Insights into cell entry and intracellular trafficking of peptide and protein drugs provided by electron microscopy

Advanced Drug Delivery Reviews 65 (2013) 1031–1038

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Insights into cell entry and intracellular trafficking of peptide and protein drugs provided by electron microscopy☆ Helerin Margus, Kärt Padari, Margus Pooga ⁎ Institute of Molecular and Cell Biology, University of Tartu, EE51010 Tartu, Estonia

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Article history: Accepted 9 April 2013 Available online 24 April 2013 Keywords: Peptide and protein therapeutics Electron microscopy Immunolabeling Intracellular trafficking Cell-penetrating peptides

a b s t r a c t For widening the arsenal of protein and peptide therapeutics that act within cells, their cell-entry mechanisms, intracellular trafficking and distribution need to be characterized in detail. Immunofluorescence microscopy has been a prevalent tool for these studies. However, due to the limited resolution, it is often complemented with other methods. This article focuses on the perspectives of electron microscopy in tracking the intracellular delivery and trafficking of proteins, peptides and their carriers. This review introduces the electron microscopy techniques and labeling methods currently used for studying the cellular whereabouts of peptides and proteins with a focus on their intracellular trafficking. Since cell-penetrating peptides have widely been harnessed as carriers for proteins and peptides, and their usage is rapidly expanding, a particular emphasis has been placed on their applications and cell-entry mechanisms. © 2013 Elsevier B.V. All rights reserved.

Contents 1. 2.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Electron microscopy as a tool in the studies of protein and peptide drugs . . . . . . . . . . . . 2.1. Preparation of cells/tissues for transmission electron microscopy . . . . . . . . . . . . 2.1.1. Conventional room temperature methods . . . . . . . . . . . . . . . . . . . 2.1.2. Tokuyasu cryosectioning method . . . . . . . . . . . . . . . . . . . . . . . 2.1.3. Fast freezing and freeze-substitution . . . . . . . . . . . . . . . . . . . . . 2.1.4. Cryo-electron microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.5. Electron tomography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Immunolabeling for transmission electron microscopy . . . . . . . . . . . . . . . . . 2.2.1. Pre-embedding labeling for transmission electron microscopy . . . . . . . . . 2.2.2. On-section labeling for transmission electron microscopy . . . . . . . . . . . . 2.3. Tagging of proteins and peptides for TEM analysis . . . . . . . . . . . . . . . . . . . 2.3.1. Direct labeling of proteins and peptides with colloidal gold . . . . . . . . . . . 2.3.2. Covalent coupling of nanogold tag to proteins and peptides . . . . . . . . . . . 3. Cell-penetrating peptides as carriers for therapeutics . . . . . . . . . . . . . . . . . . . . . 3.1. Analysis of CPP's interaction with membrane and cellular translocation by electron microscopy 3.2. Electron microscopy analysis of delivery of nucleic acids by CPPs . . . . . . . . . . . . 4. Conclusions and perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abbreviations: CPP, cell-penetrating peptide; cryo-EM, cryo-electron microscopy; EM, electron microcopy; ET, electron tomography; HPF, high pressure freezing; immuno-EM, immuno-electron microscopy; IFM, immunofluorescence microscopy; mAb, monoclonal antibody; Tat-SPIONs, superparamagnetic iron oxide nanoparticles modified with Tat peptide; TEM, transmission electron microscopy; TP, transportan. ☆ This review is part of the Advanced Drug Delivery Reviews theme issue on “Delivery of biopharmaceuticals: Advanced analytical and biophysical methods”. ⁎ Corresponding author at: Institute of Molecular and Cell Biology, 23 Riia Street, 51010 Tartu, Estonia. Tel.: +372 7 375 049; fax: +372 7 420 286. E-mail address: [email protected] (M. Pooga). 0169-409X/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.addr.2013.04.013

