering and regenerative medicine aim at promoting the regeneration of tissues or replacing the malfunctioning organs using a scaffold material. The development of scaffolds that mimic the composition and structure of natural extracellular matrix (ECM) is highly demanded in biomedical sciences. A scaffold used for tissue engineering is considered as an artificial extracellular matrix that provides mechanical support for neo-tissue formation in vitro and/or through the initial period after implantation during cell proliferation and differentiation. In addition to contributing to mechanical integrity, the ECM has an important role in signaling and regularity functions in development, maintenance, and regeneration of tissues [1]. It has long been demonstrated that the composition and architecture of a scaffold influence the cell-environment interactions that determine the effectiveness of the scaffold [2]. The scaffold material must be able to interact with cells in three dimensions and should facilitate the communication. The main goal of tissue engineering is to enable the body to heal itself by regenerating “neo-native” functional tissues [3, 4]. A highly porous scaffold is necessary to control tissue formation in three-dimensional (3D) architecture in a typical tissue engineering approach. The scaffolds provide the microenvironment (synthetic temporary extracellular matrix) for cell attachment, proliferation, differentiation, and neo-tissue genesis. Therefore, the material used for tissue scaffolding must function without interrupting other physiological processes, that is, the scaffold must not promote or initiate any adverse tissue reaction [5]. Furthermore, the scaffold must have an ability to promote normal cell growth and differentiation while maintaining a three-dimensional orientation/space for the cells.
Recently, various materials including ceramics, metals, and polymers have been exploited to develop the scaffolds for tissue regeneration. Although the inorganic/ceramic materials such as hydroxyapatite (HAP) or calcium phosphates are excellent choices for medical implants due to their good osteoconductivity and studied for mineralized tissue engineering, they are disadvantageous due to poor processability into highly porous structures and brittleness. Certain metals have superior mechanical properties and are preferred for medical implants. However, the lack of degradability in a biological environment limits their applications for tissue scaffolding [6]. In contrast, polymers have been extensively studied in various tissue engineering applications, including bone tissue engineering, due to the great design flexibility and structure which can be tailored to the specific needs [6, 7].
Recently, researches in biomaterials have turned to nanotechnology, specifically nanofibers, as the solution to the development of tissue engineering scaffolds and wound repair/care products. At present, few processing techniques are successful in producing nonwoven fibers and subsequent scaffolds on the nanoscale [8]. In basic terms, a nonwoven is a fabric or web composed of fibers. The orientation and properties of the fibers and fabric can be controlled to mimic the native cellular structures, such as collagen fibers of the extracellular matrix. Development and characterization of these nanofibrous structures for tissue engineering applications is crucial in understanding the cell-ECM interactions. Conventional polymer processing techniques are efficient in producing fibers, which are several orders of magnitude larger than the native ECM. Therefore, there has been a concerted effort to develop methods of producing nanofibers that closely mimics to the ECM geometry. In this endeavor, five distinct techniques have proven successful in producing nanofibrous tissue engineering structures: self-assembly, phase separation, melt blowing, drawing, and electrospinning.
Melt blowing and spunbond nonwovens are composed of continuous fiber filaments fabricated by forcing molten thermoplastic polymer through very fine orifices arranged in a spin beam. Melt blowing is a simple, versatile, and one-step process for the production of polymeric fibers in micrometer and smaller scale. During melt blowing and spunbond process, fibers are produced in a single step by extruding a polymer melt through an orifice die. In the melt blowing process, the filaments are drawn and accelerated toward the collector screen via hot air knives, keeping the filaments in a molten state, allowing for fine fiber attenuation. Collected fibers are still in a tacky state, allowing self-bonding between fibers. Conversely, in the spunbond process, as the filaments exit the spin beam, they are rapidly solidified by cool air before being drawn pneumatically. Spunbond and melt blowing techniques can be implemented from lab, pilot, and full production scale. Another method for generation of nonwoven fabric is carding technology. This is a mechanical process that disentangles, cleans, and intermixes fibers to produce a continuous web. In the carding process, short fibers of a few inches in length (staple fibers) are separated and entangled by a series of specialized combed rollers to form an unbonded web. Unlike spunbond and melt-blown processing that require a thermoplastic polymer for melt spinning, a variety of fiber materials can be processed via carding technology, including natural and synthetic polymers, glass, and metal fibers. The high-speed, repeatable, economical production capabilities of these techniques make them attractive candidates for the commercial production of nonwoven-based tissue engineering scaffolding materials. The main disadvantages of nonwoven webs generated by these techniques are their large fiber diameters which hinder the cell attachment, proliferation, and neo-tissue generation, thereby limiting their application as tissue scaffolding material.
Among the various techniques, electrospinning is the facile nonwoven manufacturing method and has recently gained significant interest as a method of scaffold fabrication. The size scale of electrospun fibers mimics to the native ECM and provides an ideal environment for cell proliferation, attachment, and differentiation into the target tissue. However, the fabrication of large-scale electrospun scaffolds using conventional technique is time-consuming and difficult to generate 3D microstructures. This chapter focuses on recent advances in the fabrication of the three-dimensional electrospun nanofibrous scaffolds.
