Cell Penetrating Peptides: Classification, Mechanisms, Methods of Study, and Applications

Cell‐penetrating peptides (CPPs) encompass a class of peptides that possess the remarkable ability to cross cell membranes and deliver various types of cargoes, including drugs, nucleic acids, and proteins, into cells. For this reason, CPPs are largely investigated in drug delivery applications in the context of many diseases, such as cancer, diabetes, and genetic disorders. While sharing this functionality and some common structural features, such as a high content of positively charged amino acids, CPPs represent an extremely diverse group of elements, which can differentiate under many aspects. In this review, we summarize the most common characteristics of CPPs, introduce their main distinctive features, mechanistic aspects that drive their function, and outline the most widely used techniques for their structural and functional studies. We highlight current gaps and future perspectives in this field, which have the potential to significantly impact the future field of drug delivery and therapeutics.


Introduction
Cell-penetrating peptides (CPPs) are a diverse group of short peptides with the ability to cross cell membranes without impairing their structural and functional integrity, which have been widely investigated to deliver bioactive molecules, therapeutics, and theragnostic cargoes within the cellular environment.3] Hundreds of studies and reports have shown that CPPs as are excellent tools for promoting intracellular delivery of a wide range of bioactive molecules, including oligonucleotides, siRNA [4,5] anticancer drugs, [6] bioimaging agents, [7][8][9] DNA, nanoparticles, [10][11][12] proteins, [1,13] and other peptides.This broad potential of applications is witnessed by several ongoing studies and clinical trials that confirm its success both in vitro and in vivo applications.
Historically, the first CPPs found were domains of naturally occurring proteins that showed the ability to enter cells; the sequence corresponding to Tat CPP in HIV-1 TAT protein was proven to be necessary for transactivation and subsequent gene transcription in the HIV infection process. [14]The ability of the Drosophila protein Antennapedia's homeodomain to cross cell membranes was then discovered a few years later, [15,16] confirming the existence of specific features in proteins that drive the membrane translocation.From these two seminal discoveries, a wide range of natural and natural-derived CPPs has been discovered and described; to date, the database CPPsite 2.0 [16] (http://crdd.osdd.net/raghava/cppsite/)has collected approximately 1700 CPP sequences.Intriguingly, some CPP can act as modulators of cell functions, combining delivery and bioactive properties; these peptides are commonly referred to as bioportides. [17]rom a physical-chemical perspective, CPPs are usually 4-40 amino acids in length and exhibit amphipathic and cationic character.The majority of the CPPs are indeed positively charged at physiological pH, with arginine and lysine residues being the most relevant amino acids driving membrane crossing properties and the internalization process (see dedicated section below).Structurally, some CPPs are characterized by an α-helix or a β-sheet structure, while others lack a specific conformation.Overall, the heterogeneity of these peptides is so large that a strict definition of CPPs under a structural and physio-chemical framework is nontrivial; this is further exacerbated by the multitude of possible mechanisms of internalization, which can be broadly divided into energy-dependent (endocytosis) or energy-independent (direct translocation).In turn, the internalization pathway for a given CPP may depend on several factors, including intrinsic physiochemical properties, cell type, cell membrane characteristics, CPP concentration, temperature, incubation time, and type of cargo. [18]Taken together, all these factors make CPPs a fascinating topic that is still worth investigating and that holds huge potential for application.The progression of our understanding governing their action and functionality will drive novel advancements in CPP design, with the goal of maximizing and aptly tailoring their cargo-shuttle performances to different therapeutic scenarios.

