Expert Opinion on Drug Delivery
ISSN: 1742-5247 (Print) 1744-7593 (Online) Journal homepage: http://www.tandfonline.com/loi/iedd20
Intracellular delivery of nanocarriers and targeting to subcellular organelles Aditi Jhaveri & Vladimir Torchilin PhD DSc To cite this article: Aditi Jhaveri & Vladimir Torchilin PhD DSc (2015): Intracellular delivery of nanocarriers and targeting to subcellular organelles, Expert Opinion on Drug Delivery To link to this article: http://dx.doi.org/10.1517/17425247.2015.1086745
Published online: 11 Sep 2015.
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Date: 11 September 2015, At: 06:14
Review
Intracellular delivery of nanocarriers and targeting to subcellular organelles 1.
Introduction: intracellular delivery, significance, and challenges
2.
Nanocarriers for intracellular targeting
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3.
Approaches for intracellular delivery
4.
Targeting nanocarriers to intracellular organelles
5.
Conclusions and future directions
6.
Expert opinion
Aditi Jhaveri & Vladimir Torchilin† †
Northeastern University, Center for Pharmaceutical Biotechnology and Nanomedicine, Department of Pharmaceutical Sciences, Boston, MA, USA
Introduction: Recent trends in drug delivery indicate a steady increase in the use of targeted therapeutics to enhance the specific delivery of biologically active payloads to diseased tissues while avoiding their off-target effects. However, in most cases, the distribution of therapeutics inside cells and their targeting to intracellular targets still presents a formidable challenge. The main barrier to intracellular delivery is the translocation of therapeutic molecules across the cell membrane, and ultimately through the membrane of their intracellular target organelles. Another prerequisite for an efficient intracellular localization of active molecules is their escape from the endocytic pathway. Areas covered: Pharmaceutical nanocarriers have demonstrated substantial advantages for the delivery of therapeutics and offer elegant platforms for intracellular delivery. They can be engineered with both intracellular and organelle-specific targeting moieties to deliver encapsulated or conjugated cargoes to specific sub-cellular targets. In this review, we discuss important aspects of intracellular drug targeting and delivery with a focus on nanocarriers modified with various ligands to specifically target intracellular organelles. Expert opinion: Intracellular delivery affords selective localization of molecules to their target site, thus maximizing their efficacy and safety. The advent of novel nanocarriers and targeting ligands as well as exploration of alternate routes for the intracellular delivery and targeting has prompted extensive research, and promises an exciting future for this field. Keywords: cell-penetrating peptides, cytoplasm, intracellular delivery, liposomes, macromolecules, mitochondria, nanocarriers, nuclei, polymeric micelles Expert Opin. Drug Deliv. [Early Online]
Introduction: intracellular delivery, significance, and challenges
1.
A number of therapeutics including small molecule drugs as well as biological macromolecules like DNA, siRNA, proteins, enzymes, antibodies, or oligonucleotides for the treatment of various diseases including cancer are known to act on targets localized within the cell. These intracellular drug targets include the cytosol, the nucleus, endosomes, mitochondria, the Golgi complex, lysosomes, and the endoplasmic reticulum (ER) (Figure 1) [1,2]. Drug-loaded nanocarriers also need to be delivered intracellularly to exert their therapeutic action on various intracellular organelles [1]. However, intracellular delivery of therapeutics still poses a significant challenge for drug delivery. Often, it is assumed that delivery of the drug into the cytosol will ensure its delivery to its sub-cellular target by simple diffusion and random interaction with the cell organelles. This is true in some cases, such as for siRNA, whose site of action is the cytoplasm, or for some small molecules that retain 10.1517/17425247.2015.1086745 © 2015 Informa UK, Ltd. ISSN 1742-5247, e-ISSN 1744-7593 All rights reserved: reproduction in whole or in part not permitted
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Most drugs, biologics as well as drug-loaded pharmaceutical nanocarriers need to be delivered intracellularly to their target organelles to exert their desired therapeutic effects. Extracellular as well as intracellular barriers need to be surmounted for therapeutics to reach their final destination. Escape from the endocytic pathway comprises a major bottleneck in the intracellular delivery of payloads and can be overcome by suitable modifications of nanocarriers. Size, charge, and the property of longevity of nanocarriers play a critical role in determining their fate in the circulation and eventually their accumulation at target sites within cells. Active targeting strategies that use cell-penetrating peptides (CPPs) as well as other-organelle-specific ligands and simultaneously exploit various stimuli that exist within cells play an important role in achieving the goal of targeted intracellular delivery. A number of nanocarriers are currently being investigated actively for delivery of payloads to the cytoplasm, nuclei, mitochondria, and lysosomes. Targeted delivery of payloads to specific cells and subcellular compartments results in a reduced toxicity and maximizes their therapeutic efficacy.