Electrospinning employs electrostatic force to draw a fiber from a spinneret. This fiber solidifies and lies down on a collector in the form of a nonwoven fibrous mesh. Recently, electrospinning has attracted much attention because of its low processing cost and tailorable fiber morphology and fiber diameter. Furthermore, synthetic and natural polymers can be processed into the fiber of different diameters at ambient conditions. However the electrospinning technology has been known for a long time; the practice of this technology remained largely dormant until the 1970s. Morton received the first US patent for the electrospinning of artificial fibers in 1902. Zeleny presented one of the earliest studies of the electrified jetting phenomenon in 1914 [9]. Later on, Formhals filed a series of patents on the processing and apparatus in the decades of the 1930s and 1940s to produce electrospun fibers. Only a few publications appeared in electrospinning research during the 1970s and 1980s, notably by Baumgarten and by Larrondo and St. John Manley [10, 11].
The electrospinning research has gained significant interest when Doshi and Reneker prepared submicron fibers in the 1990s [12]. Since then it has been demonstrated that almost all materials that can be spun from melt or solution by conventional methods can be electrospun into fibers. On account of the remarkable simplicity, versatility, and potential applications of this technique in a variety of fields, the number of publications in this field has been increasing exponentially during the past decade.
The principle of electrospinning involves a high voltage applied between a pendant polymer droplet and a metal target as the counter electrode [13]. Under the electrostatic force, the pendant polymer droplet is deformed from a hemispherical shape to a conical shape which is called as Taylor cone [14]. A thin polymer jet is initiated and travels toward the metal target once the electrostatic force reaches a critical value to overcome the surface tension of the polymer droplet. The charged thin polymer jet is elongated and undergoes a bending instability region where it whips swiftly in the air by the electric field [15]. The thin polymer jet is further stretched due to the evaporation of the solvent during this period and finally deposits on the metal target as a randomly oriented nanofibrous membrane as shown in Figures 1 and 2.
Schematic illustration of electrospinning setups.
(a) Photograph of electrospinning jet. (b) Schematic representation for the formation of electrospun nanofibers.
The electrospinning process is characterized by three major regions: (1) the cone region, (2) the steady jet region, and (3) the instability region. A pendant drop of a fluid is charged at the tip of the nozzle at the initial stage of electrospinning which deforms the droplet into a conical shape, just before jetting occurs. The conical shape is called the Taylor cone, named after G. Taylor who has studied this electrified fluid phenomenon [14]. At a critical electrical stress, a fluid jet is ejected from the apex of the cone. The diameter of the jet at the apex is about 100 micrometers. In the steady jet region, the jet can travel in a straight path anywhere from 1 to 20 centimeters. For a fluid that is a solution, real-time spectroscopic data shows that the loss of solvent due to evaporation in this portion of the jet is negligible. In the final region, the jet deviates from its straight path and undergoes an instability called bending or whipping instability (Figure 2) [15].
Several parameters influence the electrospinning process: polymer solution parameters, processing parameters, and ambient parameters. Extensive polymer chain entanglement is necessary for the fiber formation during electrospinning process; otherwise the polymer solution is electrosprayed into small droplets or forms fibers with large beaded polymer aggregates. Low molecular weight polymers are often difficult to electrospun because of their inability for chain entanglements, while higher molecular weight polymers often cause large changes in solution viscosity, thus increasing the surface tension of the droplet and limiting the ability to electrospun. The choice of solvent is another important processing parameter; the solvent should be nontoxic and should evaporate within the distance from the spinneret to the grounded collector. The processing parameters such as distance between spinneret and collector, solution flow rate, humidity, and voltage intensity (∼a few kV–40 kV) have substantial influence on the morphology, fiber size and uniformity, and porosity.
The tissue engineering scaffolds should closely resemble to native extracellular matrices (ECMs) that provide structural support to cells. The primary role of the scaffold is to provide the temporary support until the neo-tissue formation. The scaffolds that closely mimics to the topographies and spatial structures of ECM are efficient for the cell proliferation and differentiation. The morphologies of ECMs vary according to the functions of the target tissues. Fibrous structures with 3D orientation and random distribution are found in native ECMs in the breast, liver, bladder, lung, and many other organs and tissues [16]. Therefore, it is reasonable to fabricate scaffolds with particular morphologies and structures according to categories and functions of the original native tissues [17].
Electrospinning is a simple method to fabricate the nano−/microfibers in a continuous process. The electrospinning parameters and instrumental setups can be tailored to fabricate micro−/nanofibers with desired morphologies, such as aligned fibrous array, fiber diameter, and fibrous patterns. Therefore, electrospinning has gained significant attention to fabricate tissue engineering scaffolds composed of nano- or submicrometer fibers from numerous materials [18]. However, the conventional electrospinning method produces two-dimensional (2D) sheet-like membranes with small pore and tightly packed fibrous layers that limits the cell infiltration and growth to the depth of the scaffolds [19, 20, 21, 22]; cells mainly spread and distribute on the surface of 2D nanofibrous membrane [23]. Since most human tissues and organs possess three-dimensional microgeometry holding intrinsic functionality, developing three-dimensional structure has drawn a great interest in the search of the tissue surrogates. 3D fibrous scaffolds with macroporous, open structure facilitate the neo-tissue formation, thereby providing biomimetic environment for the cell infiltration, the cell proliferation, and the transportation of nutrients and waste.
