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New views and insights into intracellular trafficking of drug-delivery systems by fluorescence fluctuation spectroscopy Biomaterials in the nanometer size range can be engineered for site-specific delivery of drugs after injection into the blood circulation. However, translation of such nanomedicines from the bench to the bedside is still hindered by many extracellular and intracellular barriers. To realize the concept of targeted drug delivery with nanomedicines, research groups are studying intensively the extra- and intra-cellular mechanisms involved as a response to the physicochemical properties of the nanomedicines. In this review, we highlight the contributions of fluorescence fluctuations spectroscopy techniques to better understand, and in turn to bypass, the major hurdles to therapeutic delivery, focusing mostly on the intracellular dynamics of drug-delivery systems. Barriers to nanomedicine delivery to target cells In the last few decades, the development of nanotechnology for biomedical applications has become a priority. In therapeutic delivery nanotechnology involves loading the drug into nanosized materials, referred to as nanocarriers, that improve how a medicine is distributed in the body. Currently, nanocarriers have been used to deliver small drugs [1], in vivo imaging contrast agents [2,3] and biopharmaceuticals [4]. In general, they should satisfy the following requirements; low immunogenicity and toxicity alongside a high specificity to the intended target cells and subcellular compartments. A first class of nanocarriers that match these requirements is made of viral vectors [5]. Viruses have evolved to obtain optimized receptor-mediated internalization, efficient cytosolic release, directed and fast intracellular transport toward target compartments, and readily disassemble. However, viral vectors may be contaminated with the live virus and can cause fatal immune responses, toxicity and chromosomal insertion [6–8]. Another class of nanocarriers comprises nonviral vectors. They lack the evolution-driven machinery to overcome multiple barriers but present low immunogenicity/toxicity and are more easily synthesized on large scales with respect to their viral counterparts. Nonviral nanomedicines often have intracellular targets, in particular for the delivery of biopharmaceuticals such as nucleic acids (NAs), which require delivery to the cytoplasm (siRNA, mRNA, oligonucleotides) or

cell nucleus (plasmid DNA). They can be classified as lipid-polymer- and nanoparticle-based carriers such as lipoplexes [9–17], polyplexes [18], dendriplexes [19], polymeric nanospheres [20,21], nanocapsules [22], lipid nanocapsules [23] or nanoemulsions [24]. Lipoplexes and polyplexes are self-assembled complexes through electrostatic interactions between the anionic NAs and cationic molecules. Depending on sample preparation, they may range in size from 10 nm in diameter up to structures as large as 1 µm, carrying large-sized NAs [25]. The diversity and the relative ease with which nanocarriers can be modified allow great flexibility in their application [26,27]. However, despite their broad potential, moving nanomedicines into mainstream medicines still has a long way to go. Our continued poor understanding of the mechanisms involved makes the efficiency, by which these nanocarriers deliver their therapeutic payload at the required intracellular site, low. In order to improve cellular and subcellular targeting, we first need to better understand the mechanisms by which these nanocarriers might be able to overcome external and internal barriers one by one (local efficiency). Subsequently, this feedback must be used to tune the properties of nanocarriers with the aim to maximize the overall efficiency (total efficiency). This is undoubtedly a hard task because the different successful functionalities may not be integrated compatibly or behave in concert and their roles could cancel each other out. Several research groups are intensively studying the cellular processing

Stefano Coppola1 & Giulio Caracciolo*2

10.4155/TDE.13.148 © 2014 Future Science Ltd

Ther. Deliv. (2014) 5(2), 173–188

ISSN 2041-5990

Deparment of Anatomy, Histology, Forensic Medicine & Orthopedics, ‘Sapienza’ University of Rome, Piazzale A, Moro 5, 00185, Rome, Italy 2 Department of Molecular Medicine, ‘Sapienza’ University of Rome, Viale Regina Elena 291, 00161, Rome, Italy *Author for correspondence: Tel.: +39 06 49693271 E-mail: [email protected] 1

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Review | Coppola & Caracciolo Key Terms Lipoplex: Self assembled

complex of nucleic acids (e.g., plasmid DNA) and a mixture of cationic liposomes. The nucleic acids are packed within the lipid bilayers building up a multilamellar structure or may be coated with a lipid monolayer arranged in a hexagonal lattice.

Polyplex: Self-assembled

supramolecular aggregate of nucleic acids (e.g., plasmid DNA) and polymers (e.g., cationic poly-l-lisine and polyethylenimine). The formation is usually kinetically controlled, giving rise to spherical, globular or rod-like structures.

Transfection efficiency:

Reporter gene assays using green fluorescent protein, luciferase, b-galactosidase (b-gal) or secreted alkaline phosphatase allow researchers to quantitatively measure the expression of genes delivered by experimental delivery systems. Shortly, high-gene expression levels correspond to high transfection efficiency.