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1. Introduction Peptides and proteins are increasingly recognized as effective and highly specific drugs in current therapy. In case of many diseases such as enzyme deficiency and protein dysfunction disorders, cancer, and degenerative diseases, a direct introduction of therapeutic proteins or peptides has been highly beneficial. Protein and peptide drugs have several advantages over small-molecule drugs like higher specificity, lower probability of causing adverse side effects, and, as mostly being produced naturally, human or humanized proteins are less likely to elicit immune response. A diverse variety of peptide- and protein-based drugs, such as hormones, enzymes, growth factors, cytokines, vaccines and monoclonal antibodies (mAbs) are either in development or already in clinical use as therapeutic products [1–3]. Most of the protein drugs were initially utilized because of their therapeutic effect without the real understanding of their functioning mechanism. Only in the last decades we have begun to understand more about the action of administered drugs at molecular level. However, for the successful development of peptide drugs and therapeutic proteins, a detailed knowledge about the intra- and intermolecular dynamics of protein and peptide drugs and their carriers is ultimately required. During the last decades, IFM has become a prevalent tool for these studies, mainly due to its simplicity and rapidity. Indeed, IFM is an excellent tool for studying living cells, assessing a particular antigen or antigens at the whole-cell level, and can be successfully used for quantitative studies. However, it has its limitations. First, the resolution of light microscopy is about 200 nm (super-resolution methods ~20 nm) [4], which is far too low to provide a detailed information about the interaction of molecules with the plasma membrane, their exact localization in relation to intracellular compartments, and changes in cell morphology. Hence, separate structures or molecules situated in close proximity and labeled with different fluorescent dyes for distinguishing can easily be considered as co-localized. Moreover, the fluorescence may be quenched in living cells depending on a particular microenvironment that might complicate the interpretation of IFM results. Therefore, to gain more detailed information, especially in ambiguous cases, additional imaging tools should be applied in parallel. Although laborious, electron microcopy (EM) finely complements IFM results allowing the visualization of substructures of intracellular compartments with high resolution in order to study the membrane associations, uptake, intracellular trafficking, and whereabouts of molecules. Importantly, in case of tagged molecules, EM enables the visualization of single molecules within intracellular compartments, and thus, is a highly precise method for studies that require tracking of individual molecules inside cells. In addition to conventional transmission electron microscopy (TEM), more advanced techniques, such as cryo-electron microscopy (cryo-EM) or three dimensional (3D) EM can provide more comprehensive information about trafficking of proteins and reaching their targets within the native cellular environment. The current developments in these techniques also encourage the harnessing of EM in the studies of drug delivery more often. Nevertheless, not many studies have utilized the potential of EM for assessing the cellular distribution of peptide or protein drugs so far. This review aims at summarizing the applications of TEM in the studies of cell entry and intracellular trafficking of peptides and proteins. A particular emphasis is placed on the peptides that are used for transducing proteins and peptides into cells, and the respective mechanisms. 2. Electron microscopy as a tool in the studies of protein and peptide drugs Transmission electron microscopy has been an indispensable tool in delineating of subcellular organization and functioning of cells, and has also markedly contributed to unraveling the mechanisms of proteinbased drugs. Advantageously, TEM enables to combine the information of cellular substructures with sensitive immunocytochemical protein

detection methods. Hence, it is an essential and powerful tool in the studies of protein and peptide drugs to analyze their association with cells, cell-entry mechanisms and intracellular trafficking. These studies require preservation of both optimal ultrastructure and strong immunoreactivity of specimen. However, the attainment of an excellent tissue/ cell preservation without compromising immunoreactivity remains a great challenge even in modern EM. In this paragraph we will review the methods that are commonly used for preparation of cells/tissues for TEM studies. We will also provide an overview of the different approaches to conduct immunolabeling for the detection of proteins in cells and tissues, and describe different strategies available for tagging proteins/peptides with gold labels for their direct tracking. 2.1. Preparation of cells/tissues for transmission electron microscopy The most important prerequisite for the application of TEM in biomedical research is the preservation of the structures of cells or tissues in natural state. Since the preservation of ultrastructure is largely determined by the fixation of the specimen, the choice of appropriate fixation method is of a crucial importance. The methods that are applied for the preparation of TEM specimens can by principle be divided into room temperature and cryo-based techniques. 2.1.1. Conventional room temperature methods In conventional room temperature methods the specimen is fixed with aldehydes, followed by postfixation with osmium tetroxide and dehydration with organic solvents such as ethanol or acetone at room temperature, and embedding into resin. Conventional TEM has been widely used, although, it has several drawbacks. Namely, fixation with aldehydes may induce structural artifacts such as cell blebbing, vesiculation of tubular structures, alteration of shape and size of endosomes and lysosomes, and is therefore suitable only for the routine analysis of cells and tissues whose ultrastructure is known in detail [5]. However, it has been suggested that the majority of artifacts are not induced by the fixation with aldehydes but by the subsequent steps, such as postfixation with osmium tetroxide, dehydration at ambient temperature and washing [6,7]. These treatments might lead to the extraction and distortion of proteins, sugars, lipids and other molecules, which might severely interfere with their immunocytochemical detection. Despite the drawbacks, conventional room temperature methods are still broadly employed because more advanced cryo-based techniques such as high pressure freezing (HPF) followed by freeze substitution are routinely not available for many laboratories. Furthermore, as mentioned above, in case of some routine analyses, it is not necessary to employ more laborious cryofixation. Ultrastructural analysis by using conventional methods has, for example, been applied for following the progress of Gaucher disease treatment, which since 1990s can be cured with enzyme replacement therapy [8,9]. Comparison of the electron micrographs taken before and after the enzyme replacement therapy provides essential information about the treatment efficacy. Moreover, the analysis of epidermal ultrastructure provides an early and specific diagnostic tool helping to distinguish between the type 2, a rare and progressive subtype that is characterized by rapid, early-onset neurodegeneration, from other types of Gaucher disease [8]. The ultrastructural abnormalities detected by EM serve also as a basis in diagnosis of muscular dystrophies [10]. In the field of protein trafficking the conventional TEM analysis has been applied for example for assessing the effect of anti-HER2 (Human Epidermal Growth Factor Receptor 2) antibody fused to a cellpenetrating peptide (CPP) [11]. A CPP of nine arginine residues (Arg9) linked to the C-terminus of anti-HER2 single chain antibody enhanced the cell penetration and bioavailability of the antibody. Moreover, the fusion protein efficiently downregulated the tyrosine kinase activity and thereby reduced the proliferation of HER2-overexpressing cancer cells. The ultrastructural analysis revealed that the antibody construct