This box summarizes key points contained in the article.
a high metabolic stability until their eventual interaction with the organelles. In cases where the sites of drug action are organelles within the cytoplasm, achieving intracellular targeting can be challenging. Even if delivery to the cytoplasm is successful, other barriers need to be surmounted before the drug molecules can reach their target organelles [3]. It thus follows that to achieve optimal efficacy and to improve the therapeutic effects of drugs, an understanding of their intracellular disposition patterns is imperative. Furthermore, intracellular delivery can overcome or even reverse the multi-drug resistance phenomenon commonly encountered in anti-cancer chemotherapy, which further adds to its importance for targeted drug delivery [4]. Achievement of the goal of intracellular delivery is not easy, however. A major challenge for therapeutics, especially macromolecules like peptides, proteins, DNA, and siRNA is the barrier to their translocation through the lipophilic cell membranes and then the sub-cellular organelles, unless these payloads are actively transported into the cells [1]. In order to access their intracellular target receptors, therapeutic entities or nanomedicines must usually navigate the endocytic pathways in the cell. Some excellent reviews have described the endocytic mechanisms involved in the trafficking of nanomedicines [5-7]. Macromolecules and nanocarriers typically enter the cell via membrane-bound vesicles called endosomes, formed by the invagination and pinching-off of pieces of the plasma membrane in a process known as endocytosis [8]. 2
Endosomes mature from early endosomes to late endosomes, progressively become more acidic, and may ultimately fuse with the lysosomes. The particles that enter a cell via this endocytic pathway are entrapped in the endosomes and eventually end up in lysosomes where they are degraded via active enzymatic degradation by various lysosomal digestive enzymes. Since only a small fraction of endosomes degrade spontaneously releasing the contents, the majority of endocytosed material is degraded and never makes its way to the cytosol [9]. This results in a restricted delivery of therapeutic agents to their intracellular targets, and precludes them from in vivo applications due to bioavailability concerns, even though they show potential in vitro. Endosomal escape is thus a critical bottleneck for intracellular delivery of molecules [10]. A number of approaches have been investigated to allow an efficient endosomal escape of therapeutics including pH buffering, destabilization of endosomal membrane by fusogenic agents, or photochemical disruption, osmotic swelling to enable bursting of endosomes, and the use of pore-forming peptides [11]. Once nanocarriers escape the endosomal compartment, they can navigate through the cytoplasm to interact and bind various intracellular targets. Both invasive and non-invasive strategies have been developed to bypass the endocytic pathway or to facilitate endosomal escape in an attempt to deliver macromolecules or nanocarriers inside cells. The invasive techniques include electroporation, micro-injection, biolistic transfections (with a hand-held gene gun), sonoporation, and relatively recent techniques which rely on the use of microfluidic device-based electroporation [12-14]. However, many of these cause damage to the cell membrane. A relatively simpler, efficient, and non-invasive option for intracellular delivery is the use of drug-loaded nanocarriers. These are described in detail in the subsequent sections which deal with the properties, role, and modifications of nanocarriers for organelle-specific intracellular delivery and targeting. 2.
Nanocarriers for intracellular targeting
Properties and advantages of nanocarriers A host of nanocarriers have emerged as vehicles of choice for drug delivery, especially for cancer therapy. By virtue of their physico-chemical properties including size, shape, surface charge, and the density of targeting ligands on their surface, nanocarriers impart many desirable features for drug delivery. These include, but are not limited to, avoidance of rapid uptake by the mononuclear phagocyte system (MPS) or immediate renal clearance, increased stability of drugs and an improved pharmacokinetic profile, longer circulation times, tuned release of payloads, modification for active targeting to desired locations without off-target effects, and triggered release of payloads by various internal and external stimuli [15,16]. Some important features of nanocarriers are discussed in Sections 2.2 and 2.3. 2.1
Expert Opin. Drug Deliv. (2015) 13(1)
Intracellular delivery of nanocarriers and targeting to subcellular organelles
Mammalian cell Cargo
Cytoplasm
Clathrin
Caveolin Early endosome Caveosome Clathrin coated vesicle Endosomal escape
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Late endosome
Golgi
Lysosome
Mitochondrion
Nucleus
Endoplasmic reticulum
Figure 1. Target sites for intracellular drug delivery: The cytoplasm, nucleus, mitochondria, and the lysosomes constitute the major sites for intracellularly targeted drug delivery.