Among the process technologies that have been developed and successfully implemented for the design of TE scaffolds, gas foaming (GF) has recently attracted much attention. The gas-foaming technique utilizes the nucleation and growth of gas bubbles dispersed into a viscous polymer solution for the creation of porosity [24]. The gas bubbles are generated in situ either via chemical reaction or by adding inert gases to the polymer phase at different physical environments. The gas-foaming agents are generally released from a pre-saturated gas-polymer mixture resulting in the formation of 3D porous architectures [24]. A supercritical fluid, that is, a fluid above its critical point, such as carbon dioxide, is commonly used for gas-foaming purpose because of its non-flammability, non-toxicity, and moderate critical point (31.1°C and 73.8 bar). Supercritical fluids can be used to form gas-saturated polymer phase by varying the temperature and pressure [25]. In the process, high-pressure CO2 gas is subjected to solid polymer disks to allow saturation of CO2 in the polymer. In this process, the nucleation and growth of CO2 gas bubbles in the material creates the thermodynamic instability and yields mostly a nonporous surface with closed-pore structure. The main limitation of this technique is that it produces the nonporous surface with closed-pore structure with only 10–30% of interconnected pores [26]. Recently, particulate leaching technique has been combined with the gas-foaming process to improve the inter-pore connectivity, although completely eliminating closed pores remains challenging [26].
Recently, Joshi et al. have developed the novel gas-foaming technique to modify the densely packed 2D electrospun membranes into low-density three-dimensional nanofibrous scaffolds [19, 27]. In this technique, authors put the electrospun nanofibrous mat in an aqueous sodium borohydride (SB) solution where the interconnected pores of a mat were filled with the SB solution. The SB solution undergoes the hydrolysis in situ in the nano−/micropores of the nanofibrous mat and produces the hydrogen gas. As generated H2 gas molecules nucleated to form clusters that reorganize the nanofibers into multilayered 3D scaffold with low-density, macroporous spongy structure (Figure 3). In their study, nanofibrous membranes of various polymers were prepared via electrospinning and treated with sodium borohydride solution that was prepared in different solvents. They demonstrate that the solvent for sodium borohydride (either water or methanol) plays a crucial role in the fabrication of 3D scaffolds. The electrospun membranes of polar polymers were processed into 3D architecture in aqueous SB solution, while methanolic solution of SB can be used for both polar and nonpolar polymers. The fabrication process is fast in methanol solution compared to an aqueous solution which is attributed due to the rapid evolution of hydrogen gas from the methanolysis reaction compared to the hydrolysis reaction. This method forms the large pores with multilayered structure that mimics to the ECM. Similarly, Zhao et al. immersed the 2D PCL nanofibrous mat in a NaBH4/methanol solution inside a 3D-printed mold and obtained the 3D nanofibrous scaffolds with controllable geometric shapes [28]. Xie et al. also used the NaBH4 solution as gas source to expand the 2D PCL electrospun mat followed by freeze-drying and obtained 3D PCL scaffolds with highly ordered architecture with controllable gas widths and layered structure as a result [29]. Lee et al. combined electrospinning with salt leaching/gas foaming and developed micro-sized pores in poly(L-lactic acid) (PLLA)/montmorillonite (MMT) nanocomposite fibrous scaffold [30].
Schematic illustration for the formation of low-density, macroporous, spongy, and multilayered 3D scaffolds. Reproduced with permission from Ref. [
The thickness of the electrospun can be increased by increasing the spinning time during the conventional electrospinning process, leading to the 3D fibrous structure. 3D multilayered fibrous membranes of different materials can be fabricated by a sequential electrospinning or co-electrospinning, and post-processing and sometimes an auxiliary electric field is even used to converge the collected fibers into a confined space to produce 3D fibrous structures [19] (Figure 4). Pham et al. [32] prepared layer-on-layer stacks including alternating layers of PCL microfibers and PCL nanofibers. This proposed 3D structure combined the beneficial properties of nanofibers with that of microfibers. The thickness of the nanofiber layers is modulated by electrospinning the nanofiber layers for different lengths of time. They demonstrated that cell infiltration and growth in a multilayered scaffold depends upon the thickness of each layer; increasing the thickness of the nanofiber layer reduced the cell infiltration of the scaffold. Han et al. [33] fabricated the electrospun 3D scaffold of cellulose acetate with three different layers (dense layer, cellular layer, and porous layer) by varying the solutions and processing parameters. In this technique, the total number of layers in multilayered scaffold can be controlled. Furthermore, the composition, the fiber diameter, and the porosity of each electrospun fiber layer can be tailored that affects cell proliferation, migration, and/or differentiation on a scaffold. Erisken et al. [34] reported the functionally graded electrospun PCL and β-tricalcium phosphate (β-TCP) nanocomposites using a hybrid twin-screw extrusion/electrospinning process. Soliman et al. [35] fabricated 3D scaffolds of layered composites with mixed nano- and microscale PCL fibers by modifying the electrospinning setup with two parallel syringes and an actuated collector (co-electrospinning with scalability and modularity from an industrial perspective).