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was able to translocate into cells within first hour. Surprisingly, it was also routed to mitochondrial membrane where it localized specifically in the mitochondrial matrix after five hours of incubation [11]. Considering the role of mitochondria in the energy production and programmed cell death, it can be the prime target in treatment of several diseases, and Arg9 could be a useful carrier peptide for delivery of specific antibody or other macromolecules to mitochondria in anti-cancer therapy. 2.1.2. Tokuyasu cryosectioning method An alternative technique for the preparation of specimens for TEM is the Tokuyasu method that was developed for improving the immunolabeling of sections of biological samples [12]. According to his protocol, the biological sample is first chemically fixed using low concentration of aldehydes to preserve better antigenicity. Thereafter, the specimen is cryoprotected, and partly dehydrated using a solution with high concentration of sucrose, frozen in liquid nitrogen, and sectioned at below −100 °C. The immunolabeling is carried out on thawed sections which are then embedded in methyl cellulose. The Tokuyasu method is highly appreciated due to high immunoreactivity and excellent preservation of the ultrastructure of specimens. The main advantages of this method are that (1) antigens remain in a hydrated environment prior to immunolabeling, and (2) because of the open surface structure, more antigens are accessible as compared with resin section where biological structures are cross-linked in resin matrix. However, the quality of structures and antigenicity depend on the limitations of the initial chemical fixation step with aldehydes that diffuse slowly and may not properly immobilize all molecules in cells. In the field of protein therapeutics, the Tokuyasu method has been harnessed to examine the cell-entry and intracellular trafficking of mAbs. For example, Perera et al. have described the internalization and intracellular trafficking pathway of epidermal growth factor receptor-specific mAb 806 in live cells and analyzed its biodistribution also in a tumor xenografted nude mouse model [13]. Immunolabeling of mAb 806 performed on Tokuyasu cryo-sections allowed to visualize the internalized antibodies in structures morphologically resembling clathrin-coated pits and vesicles [13]. In addition, mAb 806 was detected in tubular vesicular structures resembling early endocytic compartments, and at later time points, in multivesicular endosomes. Although proteins directed to lysosomal pathway could be degraded before exerting their therapeutic effect, the accumulation and retention of mAb 806 within lysosomes could ensure a long-term effect on tumor activity when conjugated to radioisotope or toxins resistant to lysosomal hydrolases and makes it a promising tool in cancer therapy. As another example, Austin et al. thoroughly assessed the endocytic trafficking of a transmembrane receptor tyrosine kinase ErbB2 in response to treatment with a humanized mAb trastuzumab and antibiotic geldanamycin [14]. The immunogold labeling of trastuzumab, geldanamycin, or ErbB2 enabled unraveling the trafficking of ErbB2 in breast carcinoma cells upon treatment with these drugs. In contrary to the earlier observations, trastuzumab was found not to downregulate surface ErbB2 but instead to recycle passively with the internalized receptor. The geldanamycin, in contrast, downregulated surface ErbB2 by enhancing its degradative sorting in endosomes rather than increasing endocytosis [14]. The described molecular details of endosomal sorting in the maintenance of surface distribution of ErbB2 have been useful for designing the cancer therapies directed to Erb2 and other cancer biomarkers. Whereas trastuzumab (Herceptin®) was approved already in 1998 and has found wide application in clinics, geldanamycin, in contrary, displayed high hepatotoxicity and currently its analogs are under development as potential anti-cancer drugs. 2.1.3. Fast freezing and freeze-substitution The living state of a cell or tissue is the most closely preserved upon fast freezing of the biological sample [15]. Its principle is to very rapidly remove thermal energy from specimen in order to lock proteins and other structures in native state [15–18]. The freezing of