Role of size, charge, and steric stabilization of nanocarriers
2.2
Size is a critical parameter that dictates nanocarrier disposition in the body. Uptake by the MPS, tissue extravasation, renal clearance, diffusion into tissues, biodistribution, as well as the mechanism of nanoparticle internalization into cells (phagocytosis, macropinocytosis, clathrin, or caveolaemediated endocytosis) are impacted by the nanocarrier size [17,18]. A disorganized endothelium characterized by large fenestrations coupled with a defective lymphatic drainage in tumors allows the extravasation and retention of macromolecules within the tumor interstitium [19-21]. This phenomenon, termed the enhanced permeability and retention (EPR) effect, constitutes the basis of passive targeting [22]. Nanocarriers in the 10 -- 200 nm size range can optimally take advantage of the EPR effect [23,24]. Larger (> 200 nm) and smaller (5 -- 10 nm) particles are removed by the MPS and through renal clearance, respectively. The surface charge of a nanocarrier can affect its association with cells and can be modified to promote active tumor targeting as well as delivery to the cytosol. The presence of a surface charge influences the opsonization of materials, their recognition by cells of the MPS, as well as their overall plasma circulation profile [18]. Generally, positively charged nanoparticles have a higher rate of cell uptake, greater plasma protein adsorption, higher non-specific internalization rate, and a shorter blood circulation half-life compared to neutral or negatively charged formulations [18].
Imparting longevity to nanocarriers in the circulation diminishes their uptake by the MPS and allows enhanced accumulation at the tumor site via the EPR effect. Long circulating nanocarriers can undergo multiple passages through the target from the systemic circulation, which gives them more time to interact with their target. This greatly enhances retention, as well as intracellular delivery of targeted nanocarriers. Hydrophilic, flexible polymeric coatings such as poly(ethylene glycol) (PEG) and other polymers can be grafted or adsorbed on the nanoparticle surface to make them more hydrophilic or to neutralize the surface charge of the particles, thereby slowing down the process of their opsonization [25,26]. Currently, a majority of the nanocarriers are coated with PEG for biocompatibility and for extended circulation time. However, the hydrophilic surface coating of PEG may hinder intracellular uptake of the carriers by cells and endosomal escape, giving rise to a so-called “PEG dilemma” [27]. Limitations on the effectiveness of the passive targeting approach result from the pathophysiological heterogeneity in tumors, variable vascular permeability, and a weak EPR effect, particularly in the central portion of tumors [19]. Clearly, the simple modification of nanocarriers to circulate for extended periods is inadequate for optimized therapeutic outcomes. To overcome these limitations, and to enhance their binding to specific targets after extravasation, active targeting strategies must be employed.
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3.
Approaches for intracellular delivery
The most commonly employed ligands for intracellular delivery include cell-penetrating peptides (CPPs) and proteins, which can translocate through cell membranes [28], nuclear localization signals (NLS) [29], mitochondrial localization signal [30], ER signal peptide [31], and other non-peptide ligands [32,33]. Among vehicles, pH-sensitive nanocarriers as well as cationic lipids and polymers, along with many other nanosystems, have been investigated for intracellular delivery. This section discusses the major intracellular delivery approaches with a greater focus on CPPs as one of the most widely used agents to promote intracellular delivery. Active targeting of nanocarriers for intracellular delivery using ligands
3.1