SEM images showing the cross-sectional view of the multilayered scaffold. Reproduced with permission from [
With a subsequent post-process after electrospinning such as folding/rolling up, the aforementioned 3D multilayer fibrous structures, even the as-spun 2D layer-on-layer mats, can turn into a desired morphology for further application. Recently, Duan et al. [37] reported the ultralight highly porous 3D polymer sponges of extremely low-density and low-specific surface area from dispersions of short electrospun fibers in an attempt to mimic the design principle of natural sponges. Short electrospun fibers were first prepared by cutting of electrospun nonwoven and then dispersed in dioxin in different concentrations. Sponges of different densities were prepared from these dispersions by freeze-drying. Such sponge showed tunable cellular infiltration and growth. Ryu et al. [36] developed a three-dimensional scaffolds of carbonized polyacrylonitrile for bone tissue regeneration. PAN fibers were formed by electrospinning onto a Petri dish containing water, and PAN/water was lyophilized for 48 h. Performing the lyophilization step prior to the carbonization process created the micro-sized pores between the electrospun PAN fibers leading to the cotton ball-like 3D scaffolds (Figure 5). The scaffolds were carbonized under 800°C in argon atmosphere and then further modified into a 3D cylindrical geometry. Wang et al. [38] fabricated the electrospun chitosan nano−/microfiber mesh tubes by controlling the spinning parameters. The nonwoven or oriented fibers of the chitosan were deposited and reeled on the bar as the drum rotates. Furthermore, by folding the aligned electrospun nanofibrous meshes several times, a 3D nonwoven macroporous nanofibrous scaffold was manufactured. However, 3D structures generated by this method usually cannot put into use as scaffolds directly because they often have a large space or distance between adjacent fibrous surfaces. 3D electrospun poly(L-lactic acid) (PLLA) microfibrous scaffolds with 5 mm in thickness were fabricated by using a subsequent mechanical expansion process [39]. 3D scaffolds demonstrated a high level of osteoblast proliferation (1.8-fold higher than nanofibrous membranes in a week), actively penetrated the inside of the 3D scaffold, and showed a spatial cell distribution. Sintering after electrospinning is another way to fabricate 3D macrostructures. 3D electrospun scaffolds of pure PLGA and composites of PLGA/hydroxyapatite were fabricated by a two-step process: electrospun meshes were first stacked and then sintered using pressurized gas which improved the mechanical properties and porosity.
Schematic illustration showing the fabrication process of cPAN scaffolds. Reproduced with permission from Ref. [
Fabrication of fluffy nanofibrous mesh using the cosolvent that induces the charge repulsion during the spinning has been employed in recent studies [40, 41]. Lee et al. developed a novel strategy to fabricate highly porous poly(L-lactide) (PLLA)-based fibrous scaffold for bone tissue engineering. Blending of PLLA with its monomer, lactic acid (LA) produced the fluffy-type highly porous nanofibrous mesh (Figure 6). Their study revealed that the LA component in the blend solution assisted the formation of the macroporous spongy fibrous scaffold. The repulsion between the as-spun fibers occurs due to the interaction between the electric field formed by high voltage and the negative charge on LA due to the functional group (COOH), thereby generating fluffy fibrous mesh [40, 41, 42]. Similarly, Xu et al. fabricated a fluffy-type nanofibrous mesh by electrospinning the blend solution of PCL and polystyrene (PS) [42]. In another study, Lee et al. fabricated a core-sheath-type fibrous scaffold (PCL as the core and PS as shell) with fluffy-type architecture using coaxial electrospinning. The PS in the sheath was removed out to avoid its drawbacks associated with scaffold activity [43]. They reported that the negative charge accumulated on the surface of the nanofiber due to the PS (sheath) caused the repulsion between nanofibers under the influence of the strong electric field, thereby generating the fluffy-type nanofibrous mesh [43].
Diagrammatic representation showing the fabrication of three-dimensional fluffy fibrous scaffolds. Reproduced with permission from Ref. [
The highly porous three-dimensional nanofibrous scaffolds can be obtained using bath collectors containing a low surface tension solvent, and this technique is also helpful for fabricating 3D fibrous macrostructures. It is known that aligned electrospun micro−/nanofibrous arrays can be collected with the help of a water reservoir collector or a water vortex. Teo et al. [44] fabricated hierarchically organized 3D nanofibrous meshes of PCL using a water vortex. As-collected 3D PCL nanofibrous meshes were either freeze-dried or dried in a mold under ambient condition. The freeze-dried 3D meshes showed visible pores on the surface of the mesh, while the 3D meshes dried in room condition were densely packed without any apparent pores on the surface. Yokoyama et al. [45] fabricated the poly(glycolic acid) (PGA) 3D spongy nanofibrous mesh by combining electrospinning with wet spinning. The spongy-type 3D PGA nanofiber fabric showed a low apparent density and high porosity compared to the usual PGA nanofiber nonwoven mats prepared by conventional electrospinning method.