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specimen needs to be rapid in order to form amorphous ice (vitrification) and to prevent the formation of ice crystals that can distort cellular structure and integrity, make holes in the membranes, and destroy organelles [19]. The advantage of fast freezing compared to chemical fixation is the better preservation of the ultrastructure and immunoreactivity. The simplest way to vitrify the specimen is to plunge-freeze it, e.g. in liquid ethane. However, this approach is only suitable for thin samples that are up to a few micrometers thick [20,21]. Another sophisticated cryofixation method is high pressure freezing (HPF) followed by freeze-substitution that has become a highly preferred tool for cell biologists [22]. The main advantage of HPF over other cryofixation methods is the possibility to fix specimens up to 200 μm in thickness. After HPF the specimen is usually freeze-substituted to chemically fix and dehydrate the specimen by exchange of ice with an organic solvent that usually contains aldehydes and osmium tetroxide, at temperatures −78 to −80 °C. After freezesubstitution the specimen is usually embedded into resin, and hydrophilic resins are preferred since they yield better immunolabeling [23]. 2.1.4. Cryo-electron microscopy The most explicit information about the structure of biological specimen is obtained by cryo-EM analysis of vitrified sections (CEMOVIS), a method where the frozen sections are examined in TEM [24–26]. Cryo-EM is successfully used in several laboratories to study the cellular ultrastructure and their molecular components in a native hydrated state [15,27,28]. Since the specimen is kept at very low temperature during sectioning and examination, the method does not allow immunolabeling. One possibility to label structures in frozen material is to incorporate markers into living cells. Unfortunately, there are only a few such biomarkers available and their application is not widespread so far [29–31]. 2.1.5. Electron tomography Even though the conventional TEM can provide a wealth of detailed information at a nanometer resolution in two-dimensional (2-D) view, it is not sufficient to reconstruct highly complex cellular architecture or dynamic cellular processes, which are regulated in three dimensions (3-D). The evolvement of electron tomography (ET) has provided new possibilities to explore organelles, subcellular assemblies, or macromolecular complexes in their cellular context in 3-D. The principle of ET is relatively simple — the object that is rotated around an axis under different angles perpendicular to electron beam generates a large set of tilt images of the same specimen area. Thereafter, high resolution 3-D images are constructed from these 2-D projections by using computational methods. ET is especially informative at assessing the interaction of single molecules or nanoparticles with highly complex and diversely organized plasma membrane, and their cellular uptake. The structural rearrangements of complex membrane systems and exact localization of molecules or particles can only be unraveled by analysis of reconstructed cellular 3-D images that extend several micrometers into cell. Thin sections used in routine TEM, in contrary, cannot distinguish whether the membrane-surrounded structure is within cytosol or a tubular invagination of the plasma membrane reaching deeply into cell. The high potential of ET approach for drug delivery was recently demonstrated by Nair et al. to analyze the internalization and trafficking of large superparamagnetic iron oxide nanoparticles (SPIONs) modified with Tat peptide [32]. SPIONs were used as model particles which surface was modified with cell penetrating Tat peptide, an effective intracellular delivery vector for a wide range of nanoparticles and pharmaceutical agents. Although iron nanoparticles are not as electron dense and regular as gold particles, they are easily recognizable in TEM images and tomograms. Vertical and horizontal cross-sections of a tomogram allowed to clearly visualize endocytic vesicles with membranes destabilized by passages or pores, through which the Tatnanoparticles had escaped into the cytosol of glioma cells. Hence, ET could serve as a powerful tool for assessing the interaction, uptake and trafficking of various macromolecules and drugs in cells.

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ET is not only applicable for investigating the internalization and trafficking of therapeutic particles in cellular context but it could also be useful for gaining structural information for rational drug and vaccine design, or developing efficient drug delivery systems. For example, cryo-ET has been efficiently used for (1) characterizing specific structural determinants of HIV-neutralizing protein that acts by blocking the binding of viral envelope glycoprotein gp120 to the cell surface receptor CD4 [33], or for (2) assessing the encapsulation and transmigration mechanisms of therapeutic compounds into different nanostructures (e.g. liposome-based structures, vault nanocapsules) [34,35]. 2.2. Immunolabeling for transmission electron microscopy The majority of protein-sorting events occur in small, from some to tens of nanometer-sized membrane subdomains, and these can only be reliably distinguished at EM level. The most widely used approach for analyzing the protein distribution in cells at ultrastructural level is immuno-electron microscopy (immuno-EM) since it enables to easily map the protein localization in relation to cellular organelles. Immuno-EM uses electron dense markers for visualizing the antigen of interest in specimen. Gold markers/atoms are the most popular for TEM localization studies because of very high electron absorbing efficiency, making them clearly distinguishable from the cellular environment. In principle, immunostaining can be conducted either by a direct labeling technique using a primary antibody that is coupled to colloidal gold particle, or by an indirect method where the primary antibody is not labeled and the secondary antibody or protein A/G is tagged with gold particle. Alternatively, antibodies or Fab fragments can be conjugated directly to a small nanogold cluster, which size can be increased later in specimen for the better visualization by silver enhancement (see Section 2.3.2) [36,37]. The labeling methods for immuno-EM can be divided into preembedding and on-section labeling techniques depending on whether the labeling of antigen takes place in intact cells or on thin sections of cells. On-section immuno-labeling in turn is subdivided to postembedding labeling and the Tokuyasu method. In post-embedding labeling antibodies are applied onto plastic embedded and sectioned specimen, whereas in case of the Tokuyasu method antibodies are added onto thawed cryosections and the specimen is embedded in methyl cellulose later as described in the previous section. 2.2.1. Pre-embedding labeling for transmission electron microscopy In pre-embedding techniques, the primary antibody is introduced into fixed cells before embedding and sectioning. In order to facilitate the access of the antibody molecules and the gold label, the plasma membrane of cells has to be permeabilized. This, in turn, perforates at least partly cellular membranes, and hence, destroys some fine structure. Moreover, permeabilization can cause the redistribution and/or loss of soluble proteins [38], consequently leading to an incorrect labeling pattern or to the failure to detect some proteins of interest. Still, preembedding immuno-EM has been widely used for protein localization analysis, and if precautions are used to prevent harsh permeabilization, the artifacts can be avoided. For example, using Fab fragments instead of full-size antibodies, and a nanogold cluster as a label instead of much larger colloidal gold, only a slight permeabilization of cells is needed, which minimizes the extent of membrane disturbance (Fig. 1). However, pre-embedding immuno-EM is appropriate mostly only in the studies with cultured cells since tissue samples require very harsh permeabilization to ensure the diffusion of antibodies or Fab fragments deeper in the specimen, and this, in turn, might harm the quality of the studied structures. 2.2.2. On-section labeling for transmission electron microscopy On section labeling is conducted on ultrathin sections of specimen and in general two different approaches are applied: the Tokuyasu method and post-embedding immuno-EM. In the Tokuyasu method, the