Classification of CPPs
Due to the wide variety of different typologies exhibited by CPPs, a rigid and systematic classification is difficult to achieve.Different attempts of classification have been pro-posed in the last years [19][20][21] based on physicochemical properties (charge, amphipathicity, hydrophobicity etc.), structural characteristics (β-sheet, α-helical, PP-II helix, random coil or a mix of them), origin (from natural proteins, semi-or full synthetic), application (cargo delivery, bioimaging, modulators of biochemical events e. g. -bioportides-) and even mechanisms of entry. [22]Here we propose a functional taxonomy on different levels of priority bearing in mind that a fully comprehensive classification of CPPs is practically impossible, as many of the characteristics on the basis of which a classification may be done (sequence, structure, charge, function etc.) may influence other functional properties also used to categorize CPPs, leading to a de facto overlapping of the classification terms (Figure 1).
Based on their origin, CPPs are usually divided into three categories: protein-derived (natural), chimeric (or semi-synthetic) and synthetic (designed) (Figure 1).Some CPPs are fragments derived from natural proteins that have functions of membrane translocation.This function can be retained when the fragment is isolated from the entire sequence. [14,23]Oppositely, some peptides can show CPP properties only out of the parent protein (de novo function), as the case of so named cryptides. [24]Different criteria in classification relate to peptides physicochemical and structural features (Table 1).
Animal toxins, primarily isolated from arthropods and snakes, [51] and membrane active peptides, i. e., peptides that cause temporary or permanent disruption of the cell membrane, such as antimicrobial peptides (AMPs), are sources of CPPs too. [52,53](Tables 3 and 4).
It is not surprising that many AMPs can behave as CPPs as these two groups often share structural and physicochemical characteristics that, depending on the experimental conditions, are balanced between their functions: membrane disruption/ perturbation and cell penetration. [71,79]Many human proteins are also a source of CPPs, possessing the advantage of minimizing possible in vivo immunogenicity and toxicity effects.Many proteins-derived CPPs have been identified such as VectoCell, [80] Lactoferrin, [29] Sim-2, [81] 2IL-1a. [33]Tables 1-4  from natural sequences that have been opportunely modified to circumvent some negative side effects such as cytotoxicity, or to enhance penetration and stability properties.A second level of classification may be done according to the physicochemical features of CPPs which can be grouped into amphipathic and non-amphipathic CPPs with different charge  Elastin derived peptides (VPGXG) n, where X can be any amino acid except proline Elastin Hydrophobic [36]  characteristics (cationic and acid).A subclassification is generally done for amphipathic CPPs distinguishing between primary-and secondary-amphipathic CPPs.Primary peptides possess an intrinsic distribution of hydrophilic and hydrophobic residues on their molecular surface.Secondary CPPs are instead not structured in solution, but assume an α-helix or a β-sheet structure only upon interaction with a phospholipid membrane. [82]Cationic peptides have a high proportion of basic amino acid residues (mainly lysine and arginine) which are diversely distributed along their structures.Internalization mechanism may be strongly influenced by these characteristics, the binding of cationic CPPs to the lipid bilayers in cell membrane is very different from that of amphipathic CPPs, thus driving in a different way also the internalization pattern. [83]PPs beside many promising features, suffer also different drawbacks such as a poor stability in vivo conditions and a certain cytotoxicity or a poor permeability through different membranes systems.To tackle those drawbacks, structural modifications and novel design strategies have been developed to enhance the ability of CPPs to reach their targets.Such structure modifications are represented by cyclic, stapled and dendrimeric arrangements giving rise to other structural classification determinants.Linear structures are the most common, but in the CPPs panorama cyclic and dendrimeric peptides are becoming increasingly popular.Often, such kind of modifications is carried out to enhance peptide's stability to resist to protease activity in physiological conditions. [84,85]In some cases, cyclic structures also enhance the penetrating properties of these molecules as in the case of cFΦR4 cyclopeptide (Table 5) that is translocated into mammalian cells more efficiently than R9, one of the most potent linear CPPs reported to date. [86]Furthermore, cyclic peptides were observed to be highly efficient in binding to intracellular macromolecular targets thanks to their constrained conformation, specifically targeting protein-protein interactions.These features coupled with low cytotoxicity and higher stability than their linear counterparts, make cyclic CPPs promising therapeutic tools. [87,88]ationally designed peptides blocked in a stable α-helical conformation, are defined stapled peptides (Figure 2).Different chemical strategies are commonly employed using chemical linkers [89] to generally link two positions on the peptide structure allowing stabilization of the α-helical structure.
Stapled CPPs resist proteolytic degradation, mimic the binding affinities and specificities of alpha-helices in proteins, representing a new way to face challenging pharmaceutical goals such as protein-protein interactions. [90]Stapling may also modify the relative hydrophobicity leading to an enhancement of cell permeability. [91]These peptides can be obtained by molecular modeling calculations [92] starting from linear sequences known to be CPPs and opportunely modified.There are also cases of stapled peptides coupled to cyclic peptides to enhance efficiency of the resulting molecule [93] in terms of penetrability and target interaction, as in the case reported by Dougherty and coll. [94]Branched dendritic peptides are also another  approach to modulate cell entry.With such a strategy, it is possible to enrich a multipeptide structure exposing several CPP motifs to enhance cellular uptake.This strategy is often coupled to the design of peptide decorated nanoparticles (NPs) as shown in ref, [95] to efficiently deliver molecules while reducing, at the same time, the overall number of discrete peptidyl entities required per NP.98][99] Often, a nature-inspired design is used [100,101] or other methods have been employed, such as plasmid phage display. [102]ethods based on Structure-Activity Relationship (SAR) have also been exploited that allow the discovery of potent cyclic CPPs. [84] Rational design may be also afforded using bioinformatic tools specifically designed to predict cell permeability and other peptides features (charge, hydrophobicity, amphipathicity).These tools predict the cell-penetrating ability of peptides using combination of support vector machine (SVM) artificial neural networks (ANN), random forest (RF), and position-specific scoring matrix (PSSM) profiles to classify peptides as CPPs or non-CPPs, examples of these tools are CPPpred (http://bioware.ucd.ie/~compass/biowareweb/Server_pages/cpppred.php), [103]CellPPD (https://webs.iiitd.edu.in/raghava/cellppd/index.html), [104] BChemRF-CPPred (http://comptools.linc.ufpa.br/BChemRFCPPred/) [105]other, such as MLCPP 2.0 [106] and KELM-CPP [107] are based on machine learning (ML) predictors.A brief review has this topic. [108]he α-helical conformation seems to be one of the major determinants in promoting the cell penetrating power of peptides, thus the development of synthetic building blocks useful to constrain peptides and peptoids secondary structures, Synthetic designed S5-S5 stapled peptides, alpha helical [127]   TatRI qpprrrqrrkkrg Designed to deliver a iCal36 CFTR [a] stabilizing peptide Retro-inverso [128]   diLR10 LCKLLKKLCK Synthetic designed Alpha helical, self-assembling in a dimeric form [129]   [a] Cystic fibrosis transmembrane conductance regulator.
has led to the de novo production of new potent CPPs composed also of non-proteinogenic amino acids, such as α,αdisubstituted α-amino acids (dAAs), proline (Pro) derivatives, βamino acids (β-AAs), γ-amino acids (γ-AAs), inserted within sequences to stabilize helical structures in CPPs. [109,110]Retroinverso (RI) peptides, also known as retro-all-D or retroenantio peptides, incorporate D-amino acids as substitutes of L-amino acids, but presented in a reverse (retro) order compared to the parent molecule.RI analogues of L-peptides are usually employed in those harsh physiological environments to prevent proteolytic cleavages and overall improve the stability of peptides. [111]Usually, RI-CPPs maintain the main biophysical characteristics of their L-parental homologues, and hence maintain the ability to permeate cell membranes, being internalization mechanisms often independent of stereo selective interactions, [112] even if their use is limited, probably due to some potential cytotoxic side-effects. [113]Another class of CPP mimetics is the cell penetrating peptoids (CPPe).Peptoids are a family of sequence-defined oligomers of poly(N-(2-methoxyethyl) glycine) monomers, and similarly to other peptidomimetics, peptoids are highly stable against enzymatic proteolytic activity.Peptoids have an achiral backbone and due to the lack of tertiary backbone amide donors for hydrogen bonding, they generally do not show distinct secondary structures.Stable secondary and tertiary conformations may be induced by inserting proper side chains.To enhance cell permeability of CPPs and cargo transport efficiency, different strategies have been used such as the use of alternative side chains composed of cationic moieties different from the natural lysine-or arginine-like residues, [114] the synthesis of macrocyclic peptoids enriched with hydrophobic residues [115] or recently, by introducing arginine Nα-methylation, a modification demonstrated to enhance the performance of cell-penetrating peptides. [116]A generally accepted dogma regarding CPPs was that the cationic character was mandatory to exploit cell permeabilization.This aspect has been refuted by different studies in which anionic peptides perform as well as cationic counterparts, effectively broadening the landscape of molecules transportable by these peptides.One of the first examples is represented by the acidic amphipathic proline rich peptide SAP(E) where the basic residues have been substituted with the anionic function of a glutamic acid. [117]Another anionic CPP, PepNeg (sequence: SGTQEEY), was shown to be able to trespass a Brain Blood Barrier (BBB) model with high efficiency and despite its short sequence was able to transport a large protein represented by GFP. [118]P28 anionic CPP is a derivative of Azurin (residues 50-77), a member of the cupredoxin family of proteins, from Pseudomonas aeruginosa.It has been employed as a bioportide being able to penetrate different kinds of cancer cells and inducing the G2/M cell cycle arrest by modulating the expression of p53. [119]