3D fibrous macrostructures fabricated by modifying collector are common. Zhang et al. [47] obtained tubular structures through designing the collectors. The collectors used in fabricating the electrospun fibrous tubes are static 3D columnar collectors. The schematic illustration and the photos of the relevant fibrous tubes are separately shown in Figure 7A (
(A) Schematics of rotary jet-spinning process. Rotary jet spinning consisted of a perforated reservoir. Photographic image of 3D nanofiber structure produced by rotary jet spinning and corresponding SEM image. (B) (a) schematic illustration of collecting process using a cylindrical collector with equally spaced circular protrusions. (b) a fibrous tube with patterned architectures (scale bar = 5 mm). (c) Magnified image of panel b (scale bar = 200 μm). (d) Schematic illustration of collectors with two different patterns and relevant fibrous tube (pc, patterned collector; ft., fibrous tube). (e) a fibrous tube with two different patterns (scale bar = 5 mm). (f,g) Magnified images of two different patterns of panel. Reproduced with permission from Refs. [
Another technique for the fabrication of highly porous 3D structure is the use of porogens. Materials such as ice crystals, salt particles, polymers (e.g., poly(ethylene oxide) (PEO)), which acts as porogens, usually are mixed simultaneously with the precursor during electrospinning to enable rapid buildup of the nanofibrous volume and then washed away or dried after a desired thickness is reached [49]. For example, Baker et al. [50] demonstrated that inclusion and subsequent removal of a sacrificial fiber population within a fiber-aligned fibrous scaffold enhance cellular infiltration. Poly(ɛ-caprolactone) (a slow-degrading polyester) and poly(ethylene oxide) (a water-soluble polymer) were co-electrospun from two separate spinnerets to form dual-polymer composite fiber-aligned scaffolds, and PEO is washed out. Removal of these sacrificial elements (PEO fibers) preserved structural and mechanical anisotropy and tuned to generate composites with varying mechanical properties. Kim et al. [51] fabricated a 3D macroporous and nanofibrous hyaluronic acid (HA) scaffold by combining the electrospinning process with a salt leaching technique (Figure 8). The salt particulates, as a porogen, deposited during electrospinning were leached which produced water-soluble HA-based scaffold with macroporous and nanofibrous geometry. Ki et al. [52] developed 3D nanofibrous fibroin scaffold with high porosity by electrospinning. The electrospun SF nanofiber dispersion was collected in 1,4-dioxane having NaCl particles (300–500 μm) as porogen. The scaffold was cross-linked, lyophilized, and washed with PBS solution several times that was effective for cell addition and proliferation.
(A) Diagrammatic illustration showing the polymer jet evolution; (B) digital image showing the vertical growth of solidified fibers; (C) the polymer solution from the needle tip was immediately split into minijets (arrows indicate minijets); and (D) fluffy-type nanofiber mesh. Reproduced with permission from Ref. [
In conclusion, it is evident from the foregoing examples that the diversity in biomaterials is immense. The utilization of electrospun nanofibers as tissue scaffold is challenging. A great deal of effort has been put in to prepare the biomimetic 3D nonwoven scaffolds based on natural as well as synthetic polymers. Different processing routes have been proposed to produce 3D nanofibrous scaffolds with a great variety of architectures. Many clever approaches to mimicking the structure and, more importantly, the function of the ECM have been devised. Different fabrication methods have their own merits and demerits. However, despite all the extensive literature containing references to the so-called biomimetic three-dimensional scaffolds, it is the authors’ opinion that much work is still needed to obtain clinically successful materials. Various types of bioactive components, such as peptides (e.g., RGD) and hydroxyapatite, may be incorporated/integrated to enhance the cellular response and biocompatibility of the 3D nanofibrous scaffolds. More importantly, the real-time monitoring/control systems may be established by integrating either electronic components or magnetic responsive elements with 3D nanofibrous mesh, enabling remote actuation and thus with an active role in the modulation of the host response on implantation that can revolutionize human therapies toward successful regeneration. It is imperative that these important technologies continue to be investigated for their ability to interact in biological systems. It will be most interesting to follow the further progress and the expected and unexpected leaps forward that will be shaping the field in the coming years.
A novel approach to overcome cell membrane impermeability and to deliver a large variety of particles and macromolecules into cells has been recently emerged, which is called cell-penetrating peptides (CPPs), also known as protein transduction domains (PTDs) [1, 2]. CPPs are generally short (up to 30 amino acids in length) water-soluble, cationic, and/or amphipathic peptides which make them promising vectors for therapeutic delivery, leading to a considerable amount of research focused on the intracellular delivery of drugs [3, 4, 5]. There are two principal types of CPPs that have been utilized for this purpose: (i) cationic CPPs, composed of short sequence of amino acids (arginine, lysine, and histidine). The indicated amino acids give the cationic charge to the peptide and permit its interaction with anionic motifs on the plasma membrane by a receptor-independent mechanism. (ii) amphipathic peptides, which have lipophilic and hydrophilic tails that are responsible for a direct peptide translocation mechanism across the plasma membrane [6].