Fig. 1. Comparison of colloidal gold and nanogold immunoprobe size. Schematic representation of IgG adsorbed to 10 nm colloidal gold particle (on left), and IgG (in center) or Fab' antibody fragment (on right) conjugated to 1.4 nm nanogold cluster.

immunolabeling is conducted on thawed cryosections that are then embedded in methyl cellulose (see Section 2.1). In post-embedding techniques the labeling is conducted on sections of specimen that can be prepared using different strategies: by chemical fixation and embedding into hydrophobic (e.g. epoxy), or hydrophilic resin (e.g. LR White, Lowicryls), or by fast freezing fixation followed by the freezesubstitution and embedding into hydrophilic resin. The use of thawed cryosections ensures the higher labeling density because more antigens are presented to antibodies while in post-embedding labeling the detection efficiency of antigen might be strongly reduced even in the case of hydrophilic resins. On-section labeling is carried through directly on specimen slices, eliminating the need to permeabilize it, thus granting this approach several advantages. In this manner the on-section immuno-EM can be performed equally well both on cells and tissues, whereas preembedding is mostly applicable only for cells.

2.3. Tagging of proteins and peptides for TEM analysis 2.3.1. Direct labeling of proteins and peptides with colloidal gold The most widely used marker for detection by TEM is colloidal gold that has been used for tagging of proteins already from the beginning of 1970 [39]. Gold is an ideal label for EM due to its high atomic number and efficient absorption of electrons. Colloidal gold particles are prepared by reducing a solution of chloroauric acid or its salts to neutral gold atoms, and depending on the concentration of the reducing agent and used solution conditions the size of obtained gold particles can vary in range 3–30 nm. The colloidal gold particles have high affinity for proteins and bind molecules via non-covalent interactions, and one gold particle typically associates several medium-sized protein molecules [40]. Although colloidal gold can be efficiently used for labeling of various macromolecules, especially secondary antibodies and Protein A/G, and has become a golden standard in immuno-EM, it has its drawbacks. First, since the association of the molecules to colloidal gold is non-covalent, the proteins may dissociate from the gold particles, especially upon longer storage. Free protein in turn might compete with the labeled one, thereby decreasing the specific signal, for example in immunological detection. Secondly, the probes may aggregate or lose their activity due to the tight binding to the negatively charged surface of colloidal gold particles which is more common for small molecules. Such probes may non-specifically bind to other intracellular components, causing false positive signal or background labeling. Thirdly, colloidal gold can only be applied in case of the proteins and peptides that do associate stably with and do not destabilize or aggregate the colloidal gold particles. The latter tendency is especially common for proteins with high isoelectric point, like avidin and others. The strategy to directly label the active protein of interest with colloidal gold particle for the studies of protein function could hardly be practical. Association with colloidal particle might substantially interfere with the activity of protein and recognition by other proteins.