Mechanisms of CPPs internalization
Although CPPs have been the subject of numerous studies, the mechanisms by which they enter cells remain not fully under-stood, and in some cases controversial.On the other hand, understanding the mechanisms of CPP uptake into cells is crucial towards the optimization of both efficiency and safety of the intracellular delivery, which may also be aptly fit to a specific cargo.The cellular absorption pathways for CPPs include non-endocytic (or energy-independent) pathways and endocytic pathways, with each one having its peculiar characteristics (Figure 3).The uptake mechanisms experienced by individual CPPs is influenced by both its physicochemical characteristics and the chosen experimental settings. [131]Structure-activity relationship studies have highlighted the significance of positive charges, particularly arginine residues, and hydrophobic alpha helical structures in the uptake mechanism. [131,132]The majority of CPPs have several arginine residues, which are preferable to lysine for peptide delivery and CPP action.However, strong effects of arginine-free CPPs like TP10 suggest that other factors may be at play.CPP conformation and sequence length also play a role, as demonstrated by the difference in uptake between pVEC and scrambled pVEC.Non-amphipathic CPPs mostly rely on endocytosis at low doses, while primary and secondary amphipathic CPPs can directly cross the cell membrane at low micromolar concentrations.For instance, initial studies on the internalization mechanism of arginine-rich peptides indicated a direct translocation across the cellular membrane that bypassed endocytosis and the involvement of specific receptors.[135] The switch between different uptake mechanisms might be concentration dependent.It has been shown that at low concentration, arginine rich CPPs are mainly endocytosed, whereas rapid cytoplasmic entry occurred at higher concentration.Overall, experimental setups, such as CPP concentration, cell type, temperature, and incubation period, can all influence the mechanism of CPP entrance.The type, size, and binding strategy of the cargo can also affect the CPP translocation process, with smaller payloads leading to cytoplasmic redistribution of TAT.The labelling of CPPs with different fluorophores may also alter their absorption mechanism, intracellular distribution, and cytotoxicity.Here the main translocation mechanisms are briefly outlined, while comprehensive reviews on this topic can be found elsewhere.

Direct translocation
Direct translocation of CPPs is typically triggered by the interaction between the positively charged CPP with negatively charged membrane elements like heparan sulphate (HS) and the phospholipid bilayer, causing either permanent or temporary membrane instability brought on by the folding of the peptide on the lipid membrane. [135]Direct translocation is regarded as a single-step process, requiring no energy, and involving different mechanisms, such as inverted micelle creation, pore formation, the carpet-like model, and the membrane thinning model.Inverted micelle formation is observed in the early stages of cellular uptake and result in the entrapment of the peptide, favoring transport of hydrophilic compounds conjugated to the peptide. [136]For instance, this mechanism was proposed for the direct penetration of penetratin, while it is unlikely to apply to highly cationic CPPs as TAT (48-60).Direct translocation via pore formation includes instead two different models, the barrel-stave, and the toroidal model, which are proposed as mechanisms used by primary amphipathic peptides.In the barrel stave model, [137] helical CPPs create a barrel with hydrophilic residues forming the core pore and hydrophobic residues adjacent to the lipid chains.In the toroidal model, lipids bend so that the CPP is always near the headgroup, and a pore is formed by the CPP and the lipids. [138]When the peptide concentration exceeds a specific threshold, pores start to form in both processes.
The "carpet" model and the "membrane-thinning" effect have been proposed to describe the direct penetration of cationic peptides.The "carpet" model suggests that positively charged segments of the peptide lie parallel to the membrane surface binding the acidic phospholipid headgroups. [139]The peptides then self-associate in a "carpet-like" manner, and the hydrophobic sites embed in the lipid region of the membrane, while the hydrophilic parts orient towards the hydrophilic region, causing structural reorganization and internalization of the CPP.An alternative to the "carpet" model is the "membrane-thinning" effect, which results from the interaction of negatively charged lipids in the outer leaflet of the membrane with the cationic groups of the CPP. [140]This causes a lateral rearrangement of the negative charges and a thinning of the membrane, allowing for intercalation of the CPP within the membrane.In this mechanistic picture, a pivotal role is assumed for arginine guanidine groups, which can form bidentate hydrogen bonds and electrostatic interactions with sulphate, phosphate, and carboxylate moieties, all of which can be found on cell surface components.It is thought that the formation of these hydrophobic counterion complexes promotes the accumulation of CPPs on the cell surface and leads to their internalization.Upon membrane translocation, it is assumed that the peptide backbone interacts with the lipid core through hydrophobic interactions, as described for octa-arginine (R8) and the HIV TAT peptide. [141]Studies performed on the internalization of dodeca-arginine (R12) in HeLa cells suggest instead a different uptake behavior, specifically the formation of "particlelike" structures during the interaction and uptake of polyarginine. [142]It is suggested that both membrane components and R12 are involved in the formation of these "particles (1-3 μm in size).Overall, the internalization mechanism of arginine-rich peptides is still not fully understood, and recent studies suggest a combination of both direct translocation and endocytosis.