The most important characteristic of CPPs is that they are able to translocate the plasma membrane at low micromolar concentrations in vivo and in vitro without using any receptors and without causing any significant membrane damage [7, 8]. Other benefits of using CPPs for therapeutic delivery are the absence of toxicity as compared to other cytoplasmic delivery devices, such as liposomes, polymers, etc. [6]. The mechanism for the CPP-facilitated cellular uptake remains not clear and depends on cargo and cellular type [9]. Due to its high density of basic amino acid residues (Arg and Lys), the large charge at physiological pH excludes the passive diffusion of CPPs across the lipid bilayer. Furthermore, it seems that classical uptake mechanisms such as protein-based receptors and transporters are not involved. On the contrary, endocytosis was shown as a common uptake mechanism, but is controversial at the same time. For example, in a number of reports, CPP uptake was not inhibited at 4°C or in the presence of inhibitors of endocytosis; in contrast, a capture of CPPs in the endocytotic vesicles was observed when soluble heparin sulfate was added [9, 10]. Many other studies indicate that aggregation of the cell surface glycosaminoglycan heparan sulfate (HS) is an important element in the uptake mechanism [2]. The challenge of the strategy using CPPs should take into consideration the size, stability, nonspecific versus specific associations, and potency versus toxicity that all play an important role for the selection of delivery systems [5].
The CPPs are initially discovered in 1965 when it was observed that histones and cationic polyamines such as polylysine stimulate the uptake of albumin by tumor cells in culture. It was shown that the conjugation of polylysine to albumin and other proteins enhances their transport into cells. Moreover, a comparison study of different homopolymers of cationic amino acids demonstrates that medium-length polymers of arginine enter cells more effective than similar-length polymers composed of lysine, ornithine, or histidine [11]. In 1988, it was discovered that the human immunodeficiency virus type 1 (HIV-1) encoded trans-acting activator of transcription (Tat) peptide which also translocates cell membranes and gains intracellular mammalian cells [12, 13]. Covalently the conjugation of Tat peptide to proteins or fluorescent markers allowed these molecules to gain into the cell. A few years later, another discovery was followed when polycationic peptide of natural (VP22 and AntP) and synthetic origin (transportan) was used for the delivery of genes, proteins, small exogenous peptide, or even nanoparticles. Furthermore, it was demonstrated that small domains in these peptides are often responsible for cellular entry [14]. Thus, these translocation sequences could be shortened to a few amino acids in comparison with the first Tat peptide, without affecting cell penetration efficiency [13]. Since that time, the list of synthetic CPPs has increased sharply, and the number continues to rise (Table 1). In the last decade, another peptide was described named maurocalcine (MCa), a 33 amino acid residue peptide that has been isolated from the venom of the Tunisian chactid scorpion
Peptide | Sequence | Origin | Cargoes |
---|---|---|---|
Tat48-60 | GRKKRRQRRRPPQ | VIH-1 | ADN, peptide, PKC inhibitor |
Pénétratin | RQIKIWFQNRRMKWKK | HSP20 phosphopeptide | |
Transportan | GWTLNSAGYLLGKINLKALAALAKKIL | Galanin + Mastoparan | Protéine, PNA |
Pep-1 | KETWWETWWTEWSQPKKKRKV | Rich domain of tryptophan + | Enzyme |
MPG | GALFLGFLGAAGSTMGAWSQPKKKRKV | Hydrophobic motif derived from HIV-1 gp41 + | siARN, oligo-nucléotides |
CADY | GLWRALWRLLRSLWRLLWRA | Dérived from PPTG11, variant of JTS1 fusion protéin | siARN |
(Arg)x | (RRRRR)X | Synthetic peptide | siARN, Cyclosporine A |
MAP | KLALKLALKALKAALKA | Synthetic peptide | Natural CPPs |
Maurocalcine | GDCLPHLKLCKENKDCCSKKCKRRGTNIEKRCR | Doxorubicin |
Examples of four classes of CPPs and delivered cargoes. The list of cargo is not exhaustive and given for illustration. X = 7, 8, or 9 arginine residues.
This peptide possesses vector properties when coupled to fluorescent streptavidin. This complex was shown to enter various cell types within minutes and in all cell types tested, a common feature of CPPs. A variety of mutants of MCa were then designed in order to unravel the most active residues for its pharmacological and penetration activities (Figure 1) [18, 19].
Example of origin of four CPPs: Maurocalcine, penetratin, tat, and polyarginine. Maurocalcine, penetratin, and tat are derived from natural sequences, but polyarginine was produced by de novo conception in order to obtain a good cellular penetration.
Two distinct advances were shown to be used to bind CPPs to molecular cargoes. One process is non-covalently which connect CPP to its cargoes using electrostatic interactions, such as MPG and Pep-1, amphipathic peptides carriers, which link to cargoes beyond any cross linking or chemical changes [20]. The second approach is more frequent and uses a covalent relation between the two compounds. This means has been widely used by different teams and has demonstrated positive advances, especially with TAT, penetratin, or polyarginines [21].