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2.3.2. Covalent coupling of nanogold tag to proteins and peptides Nanogold tag is a small (1.4 nm in diameter) cluster of a defined number of gold atoms that is stabilized by aryl phosphine ligands. In contrast to colloidal gold, it can be covalently linked to a molecule of interest by using a chemically active group on the surface of the particle that results in homogenous well-defined labeled compounds where one gold tag marks a single labeled molecule [36,37]. In principle several nanogold tags could be coupled to a macromolecule but this approach is seldom used. The gold-molecule conjugates can be either covalently or non-covalently attached to other molecules, such as proteins (including antibodies), peptides, carbohydrates, and oligonucleotides, including molecules that do not adsorb to colloidal gold [36]. The advantages of using a smaller tag are the better penetration into tissues, greater sensitivity, and higher density of labeling [37,41]. The nanogold tag can even be directly coupled to a peptide (or protein) of interest without significant interference with its activity [42]. On the other hand, since 1.4 nm gold particles absorb too few electrons to be reliably detected in cellular environment with the magnification range routinely used for imaging cellular structures, the size of the absorbing particles needs to be increased. This adds extra steps to the specimen preparation procedure: silver is deposited onto gold enlarging the 1,4 nm gold particles to approximately 10 nm in diameter depending on the enhancement time and temperature [43]. In this process gold particles act as catalysts in the presence of silver ions and reducing agent, and reduce silver ions into metallic silver. The silver deposition has to be stabilized by toning with gold chloride in order to postfix and contrast the specimen [37]. As an example, the colloidal gold-labeled neutravidin complexes with biotinylated CPPs and nanogold-labeled peptides in the plasma membrane and within endocytic vesicles of cultured cells are presented in Fig. 2. 3. Cell-penetrating peptides as carriers for therapeutics The number of peptide, protein, or nucleic acid drugs that have reached clinical testing or application has remained moderate, partly due to their poor penetration across the plasma membrane. Different delivery systems like cationic liposomes, nanoparticles or multifunctional nanodevices have been implemented to facilitate the translocation of macromolecular drugs into the cell cytoplasm and remarkable progress has been achieved in last decades [44,45]. Among these, the CPPs have quickly gained popularity as efficient tool for intracellular delivery of macromolecular cargos like proteins, peptides, nucleic acids, antibodies and nanoparticles. CPP is a common name for a diverse class of short, mostly cationic or/and amphipathic peptides with the ability to translocate into cells [46]. When fused to a therapeutic peptide, protein or any other macromolecule, CPP drastically increases their cellular uptake, and thus, substantially augments their therapeutic potential [47–52]. Remarkably, CPPs have in vivo efficiently delivered the coupled cargo molecules to a wide variety of cells and tissues in fully functional state in preclinical studies [53,54]. Still, CPPs have not realized their full potential neither in laboratory nor in clinical settings largely due to the insufficient knowledge of their translocation mechanisms. Since the CPPs are not cell-type specific per se, many groups are screening for novel, or developing existing peptides to supplement these carriers with specific tissue-targeting ability [55–58]. The internalization mechanism and fate of CPPs and their cargos in cells have mainly been studied by using fluorescence microscopy and activity-based methods. The studies in living cells, tissues, and whole animals by noninvasive light and fluorescence microscopy have significantly expanded our understanding about the distribution and dynamics of CPPs and their cargoes. Still, the crucial questions about the interaction of peptides and cargo molecules with cell surface molecules, translocation into cells and further trafficking cannot be answered using only the light microscopy. Thus, in recent years a more detailed information about association of CPPs with cells and internalization mechanism has been acquired by TEM, which allows direct visualization of single molecules and their complexes within cells at

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high resolution [59,42]. In this section we provide some examples of CPP mechanisms and applications assessed by EM. 3.1. Analysis of CPP's interaction with membrane and cellular translocation by electron microscopy Studies of the cell penetration mechanisms of peptides have led to a consensus that CPPs use various routes for cell-entry. The prevailing mechanism depends on the properties of CPP, nature of cargo, used concentration, and targeted cell or tissue type [60,61], and both the uptake via different types of endocytosis [62–66] as well as energyindependent processes can be active [67,68]. Positive by charge, CPPs first associate with the negatively charged extracellular matrix, in particular, glycosaminoglycans [63,69–71] which might act as receptors. Upon binding to the negatively charged proteoglycans [42,72] CPPs assemble into nanoparticles that are efficiently taken up by cells. Some CPPs (e.g. SAP, Pep-1) can also self-assemble in aqueous solutions [73–75] or form nanoparticles upon complex formation with cargo molecules like CADY and MPG [74,76]. Nanoparticle formation by CPPs and/or CPP-cargo complexes in solution have been mostly assessed by dynamic light scattering which not necessarily recapitulates the real situation of interaction of CPPs with the plasma membrane, or facing the cellular milieu. Coupling of a nanogold tag covalently to a CPP allows the visualization of every peptide molecule and detailed characterization of their interaction with the plasma membrane by TEM. Using this approach we demonstrated that S413-PV peptide forms nanoparticles on cell surface, but not in solution, and the efficient translocation into cells is dependent on size and shape of formed particle [42]. Moreover, our further EM studies revealed that in analogy with S413-PV, other CPPs (Pen, Tat) form nanoparticles on the cell surface [77]. Assembly into nanoparticles for cell entry is not characteristic to only CPPs but also for the complexes of cationic CPPs with negatively charged nucleic acids (see Section 3.2) [78–80]. It can be speculated that the formation of nanoparticles is a general feature of CPPs, necessary for efficient cellular delivery of macromolecules. However, TEM studies have also helped to unravel the subsequent events in the cellular translocation process of CPPs after their initial association with cell surface. The accumulation of CPPs or CPP-cargo complexes on the plasma membrane interferes with the ordered packing of the membrane, because the distinct easily recognized lipid bilayer of the plasma membrane loses its regular organization at interaction sites and becomes undetectable in TEM (Fig. 2B) [42,78]. Ultrastructural analysis by EM in conjunction with biophysical studies indicated that S413-PV peptide can also induce transition of lipids from a lamellar to non-lamellar phase by probably recruiting certain lipids and separating phases in the plasma membrane. During this process the peptide acquires a characteristic multi-layered organization that is detected as a fingerprint-like pattern by EM after incubation of cells with higher CPP concentration [81]. Thus, the induced transition of lipids to nonlamellar phase might be one mechanism for explaining the penetration of peptides across biological membranes. Still, in general the association of peptide nanoparticles with cell surface induces uptake by endocytic mechanism, especially if cargo molecule is involved. Among CPPs a chimeric peptide transportan (TP) has extensively been examined at the ultrastructural level. The protein transduction mechanisms by TP were analyzed using streptavidin or neutravidin tagged with colloidal gold as reporter and complexed with biotinylated peptide [59,82]. This model system provides useful information about the delivery of proteins, therapeutic agents and small nanoparticles with CPPs in general. The protein complexes with TP activate several endocytosis routes in parallel to translocate into cells. TEM analysis revealed that TP-protein complexes interact first with the filopodia, inducing membrane invaginations and budding vesicles into cells [59,82]. The morphology and size of the majority of formed vesicules suggested macropinocytosis as the prevailing mechanism, which had also been proposed to mediate CPP-mediated cargo delivery earlier [69,83–86]. Similar