Active targeting utilizes various affinity ligands to modify nanocarriers, which enables them to bind molecules that are selectively or differentially expressed on the tumor cells or on the membranes of various intracellular organelles [16]. Still, EPR-mediated passive targeting remains the first step of the active uptake process. These targeting ligands include antibodies and their fragments, peptides, proteins, sugar moieties, aptamers, and small molecules like folate. To ensure delivery of nanocarriers inside cells and subsequently to their subcellular targets, they must first traverse the plasma membrane. Nanocarriers may be internalized directly via receptormediated endocytosis (RME) following their interaction with the receptors on the plasma membrane, or indirectly by associating with the lipid bilayer [34]. RME may be by either clathrin- or caveolae-mediated routes. Molecules may also be internalized via pinocytosis or phagocytosis. However, for nanocarriers, endocytosis is the major route of entry into the intracellular space. The intracellular trafficking pathway of nanocarriers is controlled by the particular endocytic pathway that it follows, which in turn depends on the ligand involved as well as the cell type [34,35]. For example, transferrin (Tf), the iron-binding protein, binds to transferrin receptors and internalizes via clathrin-mediated endocytosis, whereas viruses such as those belonging to the polyomavirus family utilize caveolae-mediated endocytosis as their entry mechanism [36,37]. Nanocarriers accumulate at their initial target (tumor) by passive targeting via the EPR effect or by specific ligandmediated active targeting. However, their subsequent intracellular delivery can be mediated by various internalizable ligands (e.g., folate, transferrin) [16]. Specific examples for various ligand-targeted nanocarriers have been discussed in section 4. Modification of nanocarriers with cellpenetrating peptides for intracellular delivery
3.2
A popular approach for surmounting the cellular barrier and enabling intracellular delivery of various therapeutic and diagnostic molecules is the use of cell-penetrating proteins 4
or peptides to modify them [16,38]. CPPs can penetrate the cell membranes by a phenomenon known as “proteintransduction” and enhance the delivery of the CPP-modified cargoes inside the cell [38]. The CPPs contain domains of less than 20 amino acids, termed protein transduction domains, which are highly rich in basic amino-acid residues [39]. This property of translocation through the plasma membrane has been found for a number of other peptides including transportan [40], VP22, a herpes virus protein [41], model amphipathic peptide (MAP) [42], synthetic polyarginines [33], DNA-binding peptides like human c-Fos (139--164), c-Jun (252--279), and RNA-binding proteins like HIV-1 Rev (34--50), BMV Gag (7--25), and FHV coat proteins (35--49) [43]. CPPs can be classified based either on their origin or their sequence characteristics [44]. On the basis of their physicochemical properties, CPPs may be divided into cationic, amphipathic, and hydrophobic CPPs [45]. Cationic CPPs such as trans-activating transcriptional transactivator (TAT) [46-48], polyarginines [33], and protamine are rich in arginine [49]. Amphipathic CPPs include peptides such as model amphipathic peptide (MAP) and transportan which are rich in lysine and arginine-rich peptides like RW9 and RL9 derived from penetratin [50]. The third class of CPPs includes those peptides that have apolar residues, including prenylated peptides [51], pepducins [52], and the signal sequences from integrin b3 [53]. A variety of mechanisms for CPP accumulation in the cytoplasm have been proposed. However, no single pathway has been accepted as a unifying mechanism for CPP-mediated uptake. This is due to differences in uptake because of peptide properties such as molecule length, their concentration, sequence characteristics, conformation, and charge delocalization, as well as cargo characteristics like size and charge [54,55]. CPPs or CPP-cargo conjugates may enter the cell using one or multiple mechanisms and may localize in different subcellular compartments. CPPs utilize both energy-dependent and energy-independent mechanisms for translocation through cell membranes. CPPs alone, or when attached to small molecules, are usually internalized via electrostatic interaction and hydrogen bonding which bring about their direct transduction through the lipid bilayer and do not require energy [56,57]. CPPs conjugated to large cargoes typically utilize the energy-dependent endocytic process of macropinocytosis, which a key mechanism for their uptake [58]. Other routes of entry include clathrin-mediated endocytosis and lipid raft-mediated caveolae endocytosis [59-61]. For any of these mechanisms of uptake, it is essential that the CPPs interact with the negatively charged cell-surface residues for successful transduction [1]. CPPs can deliver a variety of cargoes including drugs, antibodies, proteins, nucleic acids (DNA, siRNA, oligonucleotides), fluorochromes, quantum dots, nanocarriers, and contrast agents for magnetic resonance imaging (MRI), both in vitro and in vivo [38,62,63].