Endocytosis as a pathway for CPP uptake
Endocytosis is the active process by which macromolecules are transported into cells through vesicles or vacuoles pinched-off of the plasma membrane.The process involves two distinct steps: endocytic uptake followed by endosomal escape.Endocytosis is divided into two categories: phagocytosis, which involves the uptake of large particles and is restricted to specialized cells, and pinocytosis, which involves the uptake of  [130] copyright 2010, CC-BY 3.0 (https://creativecommons.org/licenses/by/3.0/).fluids and solutes and occurs in all cells.At least four different mechanisms have been described for pinocytosis: macropinocytosis, clathrin-mediated endocytosis (CME), caveolae-mediated endocytosis (CvME), and clathrin-and caveolae-independent endocytosis, all depending on distinct components and mechanisms. [18]It is now generally recognized that CPPs, when conjugated to cargo and at low concentration, are taken up by cells in an energy-dependent manner through endocytosis.The active transport of CPPs through endocytosis was suggested in 2003, [143] after Richard et al. pointed to the possible errors in the results describing direct translocation due to the experimental methods used.Since then, most studies describing the active transport of CPPs have suggested macropinocytosis as the main entry path for CPPs into cells. [18]Macropinocytosis is a receptorindependent form of endocytosis characterized by the inward folding of the plasma membrane's outer surface, embedding the molecular cargo.Eventually, membrane protrusions collapse onto and fuse with the plasma membrane to generate large endocytic vesicles called macropinosomes.Macropinocytosis was initially thought to be a non-regulated process, but it is now recognized that this uptake process is a highly organized one and consists of a series of signaling events that involve the remodeling of the cytoskeleton.Accordingly, most of the macropinocytosis regulators belong to the group of kinases, such as Src, PI3K, and GTPases, such as Rho family, Ras family, Rab proteins, which trigger the actin-driven formation of membrane protrusions.It has been described as the pathway used to deliver arginine-rich CPPs such as octa-arginine and TAT peptides into cells. [134]Receptor-mediated endocytosis relies instead on clathrin and caveolin proteins, which drive the process of membrane invagination and assist the aid in the formation of vesicles when extracellular molecules are bound to membrane receptors.Clathrin-mediated endocytosis has been described as one mechanism.CPPs can use to traverse cell membranes.It has been reported that the TAT peptide, oligoarginines, and anionic CPPs can each utilize this pathway.Although these receptors are not essential for TAT penetration, it has been hypothesized that the interaction between TAT and heparan sulfates plays a crucial role in the internalization process. [144]Nevertheless, it has been shown that there is a different absorption pathway for TAT in the presence of conjugated cargo, therefore this only applies to unconjugated TAT.Negatively charged CPPs were also proposed as oligonucleotide carriers as an intriguing alternative to the typical cationic CPPs. [145]CPPs are not generally anionic, but they do endow a nucleic acid in cell culture conditions with a negative charge.Scavenger receptors SCARA3 and SCARA5 were reported to facilitate the uptake of these anionic particles. [146]The reported CPP examples with a net neutral or negative charge are very limited, and they show cell penetration at relatively higher concentrations than CPPs with a net positive charge.As such, the mechanisms driving their ability to cross membranes are still poorly understood.Sap(E) peptide penetration was not linked to its charged nature but, rather, with a high proline content (approx.50 %).Sap(E) is coherently predicted to assume a polyproline II (PPII) type helix conformation, [147] segregating negatively charged residues from hydrophobic ones on differ-ent helix faces.The hydrophobic faces of a PPII helix can further interact with the hydrophobic faces of other helices to form more aggregated structures.As alternative mechanism, caveolae-mediated endocytosis has also been proposed in the uptake of proline-rich peptides. [146]These represent another family of cell-penetrating vehicles which have been shown to be particularly effective in driving cellular uptake while having no cytotoxicity effects.

Release from endosomes
For an effective intracellular delivery, it is essential that CPPcargo complexes can escape from endocytic organelles and reach the cytosolic space.This process seems to be a limiting step in the endocytic uptake of CPPs and, ultimately, the release from endosomes determines the efficiency with which a cargo reaches the cytosol.Frequently, endosomal release of CPPs occurs with poor efficiencies, posing significant challenges for cargo molecules that require a large number of copies to elicit a biological effect.Several mechanisms for endosomal escape of CPPs have been proposed.It is assumed that the most relevant are based on the ability of CPPs to induce pore formation and membrane leakage/disruption, easing the release of CPPs and associated cargo.According to this model, TAT has been shown to induce leakage of endosomes after interacting with negatively charged phospholipids in the endosomal membrane. [148]An alternative mechanism for escape proposed for oligo-arginines is the formation of ionic pairs between CPPs and negatively charged membrane lipids, which would then partition across the endosomal membrane. [149]Overall, endosomal escape of CPPs remains far from being fully characterized, and advancements in this field would drive the development of increasingly efficient delivery systems.Some of the emerging strategies for endosomal escape are reviewed in detail elsewhere.

Main biophysical methods to study CPPs internalization
To study CPPs from structural and functional points of view, several biophysical methods are employed.The main ones are Fluorescence (confocal microscopy, cytofluorimetry), Mass spectrometry, Circular dichroism, Nuclear Magnetic Resonance (NMR) and electron microscopy (Table 6).Often, more than one method is needed to have an almost complete framework of the events happening upon CPP interactions with cells.

Fluorescence
Fluorescent labelling of peptides is one of the mostly used methods to visualize and to monitor CPPs cellular uptake.The most widely used investigative techniques based on peptides fluorescence are represented by cytofluorimetry, fluorescence microscopy, Confocal Laser Scanning Microscopy (CLSM) and Fluorescence Correlation Spectroscopy (FCS) being cytofluorimetry and CLSM the predominant techniques reported in the literature.CLSM is a microscopy imaging technique that provides high-resolution, three-dimensional visualization of cells while cytofluorimetry, also known as flow cytometry, is a powerful analytical technique that allows for the measurement of the presence or absence of specific molecules within the cell.In all these techniques cells are incubated with labelled CPP with suitable fluorophores to allow its visualization into the cell.Often, these techniques are used in combination as they give different information regarding the CPP internalization followed pathways and peptides' fates and even because data obtained from different techniques may be not always comparable. [30,150,151]The use of fluorescent labelled peptides is one of the most accessible, but some drawbacks may arise.Firstly, the modification of a peptide with a fluorescent label may lead to an alteration of the overall hydrophobicity, hydrophilicity, and charge of the derivate thus impacting on its uptake and cytosolic distribution and hence leading to ambiguous results. [152,153]Secondly, following the action of proteases present outside and inside the cells, the moiety of the peptide linked to a fluorescent tag (usually at the N-or Ctermini), may be cut away from full-peptide giving rise to peptide fragments partially labelled and partially not, thus leading to misinterpretation of the results especially regarding peptide localization. [153]Thirdly, conditions should be adjusted and optimized based on the peptide sequence and avoiding as most as possible cell fixation in order to eliminate artefacts such as in fluorescence and confocal laser scanning microscopy.Various applications of CLSM and cytofluorimetry have been described in literature, in this section some representative examples are reported.An interesting comparative and quantitative analysis between mass spectrometry, that will be treated specifically further in this paper, fluorescence spectroscopy and cytofluorimetry has been proposed to study the internalization pathways followed by Penetratin, nonarginine and TAT in different conditions of temperature and concentration. [154]ytofluorimetry was also successfully used to compare the efficiency of a new CPP deriving from the human protein dNP2 with respect to TAT. [155] Confocal laser scanning microscopy is a consolidated powerful technique allowing the imaging of CPPs uptake and their subcellular localization also in a 3D imaging system.An interesting application of CLSM is represented by a cell penetrating peptide-based assay in which a flow chamber containing cultured cells was installed on the stage of a confocal microscope to allow for real-time observations. [156]In this way the cell penetration could be observed in a kinetic fashion conversely to many cell-penetration observations carried out as endpoint assays.Another application of CLSM is the use of CPP as probes to assay cell viability.In the study of Xia and coll.they report the synthesis of a spirolactam CPP to act as a reversible fluorescent probe sensitive to pH of cell environment. [157]The same peptide was recently employed by the same group as a ratiometric nanobiosensor for the simultaneous detection and imaging of lysosomes and extracellular pH. [158]Fluorescence Correlation spectroscopy (FCS) is a highly sensitive quantitative method that accurately and directly measures the relative efficiencies of various potentially cell-permeable molecules as they move into the cell, down to the level of individual molecules. [159]In a new experimental approach cytofluorimetry and FCS have been combined to characterize CPP properties allowing an accurate measure of CPP uptake amount. [160]Beside total cell uptake, the reported cytofluorimetry-FCS approach can give crucial information when utilizing CPPs for drug design.It can discriminate CPPs entrapped within endocytic compartments from those localized into the cytosol.Cytofluorimetry -FCS may also distinguish between healthy and dead cells allowing to quantify the amounts of CPPs that do not induce cytotoxicity or additional side effects.Furthermore, this approach can determine the fractions of CPPs that are degraded intracellularly.Fluorescence correlation spectroscopy was used also combined to fluorescence cross-correlation spectroscopy (FCCS) to detect the presence of monomers or aggregates of cationic-lipid PF14 peptide (Stearyl-AGYLLGKLLOOLAAAALOOLLK) and of peptide/ oligonucleotide complexes by labelling the peptides and the delivered siRNA with two different fluorophores in a living cells experimental system. [161,162]A through description of the methodology of FCS applied to CPPs is given by Knox and coll. [162]ble 6.Summary of useful techniques to study CPPs internalization.