Fluorescence correlation spectroscopy: Powerful technique to obtain ensemble averaged motility information from the time correlation of intensity traces of particles moving in and out an observation volume.

of nanomedicines but few general conclusions have been drawn so far. „„Extracellular

barriers In many cases where disease sites are not otherwise easy to access, systemic administration of nanomedicines is mandatory. Under these circumstances, nanomedicines have to penetrate through a series of systemic barriers in order to minimize side effects while achieving high efficiency. The first extracellular barrier is nonspecific binding of nanomedicines to negatively charged serum components, such as blood cells and serum proteins. Albumin, lipoproteins (high density lipoprotein and low density lipoprotein) and macroglobulin, complement proteins and immunoglobulins interact with nanomedicines, altering their size and surface charge [28]. These interactions can destabilize the system and provoke premature release of the payload, which could be degraded. Interactions between nanomedicines and extracellular environment can also result in aggregation. This colloidal instability is usually mentioned as the second major obstacle. In the blood circulation, they must evade uptake by macrophages, the clearance by renal filtration and degradation by endogenous nuclease [29]. Once the blood circulation hurdle is overcome, nanomedicines need to traverse from blood vessels to target tissues. Although some tissues, such as tumors, inflammatory sites and the reticuloendothelial system, RES (e.g., liver, spleen) have leaky blood vessels, the capillary vessel walls in most organs and tissues are impermeable to large particles. Furthermore, the extracellularmatrix slows down their movement to target cells due to its dense polysaccharides and fibrous proteins [30]. Even further interactions may lead to the premature payload release, triggering extracellular degradation and thus lowering the chance to be uptaken by target cells. „„Intracellular

barriers The first interaction between nanomedicines and target cells is through the plasma membrane. Cell adhesion can be nonspecific through hydrophobic or electrostatic interactions, or specific through recognition by membrane-anchored receptor proteins [31]. In all cases, the binding event can trigger transmembrane signaling and subsequent activation of the endocytic machinery, resulting in endocytosis of the nanomedicines. Nonspecific adhesion is known to depend strongly on the surface charge [32] and shape of particles [33]. Electrostatically driven adhesion 174

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can in principle occur for either cationic or anionic nanocarriers [34]. However, the adhesion of cationic nanoparticles, supposedly mediated by the highly anionic glycosaminoglycans of the cell surface proteoglycans [35], is generally much stronger and has been demonstrated to lead to higher rates of internalization [36–38]. As mentioned in the previous section, surface charge and size of nanomedicines is altered by binding of serum proteins forming the so-called ‘protein corona’ [39]. Thus, the cell adhesion and subsequent uptake may be dependent on the corona composition [40]. To minimize off-target effects due to nonspecific adhesion, nanomedicines can be functionalized with targeting ligands such as transferrin, folate, EGF, sugar moieties, ligands of cell adhesion molecules, but also monoclonal antibodies [31,41,42]. These ligands bind to cell surface receptors, which facilitate and enhance the cellular uptake. The process of cellular uptake (internalization) of nanomedicines has received a great deal of attention in the last few years. There is convincing evidence showing that after cell surface adhesion endocytosis is the predominant pathway for the internalization of nanocarriers [43–50]. For instance, uptake of lipoplexes usually involves a clathrin-mediated pathway or fluidphase macropinocytosis, while internalization of the polyplexes can occur via clathrin-mediated endocytosis, caveolae-mediated endocytosis or macropinocytosis [46,51–53]. Interestingly, these routes trigger different intracellular pathways, enhancing or reducing the final transfection efficiency [47–49,54–57]. There is clear evidence that all pathways are strongly dependent on the cell type [49,56,58] and on carrier properties, such as size [47,59], surface charge [36], conformation [37] and ligand coupling [31]. Once the uptake barrier is overcome, nanomedicines have to traverse the cytoplasm and release the payload at the required subcellular compartments. Intracellular trafficking and cargo release represent the main topics of this review and will be addressed in the following sections. The last step of intracellular therapeutic delivery is the nuclear entry. The nuclear envelope contains openings in the form of nuclear pore complexes that tightly regulate cytoplasm– nuclear trafficking and limit passive diffusion of molecules with sizes >40 kDa, corresponding to a hydrodynamic diameter of approximately 10 nm [60]. These pores are, in general, too small for free diffusion of plasmids or nanoparticles and it is still not fully understood how plasmids enter the nucleus in successfully transfected cells. In dividing cells, it has been shown that the transient future science group

Intracellular trafficking of drug-delivery systems by fluorescence fluctuation spectroscopy disassembly of the nuclear envelope facilitates the translocation of NAs and subsequent gene expression [61–63]. A better understanding of the endogenous nuclear import machinery has allowed researchers to develop strategies for nuclear import of genes. The nuclear localization sequence (NLS) is a major player that shuttles protein–plasmid complexes through the nuclear pore [64]. NLS-mediated active nuclear translocation involves a process starting from its interaction with cytoplasmic importins to binding of the NLS to the nuclear pore complex and the passage through the pore [65–67]. A first generation of nuclear-targeting nonviral vectors have been obtained, tethering NLS to pDNA (noncovalently or covalently) [64] or linking NLS and pDNA with a peptide nucleic acid (PNA) as a bifunctional linker [68,69]. A potential immune response associated with the use of an exogenous NLS has led to the emergence of a second type of nuclear targeting vector in which a DNA targeting sequence is attached to a DNA vector for active nuclear import of DNA [66,70]. „„Intracellular