Various mechanisms for CPP internalization have been suggested, but the exact one is still not well known. Yet, many data approve that the energy-dependent tool (endocytosis) and the energy-independent mechanism (direct translocation) or both are involved in the cellular uptake progress [22].
For direct penetration, various mechanisms have been described: the carpet-like model (membrane destabilization) [23] and the pore formation model (barrel-stave) [24]. Positively charged CPPs interact with negatively charged membrane components like phospholipid bilayer or heparan sulfate. Such interaction is dwelling on the first stage of all of these mechanisms, followed by destabilization of the membrane and finished by crossing of the CPP on the lipid membrane.
For endocytosis transduction or cellular digestion, pinocytosis, phagocytosis, and receptor-mediated endocytosis have been reported [25, 26]. A sum-up of CPP transduction systems is shown in Figure 2. In pinocytosis, the plasma membrane absorbs solutes, while in phagocytosis it takes great particles. In clathrin-mediated endocytosis, clathrin and also caveolin, which are receptor-mediated endocytosis and cover the intracellular part of the biomembranes, possess a key role in the uptake mechanism. These protein structures are pivotal for the membrane invagination and for the construction of the vesicles after bounding the extracellular molecule to the membrane receptor. Clathrin has a great diameter in comparison with caveolin-coated vesicles and was also considered as a selective route for the translocation of compounds into cells through specific receptors on the surface of the cell [27].
CPP translocation mechanisms.
Many determinants influence the internalization process, such as the nature of CPP or the cell type, the cargo, and the experimental conditions (temperature and pH) [22].
Chemotherapy used for treatment of cancer has a lot of defects because of the toxicity of the drugs to normal healthy cells and also to resistance developed by tumor cells to the anticancer drug [28]. The major inconveniences with used cancer chemotherapy are the absence of specificity target to tumor cells and thus poor antitumor effect. The challenge in cancer therapy is to know how to deliver a drug intact to the cytosol of every cancer cell, sparing healthy cells.
It was shown that polyarginines carry cargoes that exceed 500 Da by molecular electroporation across the cell membrane which may solve part of the drug delivery problem [29]. However, the use of well-chosen linkers and anions can help target cancer cells and contribute to successful conjugation process. For example, the CXC chemokine receptor 4 (CXCR4) is overexpressed in different types of cancer, including prostate, breast, colon, and small-cell lung cancer. Snyder et al. linked the CXCR4 receptor ligand, DV3, to two transducible anticancer peptides: a p53-activating peptide (DV3-TATp53C′) and a cyclin-dependent kinase 2 antagonist peptide (DV3-TAT-RxL). Treatment of tumor cells expressing the CXCR4 receptor with either the DV3-TATp53C′ or DV3-TAT-RxL targeted peptides resulted in an enhancement of tumor cell killing compared with treatment with nontargeted parental peptides [30]. Furthermore, hypoxia-inducible factor-1 (HIF), the transcription factor central to oxygen homeostasis, is regulated via the oxygen-dependent degradation domains (ODD) of its α isoforms (HIFα). The amino- and carboxyl-terminal sequences of ODD (NODD and CODD) were fused to TAT and injected into sponges implanted subcutaneously (s.c.) in mice by William et al. They demonstrated that this injection causes a markedly accelerated local angiogenic response and induction of glucose transporter-1 gene expression, thus opening additional therapeutic avenues for ischemic diseases [31].
In some cancer cells, such as melanoma (common eye cancers in adults), p53 seems to be inhibited by overexpression of HDM2. A transducible peptide that inhibits HDM2 and Bcl-2 for their ability to induce tumor-specific apoptosis in these cells was tested [30]. In this study, it was demonstrated that the anti-Bcl-2 peptide induced apoptosis in tumor cells but also caused variable levels of toxicity in normal cells and tissues. On the contrary, the anti-HDM2 peptide induced apoptosis in tumor cells, with little effect on normal cells in a therapeutic dose range. This peptide also caused regression of retinoblastoma in rabbit eyes, with minimal damage to normal ocular tissues. They conclude that the inhibition of HDM2 may be a promising strategy for the treatment of uveal melanoma and retinoblastoma, and that strategy may be an effective technology for local delivery of anticancer therapy to the eye.
Most of the patients with sporadic renal cell carcinomas (RCCs) exhibit mutation of the Hippel-Lindau (VHL) tumor suppressor gene. Conjugation of the protein transduction domain of HIV-TAT protein to the amino acid sequence (104–123) in the beta-domain of the VHL gene product (pVHL) arrested and then reduced proliferation and invasion of 786-O renal cancer cells in vitro. Besides, daily i.p. injections with the conjugate put off and, in some cases, caused partial regression of renal tumors that were implanted in the dorsal flank of nude mice [32].