Techniques
Result provided Drawbacks Ref.
Fluorescence Peptide uptake and distribution Peptide labeling with fluorescent tag.[146-156]   Mass Spectrometry Peptide internalization and distribution Discriminate between internalization within cells and association to cell surface.
[ 157-161]   Circular Dichroism Peptide secondary structure Distinguish between internalization process and interaction with membrane surface.
Discriminate between internalization within lipid bilayer or surface [170-179]   Electron Microscopy Peptide distribution and membrane modification induced by the peptide.

Mass spectrometry
Mass spectrometry is one of the most reliable and sensitive techniques that allow the study of cell internalization and distribution giving qualitative and quantitative data on CPPs uptake.One of the advantages of Mass Spectrometry is the possibility to study peptides in their native form thus avoiding many of the potential artefacts deriving from peptides' chemical modification.One of the first studies reporting this methodology was from Elmquist and Langel. [31]Matrix Assisted Laser Desorption Ionization-Time of Flight (MALDI-TOF) is probably the most diffuse mass spectrometry technology used to study CPPs cell internalization.MALDI-TOF was also employed to give quantitative information about peptides uptake.Generally, due to the peculiar preparation of the sample (cocrystallization with the matrix leading to deposit heterogeneity) and due to the signal intensity depending on peptides structural features, MALDI-TOF can hardly be used as a quantitative technique, so in their study, Burlina and coll.developed a method to rapidly quantify cell internalization by combining a capture step of peptides on biotin functionalized beads enabling concentration of the internalized CPP and employing the same peptide labelled with a stable isotope as an internal standard. [163]Similarly, MALDI-TOF has been employed to verify cargo internalization, [164,165] indeed, one of the problems with the use of mass spectrometry is to distinguish between peptides really internalized within cells and those associated to cell surface.To face this issue, a simple experimental approach is represented by that reported by Henriques and coll. in detecting the amount of the Cyclotide Kalata B1 within HeLa cells. [76]After peptide treatment, cells were collected and worked differentially to obtain a total soluble cell fraction and a membrane associated fraction, and the resulting samples analyzed via a LC mass spectrometry quantitative methodology using multiple reaction monitoring (MRM).In a recent approach Makarov and coll., applied a cell based high-throughput label-free methodology called Cellbased Approach Membrane Permeability Assay (CAMPA) based on a MALDI-hydrogen-deuterium exchange mass spectrometry (MALDI-HDX-MS) approach, to classify peptide cell membrane permeability using live THP-1 and AsPc-1 cells.In order to prevent unwanted HDX back-exchange, an aprotic matrix solution (DHAP in acetonitrile with NBA) was utilized.A differential hydrogen-deuterium exchange approach was used to distinguish the peptides outside of the cells from internalized ones.The peptides on the outside of the cells were labelled using sufficiently short exposure to deuterium oxide, while the peptides inside of the cells were protected from labelling because of permeation into the cells. [166]MALDI-TOF was recently used to monitor the uptake of different CPPs linked to phosphorodiamidate morpholino oligomer (PMO) a therapeutic macromolecule employed as an antisense "exon skipping" therapy for Duchenne muscular dystrophy (DMD).In this study, the uptake of biotinylated CPPs and PMO-CPPs was calculated to determine their relative concentrations in the whole cell and cytosol via MALDI-TOF mass spectrometry analysis. [167]