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Intracellular delivery of nanocarriers and targeting to subcellular organelles
Recently, TATp-modified liposomes (TATp-L) were shown to deliver DNA into dendritic cell (DC) cultures [46]. TATp increased the uptake and internalization of liposomes in DC and enhanced the in vitro transfection efficiency as was evidenced from EGFP and bovine herpes virus type 1 (BoHV-1) glycoprotein D (gD) expression. Although DCs treated with plain-L or TATp-L loaded with pCIgDA (plasmid for eukaryotic expression of BoHV-1) both expressed gD, the transfection with TATp-L resulted in a two-fold higher protein expression than that of plain-L. The de novo synthesized gD was immunologically active only when DCs were transfected with TATp-L, and surpassed the threshold needed to trigger the secretion of the interleukin-6 (IL-6) cytokine [46]. Another CPP, octa-arginine (R8) was used to modify the surface of PEGylated doxorubicin (DOX)-loaded liposomes, commercially available as Doxil or Lipodox using a polyethylene glycol-dioleoyl phosphatidylethanolamine (PEG-DOPE) amphiphilic co-polymer [33,64]. The R8-modified liposomes enhanced the anti-cancer activity, improved intracellular delivery, and endosomal escape of DOX relative to the unmodified liposomes, resulting in a higher DOX delivery to the cytoplasm and its eventual accumulation in the nucleus. The higher accumulation of DOX led to greater apoptosis and cytotoxicity in vitro, and translated into effective tumor growth suppression in BALB/c mice bearing subcutaneous (s.c) 4T1 tumors [64]. Similar results with R8-modified Lipodox were also seen both in vitro in A549 (non-small cell lung cancer) cells and in vivo in A549 tumor xenograft-bearing nude mice [33]. CPPs have also been used for the intracellular delivery of imaging and contrast agents or both simultaneously [63]. Liu et al. reported the synthesis of water-soluble, biocompatible, carboxyl-, and PEG-functionalized indium phosphide (InP)/zinc sulphide (ZnS) quantum dots (QDs), designated QInP, and evaluated their uptake into cells using four CPPs [65]. The CPPs evaluated included synthetic nonaarginine (SR9), R9 modified with polyhistidine (HR9), a CPP composed of nona-arginine and the penetration accelerating peptide sequence (Pas) (PR9), and a CPP composed of an INF7 fusion peptide and nona-arginine (IR9). All the CPPs interacted non-covalently with QInP in vitro to form stable CPP/QInP complexes. These complexes efficiently delivered QInP into human non-small cell lung cancer (A549) cells. Moreover, the complexes were non-toxic when 500 nM of CPP was complexed with QInP at a molecular ratio of 30, and QInP did not cause significant cytotoxicity when the concentrations were £ 1 µM [65]. Table 1 lists additional examples of nanocarriers modified with CPPs for intracellular delivery of drugs or biologicals. CPPs can target diverse cell types in heterogenous patient populations due to their ability to facilitate non-specific and receptor-independent cellular uptake of various carriers, and thereby overcome limitations of many active targeting agents. Moreover, they can guide cellular internalization through less degradative pathways than RME to minimize the breakdown
of labile therapeutic cargoes [66]. However, the non-specificity of CPPs is a major deterrent to their systemic application and hinders their utility to target specific sites in vivo, thus increasing the risk of toxicity and off-target effects. Other limitations of CPPs include their susceptibility to proteolytic cleavage under physiological conditions and charge-mediated blood clearance, both of which result in a shorter duration of action due to rapid renal clearance [45,48]. A number of approaches have been suggested to overcome challenges associated with the systemic administration of CPPs. These typically involve control of the presentation of CPPs only at a target site in vivo. These have been summarized very well in a recent review by MacEwan and Chilkoti [66]. To achieve control over CPP presentation, “smart” or stimuli-responsive systems have been developed based on the physiological or microenvironmental features peculiar of the targeted tissue or cell type [44,66]. The triggers for CPP activation may be intrinsic to the tumor environment (low pH or over-expression of enzymes like proteases) or extrinsic such as local application of heat or light. For the controlled presentation of CPPs, five approaches have been investigated including: i) controlled display of CPPs by removal of “stealth” polymers [48], ii) triggered display of CPPs by actuation of molecular tethers [67], iii) use of ionizable residues to manipulate CPP charge [68], iv) presentation of CPPs by dissociation from ionic inhibitors [69], and (v) modulation of the interfacial density of critical CPP residues by temperature-triggered micelle assembly [66,70].
Cationic molecules Other approaches for promoting intracellular delivery make use of cationic molecules, which interact with the negatively charged plasma membrane of the cells. These include amine containing polymers, cationic lipids (e.g., DC-Cholesterol, DOTAP), dendrimers, and polypeptides. For example, polyethyleneimine (PEI) is widely used for its ability to induce membrane permeability, and allow the nanoparticle entry into cells. It finds applications in gene delivery because it binds and condenses nucleic acids very efficiently. However, cationic molecules can cause concerns about cytotoxicity and poor stability in biological buffers. To circumvent this, they are often modified or shielded with PEG. PEG-modified amines, for example, exhibit stability in biological buffers, penetrate cell membranes, can disrupt endosomes, and are able to access the cytosolic space [34]. The endosomedisrupting ability of amine-containing polymers results from the “proton-sponge effect.” The amine groups of these polymers are able to absorb protons generated by the ATPase, causing osmotic pressure build-up within endosomes. This leads to the destabilization and rupture of endosomes releasing their contents into the cytosol [71,72]. Another mechanism for the membrane permeability by cationic molecules is their ability to form nanoscale holes in the plasma membrane [73]. 3.3
Expert Opin. Drug Deliv. (2015) 13(1)
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Table 1. CPPs for intracellular delivery of nanocarriers. CPP (sequence)
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CKRRMKWKK (derived from penetratin) RRRRRRGGRRRRG derived from protamine CGRRMKWKK
Peptide for ocular delivery (POD) CGGG[ARKKAAKA]4 or HIV-Tat (CGGGGYGRKKRRQRRR) R8 (octaarginine) R9 (nonaarginine)
TAT
ACPP (Suc-e8-(Aop)-PLGC(Me)AGr9-c-NH2)
Nanocarrier
Thermo-sensitive liposomes Lipid nanoparticles
Cargo
Method of CPP activation/ exposure
Ref.