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Fig. 2. Visualization of the colloidal gold-labeled protein complexes with cell-penetrating peptide (A) and nanogold-peptide conjugate (B) in cells by transmission electron microscopy. A. Binding of 10 nm colloidal gold labeled neutravidin complexes with biotinylated TP to plasma membrane (arrows) and localization in endosomal structures (magnified view) in HeLa cell. B. Association of 1,4-nm nanogold-tagged S413-PV peptide into nanoparticles on the plasma membrane of HeLa cells (magnified view) and localization in endosomes (arrows).

to TP the peptide derived from the N-terminus of bovine or mouse prion protein [87,88] in complex with biotin-tagged DNA and streptavidin gold-conjugate was demonstrated to bind to the cell-surface proteoglycans and being taken up by macropinocytosis as revealed by TEM [88]. In addition to morphological analysis by conventional TEM, immuno-EM has been utilized for further analysis of cellular uptake mechanism of CPP-protein constructs. TP-protein complexes were shown also to localize in caveolin-positive vesicles [82]. Moreover, quantitative analysis of TEM images that would not have been possible to perform at LM resolution indicated that the overall number of caveosomes in control cells was about 2-fold lower than in cells incubated with CPP-protein complexes, suggesting strong induction of caveolae internalization by CPPs. TEM analysis also demonstrated that flotillin-dependent pathway, which uses vesicles that are morphologically very similar to caveolae [89], is not involved in the cellular delivery of proteins by TP, whereas confocal microscopy had suggested a putative co-localization of TP-protein complexes with flotillin earlier. The endosomal pathway leads to the entrapment of biologically active cargo in endosomal vesicles and further targeting into lysosomes for degradation, which might limit the efficacy of drugs delivered by CPPs. Still, the cargo molecules delivered into cells by CPPs typically reach the cytoplasm or cell nucleus retaining the functionality and sufficient activity [79,83,90]. How the cargo molecules escape from the endosomes to retain their biological activity is not completely understood. By TEM we have observed that endocytosed nanoparticles of CPPs (and CPP/oligonucleotide or CPP/protein) associate with endosomal membrane. In some vesicles the membrane is disorganized and ruptured, and peptides or peptide-cargo nanoparticles have escaped into cytosol. Moreover, the characteristic fingerprint-like organization of peptide nanostructures, indicative of non-lamellar organization of lipids, can be found in endosomes even at low micromolar concentration of CPPs [81]. This data allows to speculate that CPPs also interfere with the

regularity of endosomal membrane packing and thereby facilitate the escape of the cargo into cytosol in a functional and active form. 3.2. Electron microscopy analysis of delivery of nucleic acids by CPPs Electron microscopy has been applied as a complementary method in addition to fluorescence microscopy and FACS analysis to provide ultrastructural information about the mechanism of nucleic acids delivery by CPPs. For example, the nanogold-labeled splice-correcting oligonucleotides complexed with a novel chemically modified stearyl-TP10 peptide-based delivery vectors, called NickFects, form electron dense spherical particles, which associate with the cell surface and are efficiently engulfed by cells [79]. The density and regularity of peptideoligonucleotide particles were found to be in correlation with their efficiency to enter the cells, with ability to escape from endosomes, and translocate into nucleus. Another amphipathic peptide CADY that was designed for delivery of nucleic acids formed analogous small nanoparticles with siRNA [78]. However, the obtained particles were less electron dense compared to NickFect particles. CADY–siRNA particles also aggregated to form bigger complexes, and associated avidly with cell surface inducing both endocytic and non-endocytic translocation into cells. Although unexpected, the non-endocytic internalization of siRNA–CADY complexes is well in line with direct translocation of Tat-peptide-modified gold nanoparticles into cells [91]. Another group of new generation CPPs developed on the basis of stearyl-TP10, called PepFects (PF), have been used extensively for the delivery of oligonucleotides and plasmids, which have also been characterized on ultrastructural level by TEM [92–94]. One of those novel CPPs, PF14 forms stable nanocomplexes with splice correcting oligonucleotides with a diameter of about 100 nm, as revealed by TEM [80]. Although no specific receptor is identified for CPPs or CPP-cargo conjugates so far, the uptake of PF14-oligonucleotide