Circular dichroism
Circular dichroism (CD) has become increasingly recognized as a valuable structural technique to characterize the secondary structure of CPPs in solution and perform structural studies in relation to membrane interactions.CD techniques are based on absorption differences of the circularly polarized light by chiral molecules.Light absorption is mainly due to the π-electrons involved in peptide bonds and usually falls in the interval between 190 and 260 nm.Typically, peptides are organized in α-helix, β-structure, and random coil secondary structure, characterized by well-defined CD signatures.Changes in the environment can be reflected in the secondary structure and can be monitored by CD analysis.Moreover, specific interactions with different guests (membranes, receptors, lipids, and CPP cargoes) affect the CD spectrum, detecting an effective recognition process.To explain the mechanisms that drive CPP to cross the lipid bilayer and monitor structural differences, CD analysis are usually performed using phospholipid liposome model.From the reported literature, there is not a specific secondary structure attributable to an efficient passage through the membrane, but the uptake process was obtained with peptides characterized by an α-helical, β-sheet, or a random coil structure.Despite this, it is sometimes suggested that peptides with α-helical structure, have some advantages in passing the lipid bilayer.This arises from the structural determination of the secondary structure of the first known CPPs, Tat (38-60). [168]Actually, it becomes clear that the ability to penetrate the cellular membranes is driven by a combination of different factors, including hydrophobicity, charge, flexibility and ordered structure of the peptide. [169]Many recent studies on CPPs secondary structures have focused on arginine rich CPPs, owing to their ability to deliver various cargos into cells across the hydrophobic barrier both in vitro and in vivo. [6,170]ne such study, that compared the membrane interaction with hydrophobic properties and amphipathicity of the peptides, showed that cell penetration efficiency tends to increase with their α-helical and amphipathicity content, but, with the highest hydrophobic moment, CPPs predominantly remain on plasma cell membranes. [171]Another group has explored the neuroprotective effects of two arginine rich CPPs, containing TAT fragments, by CD experiment aimed to characterize the interaction of these peptides with an artificial neuronal membrane.Modification in the intensity of the peptide CD signal in combination with the redshift effect was symptomatic of the formation of ordered structures during the interaction with neuronal mimicking liposomes. [172]Generally, peptide-lipid interactions of amphiphilic peptides stabilize their helical structural conformation via hydrophobic forces, [173] inducing an efficient crossing of the membrane.This behavior has been successfully exploited to design a trimeric epsin-peptide able to induce positive curvature and promote loosening of the lipid packing by inserting its hydrophobic moieties into the lipid bilayer.CD studies demonstrated that interaction with lipid membrane stabilizes the helical conformation of the peptide, inducing a more pronounced capacity of melting with the lipid packing. [169]Another strategy to obtain smart CPPs is to synthesize topologically constrained amphipathic peptides, unordered in solution but in α-helix conformation when in contact with the membrane.For example, Maculatin 1.1 (Mac1 GLFGVLAKVAAHVVPAIAEHF-NH 2 ) is a cationic peptide with a well-known antibacterial activity. [174]Mac1 is unstructured in an aqueous solution but, upon interaction with neutral and anionic membranes, adopts an α-helix conformation.Its insertion into lipid membranes induces a modification of the thickness and the order of the bilayer, inducing leakage in both types of membranes.Of note, Mac1.1 showed higher affinity for anionic membranes while greater disruptive efficiency for neutral membranes.In recent work, Sani and co-workers have elucidated the orientation of the peptide within a model lipid bilayer and the process for membrane insertion and disruption.To investigate the peptide topology in different mimics, the Authors performed CD analysis supported by NMR techniques that exploited both DPC isotropic and DMPC anisotropic micelles.The results show that, in the presence of both the micelles, Mac1 shifts the secondary conformation from a random coil to an α-helix.Moreover, the peptide retains a superficial location on the surface of the vesicles but was oriented in a transmembrane fashion within a lipid bilayer, inducing changes in the bilayer structure, pointing toward a crucial role of the membrane curvature in the molecular mechanism of peptide insertion. [175]

Nuclear magnetic resonance
Nuclear magnetic resonance (NMR) spectroscopy is a powerful technique to provide important information at the atomic level on the structure of a biomolecule, such as proteins, peptides, nucleic acids, and lipids.In NMR, a magnetic field and radio frequency pulses are applied to an atomic nucleus, characterizing their resonant frequency according to its chemical or environmental surroundings.Beyond the molecular structure, NMR spectroscopy can determine phase changes, conformational modifications, solubility, and diffusion potential.For NMR spectroscopy, the most common valuable are atoms 1 H, 19 F, and isotopes, such as 13 C, 15 N, 31 P thanks to their best magnetic properties.In the field of CPPs characterization, NMR, coupled with CD experiments, has been successfully exploited to reveal the three-dimensional structures of CPPs and their behavior towards lipids. [176]In this context, most NMR studies on CPPs have focused on the interaction between CPPs and the membrane lipid bilayer, revealing the interaction mechanism and the affinity between them. [177]For example, a model system containing CPP, glycan, and lipid has been monitored by different NMR techniques among which 1 H-NMR, proton-proton correlated spectroscopy ( 1 H À 1 H COSY), total correlation spectroscopy (TOCSY), rotating frame overhauser enhancement spectroscopy (ROESY), and 13 C to determine cell penetration mechanism and heparin recruitment, exploiting DPC vesicles ad model membrane.[180] Many NMR studies have inves-tigated the interaction of Penetratin with negatively charged mimics, revealing that Penetratin strongly interacts with anionic membranes. [38,83,181]In particular, exploiting liposomes containing anionic lipid, detailed molecular insights into the interaction between Penetratin and membranes have been elucidated through NMR experiments (2D 1 H-1 H TOCSY and 2D 1 H-1 H NOESY).The Authors demonstrated that in the presence of multiple negative charges, Penetratin adopts a stable α-helical structure, oriented in a parallel fashion to the lipid bilayer, improving the internalization process. [182]Static solid-state NMR is a technique commonly used with model lipid systems to study perturbations, including alterations in lipid acyl chain order.Since CPP-induced permeabilization of the lipid bilayer affects the lipid acyl chains, this technique can be used to assess changes in lipid orientation resulting from the membrane disruption by CPPs. [183,184]In a recent study, the influence of lipopolysaccharide on the ability of the CPP to cause disordering of the lipid membranes of the E. coli has been studied with deuterium solid-state NMR ( 2 H-NMR) on intact bacteria cells.Experimental observations are consistent with a CPP-induced increase in the angular amplitude of lipid chain motions, diagnostic of enhanced permeabilization of the membrane. [176]Stability monitoring of CPPs can also be investigated using 19 F-modified amino acids, recording 19 F NMR spectra under proton decoupling.This technique allows the monitoring of changes in fluorinated peptides in complex biological mixtures following the chemical shifts of the signal.This method was successfully exploited to monitor the stability and the antimicrobial activity CPP stability in real serum samples. [185]