Doxorubicin
Mild hyperthermia
[143]
–
[144]
Liposomes
siLuc2 (against luciferase), and siGFP c-myc siRNA
[145]
PLGA nanoparticles (NPs)
Fluorbiprofen
NIR triggered two-photon absorption and cleavage of photoresponsive group from inactive CPP --
Graphene oxide sheets Phenyl boronic acid targeted mesoporous silica NPs Micellar NPs
pEGFP Doxorubicin
-Cleavage of MMP-2 responsive peptide PVGLIGK(Cy5)G
[147] [148]
Plk-1 siRNA
[149]
Dendrigraft poly-L-lysine (DGL-G3) NPs
pEGFP
Shell cross-linked knedellike NPs
Cleavage of MMP-2 responsive peptide PLGLAG Low tumor extracellular pH and MMP-2 triggered activation of CPP –
Peptide nucleic acid for iNOS mRNA and alexa fluor 633 for imaging Doxorubicin Cathepsin B triggered activation of TAT Gadolinium-DOTA and/or MMP 2/9 triggered release of Cy5 the ACPP and cargo
Mesoporous silica coated QD NPs PAMAM generation 5 dendrimer
[146]
[150]
[151]
[152] [63]
Aop: 5-amino-3-oxapentanoyl; ACPP: Activable cell penetrating peptide; C(Me): S-methylcysteine; c-NH2: C-terminal amide; lower case letters: D-amino acids; Suc: Succinyl.
Targeting nanocarriers to intracellular organelles 4.
Agents need to be delivered to specific locations within the cell to elicit their therapeutic effects. For example, genes and anti-sense therapy preparations, siRNA, pro-apoptotic drugs, and lysosomal enzymes need to be delivered to the nucleus, cytoplasm, mitochondria, and the lysosomal compartment, respectively, to exert their therapeutic actions [1]. To ensure the delivery of therapeutics to these intracellular organelles, nanocarriers can be modified with suitable ligands to direct them to specific compartments and avoid chemical modification of the therapeutic molecule itself. This section includes discussion of targeted delivery of therapeutic, diagnostic, or imaging payloads to specific intracellular compartments including the cytosol, nucleus, mitochondria, and lysosomes. Delivery of nanocarriers to cytoplasm A number of therapeutics require delivery into the cytosol either because their receptors are present in the cytosol or because their target is an intracellular organelle accessible through cytosolic transport [74]. Nanocarriers, which deliver a variety of cargoes from small molecules to biologics 4.1
6
(DNA, siRNA, proteins, and antibodies) have been developed and employed for cytosolic delivery. Liposomes are the oldest and by far, the most widely investigated nanocarriers. Various strategies have been employed to utilize liposomes for cytosolic delivery of therapeutics. One of the strategies includes the development of “fusogenic” or pHsensitive liposomes [75,76]. Fusogenic lipids undergo a phase transition under acidic conditions, which facilitates their interaction and fusion or destabilization of the endosomal membranes, resulting in the release of the encapsulated cargo into the cytoplasm [74]. Depending on the mechanism for triggering the pH-sensitivity, various strategies may be utilized for the formulation of pH-sensitive liposomes. Typically, combinations of DOPE with compounds containing an acidic group (e.g., carboxylic groups in oleic acid, methacrylic acid, cholesteryl hemisuccinate (CHEMS), and N-isopropylacrylamide) that can stabilize PE at a neutral pH are utilized [77]. Fusogenic properties may also be imparted to liposomes by the incorporation of synthetic fusogenic peptides or polymers in the lipid bilayers [78,79]. Serum or plasma proteins are known to hamper formulations by destabilizing them, causing the premature leakage of their contents and also reduce their pH-sensitivity, thus rendering them inappropriate as carriers for in vivo delivery to the cytoplasm [79]. Long-circulating,
Expert Opin. Drug Deliv. (2015) 13(1)
Intracellular delivery of nanocarriers and targeting to subcellular organelles
Table 2. Nanocarriers for cytoplasmic delivery of payloads. Nanocarrier Liposomes
Liposomes
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Micelles (mixed micelles) Doxil
Core-shell colloids
Multi-layered polyion complex (PIC)
Composition
Payload(s)
(DPPC/Chol or DPPC/DOPE) liposomes modified with fusogenic polymers PEG-PMMICholC6 or PMMI-co-PDMAEMA DOPE/CHEMS/DOPE-PEG1K liposomes modified with R8 DSPE-PEG2000/DSPE-3400--2C5/ PHIS-PEG2000 DOX-loaded liposomes modified with MCF-7 cell specific chimeric phage fusion coat protein (Phage-Doxil) Proton sponge-based core made of PEGDMA crosslinked PDEAEMA and shell of PAEMA
PEG-SS-PAsp(DET)/silica-coated PAsp(DET)/siRNA PIC
Mechanism of cytoplasmic delivery
Ref.