The tumor suppressor gene
Recently, it was shown by Sonia et al. that coupling doxorubicin (Dox) to three cell-penetrating peptides Tat, penetratin, and maurocalcine (Dox-CPPs) is a good strategy to overcome Dox resistance in MDA-MB 231 breast cancer cells and CHO cells (Figure 3) [3, 34]. We also reported that all conjugates are able to promote cell apoptosis in the breast cancer-resistant cells MDA-MB 231 at lesser concentration needed for Dox alone. Indeed, apoptosis death was shown to be correlated with ladder-internucleosomal degradation, chromatin contraction, caspase activation, Bad and Bax activation by oligomerization on the mitochondrial membrane, and liberation of cytochrome c. Despite the effective Bcl-2 overexpression in apoptosis induced by the Dox alone, such potency was shown to be insufficient in case of Dox-CPP-triggered cell apoptotic death. Otherwise, these results suggest that there are other apoptotic signaling pathways, independent of mitochondrial one, which are implicated in Dox-CPP apoptosis. Moreover, greater effectiveness of Dox when coupled to CPPs is not due only to its higher accumulation on the cells but also to the incitement of other signaling pathways. These pathways include death receptors and activation of the JNK pathway [4, 35].
Cellular internalization of Dox by MCa. MDA-MB231 cells treated with (a) RPMI, (B) Dox alone (red), and (C) Dox coupled to Dox at the same concentration (red).
Another study led by Leslie Walker et al. showed that conjugated Dox to both ELP and SynB1 prevents tumor development in mice. In fact, conjugation of Dox to SynB1-ELP was more efficient in tumor inhibition under hyperthermic condition than Dox alone, which was twofold higher. Such conception was considered hopeful peptide candidates for drug delivery [36]. The anticancer activity of Dox was also enhanced when constructed a drug delivery system by developing 25 nm gold nanospheres (GNSs) conjugated to four α-helical CPPs [37].
A thermally sensitive quantum dot that exhibits an “on-demand” cellular uptake behavior via temperature-induced “shielding/deshielding” of CPP on the surface was synthesized. Poly(N-isopropylacrylamide) (PNIPAAm) and CPP were biotinylated at their terminal ends and co-immobilized onto the surface of streptavidin-coated quantum dots (QDs-Strep) through biotin-streptavidin interaction. Namely, under a lower critical solution temperature (LCST), the hydrated PNIPAAm chains blocked CPP cellular uptake. This effect was broken down when the LCST was raised to allow CPP moieties to be exposed on the cell surface, leading to QD cellular uptake.
Additionally, the “shielding/deshielding” temperature of CPP was also used for siRNA delivery system. Biotinylated siRNA was coupled to the surface of TSQDs. Indeed, the amount of corresponding gene silencing was increased due to the surface exposure of CPP within a rising temperature above the LCST [38].
Over the last decade, a great attention has been assigned to the importance of CPP on drug transportation of bioactive molecules in various preclinical studies. In fact, novel computational basics have been made in order to develop knowledge on CPPs [39].
Previously, different researchers have developed a few in silico algorithm approaches for CPP prediction (CPPpred) and screening to facilitate throughput CPP-based research. The in silico screening/prediction methods aimed on the use of scales of chemical characteristic, such as z-descriptors [40, 41]. It is generally followed by experimental validation to make it reliable with less cost and time-consuming approach. Later on, other CPP prediction applied neural network (NN) strategies were developed and consist on introducing an N-to-1 NN. The network proceeds by a sequence of 5 to 30 amino acids in length, as input, and gives a prediction of how probably each peptide is to be cell penetrating [42]. This CPPpred offers an advantage since it was developed with repetition-reduced training and test sets.
Over the years, the commitment therapeutic importance of CPPs motivated other teams to develop the first version of CPP database, i.e., CPPsite which supports broad information on the promising use of CPPs [43]. The CPPsite manually created database of 843 experimentally described CPPs. Each consulting gives us data of the peptide involving peptide sequence, peptide name, nature of peptide, origin, chirality, uptake efficiency, subcellular localization, etc. A deep area of user-friendly tools has been integrated in this database like analyzing and browsing tools. Moreover, they have introduced other informations concerning peptide sequences such as secondary/tertiary structure and physicochemical properties of peptides.
This database version was then developed and updated as a CPPsite 2.0 and holds 1855 entries, including 1012 recent new entries [44]. The renovated version contains further data concerning chemically modified CPPs used on the in vivo model. In addition to other informations on delivered cargoes by CPPs (proteins, molecules, nanoparticles, DNA, RNA, etc.), secondary and tertiary structures of natural and chemical CPPs (including CPP with D-amino acids) were also predicted in view of their important role in the functionality of CPPs and stored in the database. Numerous tools for information browse and analysis are combined in this database and considered as a useful resource since it is compatible for all users, including smartphone and tablet.
CPP prediction sites are a promising assist to the researchers to design cell penetrating peptide, as well as making different modification and to investigate their effect on cell penetration potency [45].
The progressive and continuous application of CPPs shows that they are efficient delivery vectors. Because of the need to ameliorate the drug delivery, a great number of CPP-based applications are still drawing the attention of researchers.
In this review, the current tendency in drug delivery by CPPs is summed up. Conjugation with CPP increases cell-surface affinity and eventual cellular uptake of bioactive molecules.
Authors are listed below with their open access chapters linked via author name:
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\n\n\n\n\n\n\n\n\n\nJocelyn Chanussot (chapter to be published soon...)
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