Electron microscopy
Electron microscopy is a valuable tool for the morphological characterization of biological materials at high resolution.It can provide useful information at the structural level about the interaction and arrangement of CPPs in the membrane cell, the entrapment in cellular organelles and the translocation to the cytoplasm.Two types of electron microscopy setup can be exploited to discern CPPs mechanism: transmission electron microscopy (TEM) and scanning electron microscopy (SEM).In TEM, electrons cross the biological sample, revealing the inner structures.In SEM, atoms in the sample scan the electron beams, producing a surface topography image.Both TEM and SEM techniques are in use for studying CPPs, offering useful information on membrane-peptide interactions and peptide distribution in cells.188] Alternatively, TEM images could also be obtained incorporating CPP in fusion protein-like architectures [189] or using self-assembling CPPs molecules that organize in hierarchical supramolecular species. [190,191]Such an example is the study of the branched peptide dendrimers, G3KL, rich in lysine and leucine, that exerts a strong activity against Gram-negative bacteria including Pseudomonas aeruginosa and Escherichia coli.
TEM images of P. aeruginosa cells, exposed to the branched peptide, show the accumulation of the peptide in the bacterial cytosol causing shape change, membrane damage, vesicle structure formation and peptide aggregation inside the cells (Figure 4).Moreover, while active disrupts bacterial membranes with fast kinetics, the branched CPP has no effect on mammalian cells. [191]ven if TEM images show that the CPP dendrimer induces the permeabilization of bacterial cells through damage at both the outer and inner membrane, internalization studies need to be supported by fluorescence experiments.On the other part, the uptake mechanisms of CPPs conjugated with electrondense vector could be followed by TEM experiments more effectively.Moreover, studies on these hybrid nanovectors show improved cellular entry of peptides and gold nanoparticles (GNPs) make allowing a precise localization by TEM within organelles.For example, gold nanoparticles (GNPs) functionalized with arginine-rich or hydrophobic fragments (CALNNPFVYLI, CALRRRRRRRR) through sulfhydryl PEG, showed increased stability, bioavailability and enhanced cellular uptake in cancer cell lines.TEM data show that uptake of CPP-modified GNP is mainly driven through caveolae-mediated endocytosis and micropinocytosis. [192]Similarly, TEM has been exploited to perform ex-vivo studies on rat glioblastoma cells treated with multifunctional nano-vectors composed of biotinylated CPPs, and biocompatible PEG-GNPs (BI-OT-NFL-PEG-AuNPs), assessing their localization in vacuoles. [193]o study interaction in physiological conditions a variant of TEM, cryo-TEM, can also be used to further support classical dry techniques, allowing to observe CPPs membrane interaction in hydrated conditions. [194,195]Recently SEM analysis was also used for studying CPP mechanisms in combination with other techniques, to assess how CPPs affect the morphology of the treated cells. [196,197]For example, a family of peptides containing N-trifluoro acetyl lysine and N-thioacetyl lysine, are designed to promote cell penetration and antimicrobial activities and they have been tested with Gram-negative and Gram-positive pathogens. [198]The antimicrobial effect on biofilm formation and eradication of the peptide has been monitored with SEM ad TEM showing bacterial membrane disruption.A smart CPPderived-peptide containing dual cancer-targeting functions has been developed and tested on HepG2 cancer cells.The killing properties have been monitored using TEM, fluorescence, and Cryo-SEM.In detail, Cryo-SEM measurements are performed to obtain the morphological changes of HepG2 cells before and after peptide treatment.The cell membrane surface appeared lysed, or structural disruption was clearly visible, indicating severe membrane permeation caused by peptide interactions. [199]

Clinical and diagnostic applications involving CPPs
From their first discovery in late 80 s, [14,200] CPPs have evolved in their applications as potential clinical and diagnostics tools, till reaching clinical trials for different pathologies.Especially in cancer, many examples of CPPs employed as therapeutic tools are present and some of them are involved in pre-clinical and clinical trials. [170,201,202]Despite the large amount of research and pre-and clinical studies carried out till now (Table 7), no CPPs or CPP/cargo formulations have been approved by EMA or FDA and several clinical trials have been unsuccessfully terminated.In fact, many issues may hamper translation of CPPs into clinics, even if many of them may be overcome in different ways. 1) Stability in physiological conditions (in vivo).For many years the use of peptides as drugs has been hampered by the general knowledge that peptides were unstable molecules especially if employed in a natural environment (e. g.: human body), mainly due to spontaneous chemical modifications acid/base hydrolysis, deamidation, oxidation, disulphide exchange and the action of proteases leading to peptide degradation and consequent inactivation. [188]In many studies it was observed that CPPs had a short plasma half-life.To circumvent this critical issue diverse strategies are currently employed such as the conversion of L-amino acids in the corresponding D-forms, the use of delivery nanosystems, [203] polymerization techniques (e. g.: dendrimers peptides), [204] design of cyclic (or bicyclic) structures, [205] chemical modification of peptides [206] 2) Immunogenicity.If on one side a promising strategy based on covalently linking peptide antigens to cell-penetrating peptides may be used to enhance peptide vaccine efficacy, [207] undesirable immunogenic effects may arise from their use in in vivo conditions.The use of CPPs deriving from viruses or bacteria or obtained through a rational design, may lead to potential severe immunogenicity and thus neutralizing the therapeutic effects of their cargoes or of themselves.The immunogenicity of the CPPs can also be due to their different physicochemical properties such as charge, amphipathicity, size, structure, and the type of delivered cargo.To avoid or diminish these issues some parts of a peptide sequence may be changed also introducing non-natural amino acids, to do not represent potential T-or B-cell epitopes or even modifying the type or route of administration and dosage [208] 3) Potential cellular toxicity.As for the point 2) the structural characteristics of CPPs Reprinted with permission from. [191]Copyright 2019 American Chemical Society.may provoke injuries to target and non-target cells.Amphipathicity may represent one of the major critical points in using CPPs, depending on the concentrations of use they may in fact lead to a damage or even a disruption of the cells, as seen for MAP and TP10, causing important cell membrane leakage. [209]n this view it is crucial to develop delivery systems employing CPP or CPP-conjugates at lowest concentrations possible.Some promising examples are reported in literature showing CPPs applied at nanomolar ranges of concentrations.In the study of Oh and coll.linear multimeric forms of LK peptide (LKKLLKLLKKLLKLAG) linked to Enhanced green fluorescence protein (eGFP) used as a model protein were seen to be efficiently internalized within at 100-1000-fold lower concentrations than Tat peptide. [210]Another example from the same research group is represented by a pH-activable cellpenetrating peptide dimer LH2 composed of histidine residues where the dimerization was obtained through disulfide bridges between two LH peptides. [211]This compound conjugated with methotrexate and paclitaxel showed to be effective in decreasing cell viability of MDA-MB-231 breast cancer cells at concentration below micromolar ranges.Remaining in the cancer therapy field a dendrimeric construct based on an amphiphilic penetrating peptide (FKKFFRKLL) and sensible to enzyme-proteolysis, was armed with a camptothecin warhead showing to be highly selective and effective with IC50s in the 31-747 nM range. [204]ther undesired interactions may arise once within cells, with molecular entities structures thus leading to uneven side effects.The modification of peptide's sequences and structures (substitution of key residues, synthesis of dendrimeric or cyclic peptides, employ of nanosystems) may lead to less cytotoxic effects.4) Low specificity.When using CPPs in delivering drugs a crucial point is to address the desired effect to specific cell populations or tissues.Lack of specificity has hampered many clinical or pre-clinical studies that require a high degree of drug delivery specificity to the target site to avoid systemic toxicity. [212]Moreover, the penetration ability of CPPs to different cells is variable, adding a difficulty in selecting the right sequence for a specific cell line. [190]This issue may be avoided by designing fusion peptides composed of a CPP and a celltargeting amino acid sequence, [213] for example by producing a peptide-phage display library towards a specific receptor. [214]In treating cancers these specific sequences are best known as Homing peptides. [8]The use of Activable cell penetrating peptides (ACPPs) is another emerging strategy in which peptides can deliver their cargoes only if submitted to specific stimuli (specific light wavelengths, ultrasounds, electric triggers etc.) [215] 5) Endosomal degradation.Endosomal pathway is one of the ways that lead to an inactivation of the therapeutic effects of peptides in general and of CPPs-cargoed drugs.Endosomes are organelles in which external molecules or cells are destroyed, thus an endosomal escape route should be favored by modifying CPPs in different ways.One is to insert appropriate sequences, [216] with a cationic character [217] or by using fusogenic lipids to improve the endosomal release. [218]ome other issues are more difficult to avoid, due to the lack of a deep knowledge of the mechanisms of penetration, leading to an under-or overestimate CPPs efficiency, lack of information regarding their ability to cross some physiological barriers and even having reliable information about their stability.So, a long road should be paved before manipulating these molecules in the most efficient way for a clinical translation.Beside their potential in aiding therapeutics delivery, CPPs are becoming a promising tool in different bio-imaging applications.In cancer diagnostic for example, CPPs have been successfully employed to deliver contrast agents for different imaging diagnostic applications such as Positron Emission Tomography (PET), [219,220] Magnetic Resonance Imaging (MRI), [221][222][223][224] optical imaging and Single Photon Emission Computed Tomography (SPECT), [225] near-infrared (NIR) fluorescence. [226]