Mitoxantrone
pH-sensitivity and fusogenic ability
[79]
Bleomycin
Fusogenic ability and CPP mediated cytoplasmic delivery pH-sensitivity and fusogenic ability of PHIS pH-dependent membrane fusion property of phage-Doxil
[153]
Paclitaxel Doxorubicin
Streptavidin coated QDs (605-SA-QDs) Luminescent nanocrystals for in vitro bioimaging siRNA against VEGF or luciferase
[154] [155]
[81] Proton-sponge core driven volume expansion (~50-fold) and accompanying increase in zeta potential at pH
ISSN: 1742-5247 (Print) 1744-7593 (Online) Journal homepage: http://www.tandfonline.com/loi/iedd20
Intracellular delivery of nanocarriers and targeting to subcellular organelles Aditi Jhaveri & Vladimir Torchilin PhD DSc To cite this article: Aditi Jhaveri & Vladimir Torchilin PhD DSc (2015): Intracellular delivery of nanocarriers and targeting to subcellular organelles, Expert Opinion on Drug Delivery To link to this article: http://dx.doi.org/10.1517/17425247.2015.1086745
Published online: 11 Sep 2015.
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Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=iedd20 Download by: [Texas A & M International University]
Date: 11 September 2015, At: 06:14
Review
Intracellular delivery of nanocarriers and targeting to subcellular organelles 1.
Introduction: intracellular delivery, significance, and challenges
2.
Nanocarriers for intracellular targeting
Downloaded by [Texas A & M International University] at 06:14 11 September 2015
3.
Approaches for intracellular delivery
4.
Targeting nanocarriers to intracellular organelles
5.
Conclusions and future directions
6.
Expert opinion
Aditi Jhaveri & Vladimir Torchilin† †
Northeastern University, Center for Pharmaceutical Biotechnology and Nanomedicine, Department of Pharmaceutical Sciences, Boston, MA, USA
Introduction: Recent trends in drug delivery indicate a steady increase in the use of targeted therapeutics to enhance the specific delivery of biologically active payloads to diseased tissues while avoiding their off-target effects. However, in most cases, the distribution of therapeutics inside cells and their targeting to intracellular targets still presents a formidable challenge. The main barrier to intracellular delivery is the translocation of therapeutic molecules across the cell membrane, and ultimately through the membrane of their intracellular target organelles. Another prerequisite for an efficient intracellular localization of active molecules is their escape from the endocytic pathway. Areas covered: Pharmaceutical nanocarriers have demonstrated substantial advantages for the delivery of therapeutics and offer elegant platforms for intracellular delivery. They can be engineered with both intracellular and organelle-specific targeting moieties to deliver encapsulated or conjugated cargoes to specific sub-cellular targets. In this review, we discuss important aspects of intracellular drug targeting and delivery with a focus on nanocarriers modified with various ligands to specifically target intracellular organelles. Expert opinion: Intracellular delivery affords selective localization of molecules to their target site, thus maximizing their efficacy and safety. The advent of novel nanocarriers and targeting ligands as well as exploration of alternate routes for the intracellular delivery and targeting has prompted extensive research, and promises an exciting future for this field. Keywords: cell-penetrating peptides, cytoplasm, intracellular delivery, liposomes, macromolecules, mitochondria, nanocarriers, nuclei, polymeric micelles Expert Opin. Drug Deliv. [Early Online]
Introduction: intracellular delivery, significance, and challenges
1.