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complexes is dependent on the presence of class-A scavenger receptors (SCARAs) in cells [80]. 4. Conclusions and perspectives Here we analyzed the potential of transmission electron microscopy for studying the mechanisms of protein and peptide drugs with the emphasis on their association with molecules on plasma membrane and localization within cells. So far, electron microscopy has been applied for these studies quite seldom. However, since it enables the detection of single molecules in cellular subdomains that are recognizable without specific staining, electron microscopy provides detailed information about the cell-entry routes and especially about intracellular traffic of proteins and peptides with the emphasis on their association with molecules on plasma membrane and localization within cells. The better understanding of the trafficking of therapeutic proteins, peptides or nucleic acids might, in turn, result in the development of more efficient drugs and their delivery systems [95]. Acknowledgments H.M., K.P. and M.P. were supported by grants from the Estonian Science Foundation (ESF 8705), the Estonian Ministry of Education and Research (0180019s11), and the European Union Regional Development Fund (grant EU30020) through the Competence Centre on Reproductive Medicine and Biology. References [1] B. Leader, Q.J. Baca, D.E. Golan, Protein therapeutics: a summary and pharmacological classification, Nat. Rev. Drug Discov. 7 (2008) 21–39. [2] S. Swain, D. Mondal, S. Beg, C.N. Patra, S.C. Dinda, J. Sruti, M.E. Rao, Stabilization and delivery approaches for protein and peptide pharmaceuticals: an extensive review of patents, Recent Pat. Biotechnol. (2013) (in press). [3] P. Agarwal, I.D. Rupenthal, Injectable implants for the sustained release of protein and peptide drugs, Drug Discov. Today 18 (2013) 337–349. [4] L. Schermelleh, R. Heintzmann, H. Leonhardt, A guide to super-resolution fluorescence microscopy, J. Cell Biol. 190 (2010) 165–175. [5] J.L. Murk, G. Posthuma, A.J. Koster, H.J. Geuze, A.J. Verkleij, M.J. Kleijmeer, B.M. Humbel, Influence of aldehyde fixation on the morphology of endosomes and lysosomes: quantitative analysis and electron tomography, J. Microsc. 212 (2003) 81–90. [6] G.E. Sosinsky, J. Crum, Y.Z. Jones, J. Lanman, B. Smarr, M. Terada, M.E. Martone, T.J. Deerinck, J.E. Johnson, M.H. Ellisman, The combination of chemical fixation procedures with high pressure freezing and freeze substitution preserves highly labile tissue ultrastructure for electron tomography applications, J. Struct. Biol. 161 (2008) 359–371. [7] G.E. Sosinsky, T.J. Deerinck, R. Greco, C.H. Buitenhuys, T.M. Bartol, M.H. Ellisman, Development of a model for microphysiological simulations: small nodes of Ranvier from peripheral nerves of mice reconstructed by electron tomography, Neuroinformatics 3 (2005) 133–162. [8] A. Chan, W.M. Holleran, T. Ferguson, D. Crumrine, O. Goker-Alpan, R. Schiffmann, N. Tayebi, E.I. Ginns, P.M. Elias, E. Sidransky, Skin ultrastructural findings in type 2 Gaucher disease: diagnostic implications, Mol. Genet. Metab. 104 (2011) 631–636. [9] D.J. Fowler, M.A. Weber, G. Anderson, M. Malone, N.J. Sebire, A. Vellodi, Ultrastructural features of Gaucher disease treated with enzyme replacement therapy presenting as mesenteric mass lesions, Fetal Pediatr. Pathol. 25 (2006) 241–248. [10] A. Nadaj-Pakleza, A. Lusakowska, A. Sulek-Piatkowska, W. Krysa, M. Rajkiewicz, H. Kwiecinski, A. Kaminska, Muscle pathology in myotonic dystrophy: light and electron microscopic investigation in eighteen patients, Folia Morphol. (Warsz) 70 (2011) 121–129. [11] Y. Hu, C. Qiao, M. Lv, J. Feng, M. Yu, B. Shen, Q. Zhang, Y. Li, Arg9 facilitates the translocation and downstream signal inhibition of an anti-HER2 single chain antibody, BMC Res. Notes 5 (2012) 336. [12] K.T. Tokuyasu, A technique for ultracryotomy of cell suspensions and tissues, J. Cell Biol. 57 (1973) 551–565. [13] R.M. Perera, R. Zoncu, T.G. Johns, M. Pypaert, F.T. Lee, I. Mellman, L.J. Old, D.K. Toomre, A.M. Scott, Internalization, intracellular trafficking, and biodistribution of monoclonal antibody 806: a novel anti-epidermal growth factor receptor antibody, Neoplasia 9 (2007) 1099–1110. [14] C.D. Austin, A.M. De Maziere, P.I. Pisacane, S.M. van Dijk, C. Eigenbrot, M.X. Sliwkowski, J. Klumperman, R.H. Scheller, Endocytosis and sorting of ErbB2 and the site of action of cancer therapeutics trastuzumab and geldanamycin, Mol. Biol. Cell 15 (2004) 5268–5282. [15] J. Dubochet, M. Adrian, J.J. Chang, J.C. Homo, J. Lepault, A.W. McDowall, P. Schultz, Cryo-electron microscopy of vitrified specimens, Q. Rev. Biophys. 21 (1988) 129–228.

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