itinerary of nanoparticles As introduced in the previous section, after internalization nanomedicines have to traverse the cytoplasm and release their payload in the desired subcellular compartments. The intracellular pathway has been demonstrated to be dependent on the endocytic route and could lead nanomedicines to the acidic/digestive route [47,55]. Degradation by lysosomes, which highly reduces the transfection efficiency, has been correlated with active transport along microtubules [71–73]. Microscopy colocalization studies have been extensively performed to elucidate interactions between nanomedicines and intracellular structures [74], endosomal escape of the payload and eventually degradation inside lysosomes [71,75]. However, colocalization methods (intensity or object based) have the fundamental limitation that they entirely rely on the information from a single image, which makes it difficult or sometimes impossible to distinguish, for example, coincidental colocalization from real interaction. Since intracellular trafficking and endosomal escape are highly dynamic processes, several advanced fluorescence techniques have played an important role in this research during the last decade. Stability of nanomedicines in living cells has been addressed by means of fluorescence correlation spectroscopy (FCS), while transport has mostly been studied by single particle tracking (SPT) [76–83]. Image correlation future science group

| Review

spectroscopy (ICS) techniques [84,85] represent a powerful alternative to SPT, even if until now they have been scarcely employed in studying intracellular trafficking of nanomedicines [86–89]. While SPT is performed to reconstruct the trajectory of single molecules, ICS methods rely on correlation of images containing fluorescence fluctuations of many single molecules and therefore the output is an ensemble average information. A detailed comparison study of the performance of SPT and ICS has recently shown that both techniques are suitable and complementary in retrieving mobility parameters of nanomedicines [90]. The main aim of the present review is to discuss recent advancements in our understanding of the intracellular trafficking and cargo release of nanomedicines, using ensemble fluorescence-correlation techniques, such as FCS and ICS. „„Ensemble

fluorescence-correlation techniques In fluorescence correlation techniques such as FCS and ICS, the fluorescence fluctuations are originating from single molecules but the mathematical analysis is performed on a (spatio-) temporal trace consisting of many fluctuations so that the results resemble the ensemble average of many hundreds or thousands of molecules. FCS is a powerful technique that was developed during the 1970s (Figure 1) [91]. A FCS experiment is based on a confocal laser scanning microscope (CLSM)-type of instrument so that only light from the focal spot, the so-called confocal detection volume, can reach the detector. The fluorescence intensity is monitored with very high sensitivity and temporal resolution using avalanche photo diode detectors, holding the focused laser stationary at one particular location of interest. The raw output from a FCS experiment is, therefore, a fluorescence time trace with fluctuations originating from fluorescent molecules moving in and out of the confocal detection volume. Motility parameters can be retrieved from the analysis of autocorrelation profiles of those time traces. FCS is able to detect diffusion, biased diffusion and other kinds of processes by single or multiple populations. Quantitative information on association and dissociation is of fundamental importance for the study of cargo release and degradation processes. It can be achieved by performing fluorescence cross-correlation spectroscopy from fluorescence time traces of two different labeled species [92,93]. If the two species are associated, www.future-science.com

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Review | Coppola & Caracciolo Key Term

S

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Spatio-temporal image correlation spectroscopy:

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se r

Robust tool to get ensemble averaged dynamical information from the temporal evolution of spatial correlations of raw 2D images acquired with a fluorescence microscope.

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ACF green ch ACF red ch CCF 10-1

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Figure 1. Fluorescence correlation spectroscopy experiment. A laser beam passes through a BE and is focused by the O to a diffraction limited volume in the S after being reflected by the TM. The fluorescence signal, generated by fluorescent molecules/particles entering the acquisition volume, is collected again by the objective lens and is split up into different spectral channels (e.g., green and red) by a dichroic mirror and proper F. The signal then reaches the detectors (detector 1 & 2), after going through P. The AC and CC functions give the motility parameters of the molecules/particles when fitted to the proper dynamical model. AC: Auto-correlation; BE: Beam expander; CC: Cross-correlation; F: Emission filters; O: Objective lens; P: Confocal pinholes; S: Sample; TM: Trichroic mirror. This figure can be viewed in full color at: www.future-science.com/doi/full/10.4155/TDE.13.148

their motion in and out of the focal volume is concomitant and concerted. The fluorescence traces from the two acquisition channels are 176

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therefore positively cross-correlated in time. If the emission spectrum of one labeled species (called a donor) overlaps with the excitation future science group

Intracellular trafficking of drug-delivery systems by fluorescence fluctuation spectroscopy spectrum of the other one (called an acceptor), excitation energy can be transferred from the donor to the acceptor fluorophore (for mutual distances

New views and insights into intracellular trafficking of drug-delivery systems by fluorescence fluctuation spectroscopy.

Biomaterials in the nanometer size range can be engineered for site-specific delivery of drugs after injection into the blood circulation. However, tr...
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