Summary and Outlook
In conclusion, Cell Penetrating Peptides (CPPs) hold great promise as a therapeutic and diagnostic tool due to their ability to cross biological membranes and deliver various types of cargo, including drugs, nucleic acids, and imaging agents, to the target cells and tissues.CPP-based therapeutics have shown promising results in preclinical studies for the treatment of various diseases, including cancer, genetic disorders, and neurodegenerative diseases.In the field of pre-clinical trials is advisable to use more appropriate models such as cellular 3D models to test CPPs in a more reliable way.Moreover, CPPbased diagnostic tools offer a non-invasive and highly sensitive approach for disease detection and monitoring.However, further research is needed to optimize the efficacy, specificity, and safety of CPP-based therapeutics and diagnostics.In particular, more research is needed to shed light on the molecular mechanisms behind CPP-mediated uptake, including the involvement of cell surface receptors, lipid rafts, and endocytic pathways.In addition, endosomal escape is a critical step for the successful delivery of CPP cargoes, to address this issue many strategies have been developed to enhance this process.Furthermore, peptide stability, playing a crucial role in determining the efficacy of CPP-based therapeutics and diagnostics, is another critical point which is considered in numerous studies to be faced applying various modifications to improve the stability and half-life of CPPs.Overall, CPPs represent a valuable and versatile tool that has the potential to revolutionize the field of medicine and patient outcomes.
list some sequences derived from natural proteins or fragments used as CPPs without any modification.Many CPPs also derive Greta Bergamaschi is a Senior Researcher at the National Research Council of Italy at SCITEC-CNR in Milan.She received her PhD from the University of Pavia (2014) working on the design of fluorescent receptors for the recognition of physiologically relevant charged species.She spent research periods as a visiting scientist in Germany (TUM-Munich) and Switzerland (EPFL, UZH).She completed postdoctoral training at the Politecnico di Milano approaching the field of peptide nanoscience.Her research interests are focused on new strategies for peptide structural control, exploration of the role of conformation and dynamics in bioactivity, and development of new peptide-based (bio) materials.Alberto Vitali obtained his degree in Biological Science and since 2001 is a Researcher at the National Research Council of Italy at SCITEC-CNR in Rome's seat.For several years he worked in the field of proteomic and peptidomics characterization of biological fluids spending a period in France (CNRS-Strasbourg) as visiting researcher.Currently his scientific interests focus on bioactive peptides obtained from natural sources and from proteome mining to be applied in diverse therapeutic applications ranging from cancer to antimicrobial resistance.Alessandro Gori is a Senior Researcher at the National Research Council of Italy in Milan (SCITEC-CNR, www.ctbio.eu).He received his PhD from the University of Milan, working on the conformational stabilization of peptides and peptidomimetics.His research interests encompass many aspects of peptide science, including bioactive peptides, peptides as delivery agents, peptide nanomaterials and peptides in microanalytic applications Giulia Lodigiani graduated from the Università Statale di Milano in December 2021 with a degree in Chemical and Pharmaceutical Technologies.She worked as a research fellow under the supervision of Prof. Ermanno Valoti from April to December 2022, focusing her work on pharmaceutical chemistry.She has been a research fellow at SCITEC-CNR in Milan for the National Research Council of Italy since January 2023.She is currently working on developing new peptide-based (bio) materials, studying the impact of conformation and dynamics on bioactivity, and synthesising novel peptide-based (bio) materials.Stella G. Colombarolli is a postdoctoral researcher at National Research Council of Italy -SCITEC, Rome, Italy.PhD and MSc in Cellular and Molecular Biology, Genetics and Bioinformatics.She has expertise in peptide screening using phage display technology, peptide microarrays and bioinformatic tools.Working mainly on the following subjects: use of peptides for new therapeutic and diagnostic methods development; antimicrobial resistance; arboviruses; and extracellular vesicles to medical applications.

Figure 1 .
Figure 1.A possible functional classification of CPPs.A first division can be made based on the origin of the CPPs.Within these classes many different sequences, length and structures can be found.Because of the structural features, other properties, such as target specificity and mechanism of transport, may derive.These last are also dependent on the cell type used to test the CPPs.

Figure 2 .
Figure 2. Graphical sketch of a stapled peptide.In this example a single stapled peptide blocked in two positions, is shown, evidencing the alphahelical conformation.

Figure 4 .
Figure 4. TEM imaging of P. aeruginosa cells exposed to G3KL.The arrows represent different damages to the bacterial cells.Red: aggregation inside the cell.Blue: bacterial shape change.Light blue: visible broken inner membrane.Yellow: vesicle-like structures from the outer membrane.Reprinted with permission from.[191]Copyright 2019 American Chemical Society.

Table 1 .
Some examples of CPPs derived from human proteins.

Table 2 .
Some examples of CPPs derived from viral proteins.

Table 3 .
Some examples of CPPs derived from venom toxins.

Table 4 .
Some examples of CPPs derived from AMPs.

Table 5 .
Some examples of different types of synthetic and designed CPPs.

Table 7 .
Some examples of Clinical trials involving CPPs as drug delivering and bio-imaging tools.