A number of therapeutics including small molecule drugs as well as biological macromolecules like DNA, siRNA, proteins, enzymes, antibodies, or oligonucleotides for the treatment of various diseases including cancer are known to act on targets localized within the cell. These intracellular drug targets include the cytosol, the nucleus, endosomes, mitochondria, the Golgi complex, lysosomes, and the endoplasmic reticulum (ER) (Figure 1) [1,2]. Drug-loaded nanocarriers also need to be delivered intracellularly to exert their therapeutic action on various intracellular organelles [1]. However, intracellular delivery of therapeutics still poses a significant challenge for drug delivery. Often, it is assumed that delivery of the drug into the cytosol will ensure its delivery to its sub-cellular target by simple diffusion and random interaction with the cell organelles. This is true in some cases, such as for siRNA, whose site of action is the cytoplasm, or for some small molecules that retain 10.1517/17425247.2015.1086745 © 2015 Informa UK, Ltd. ISSN 1742-5247, e-ISSN 1744-7593 All rights reserved: reproduction in whole or in part not permitted
1
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Most drugs, biologics as well as drug-loaded pharmaceutical nanocarriers need to be delivered intracellularly to their target organelles to exert their desired therapeutic effects. Extracellular as well as intracellular barriers need to be surmounted for therapeutics to reach their final destination. Escape from the endocytic pathway comprises a major bottleneck in the intracellular delivery of payloads and can be overcome by suitable modifications of nanocarriers. Size, charge, and the property of longevity of nanocarriers play a critical role in determining their fate in the circulation and eventually their accumulation at target sites within cells. Active targeting strategies that use cell-penetrating peptides (CPPs) as well as other-organelle-specific ligands and simultaneously exploit various stimuli that exist within cells play an important role in achieving the goal of targeted intracellular delivery. A number of nanocarriers are currently being investigated actively for delivery of payloads to the cytoplasm, nuclei, mitochondria, and lysosomes. Targeted delivery of payloads to specific cells and subcellular compartments results in a reduced toxicity and maximizes their therapeutic efficacy.
This box summarizes key points contained in the article.
a high metabolic stability until their eventual interaction with the organelles. In cases where the sites of drug action are organelles within the cytoplasm, achieving intracellular targeting can be challenging. Even if delivery to the cytoplasm is successful, other barriers need to be surmounted before the drug molecules can reach their target organelles [3]. It thus follows that to achieve optimal efficacy and to improve the therapeutic effects of drugs, an understanding of their intracellular disposition patterns is imperative. Furthermore, intracellular delivery can overcome or even reverse the multi-drug resistance phenomenon commonly encountered in anti-cancer chemotherapy, which further adds to its importance for targeted drug delivery [4]. Achievement of the goal of intracellular delivery is not easy, however. A major challenge for therapeutics, especially macromolecules like peptides, proteins, DNA, and siRNA is the barrier to their translocation through the lipophilic cell membranes and then the sub-cellular organelles, unless these payloads are actively transported into the cells [1]. In order to access their intracellular target receptors, therapeutic entities or nanomedicines must usually navigate the endocytic pathways in the cell. Some excellent reviews have described the endocytic mechanisms involved in the trafficking of nanomedicines [5-7]. Macromolecules and nanocarriers typically enter the cell via membrane-bound vesicles called endosomes, formed by the invagination and pinching-off of pieces of the plasma membrane in a process known as endocytosis [8]. 2
Endosomes mature from early endosomes to late endosomes, progressively become more acidic, and may ultimately fuse with the lysosomes. The particles that enter a cell via this endocytic pathway are entrapped in the endosomes and eventually end up in lysosomes where they are degraded via active enzymatic degradation by various lysosomal digestive enzymes. Since only a small fraction of endosomes degrade spontaneously releasing the contents, the majority of endocytosed material is degraded and never makes its way to the cytosol [9]. This results in a restricted delivery of therapeutic agents to their intracellular targets, and precludes them from in vivo applications due to bioavailability concerns, even though they show potential in vitro. Endosomal escape is thus a critical bottleneck for intracellular delivery of molecules [10]. A number of approaches have been investigated to allow an efficient endosomal escape of therapeutics including pH buffering, destabilization of endosomal membrane by fusogenic agents, or photochemical disruption, osmotic swelling to enable bursting of endosomes, and the use of pore-forming peptides [11]. Once nanocarriers escape the endosomal compartment, they can navigate through the cytoplasm to interact and bind various intracellular targets. Both invasive and non-invasive strategies have been developed to bypass the endocytic pathway or to facilitate endosomal escape in an attempt to deliver macromolecules or nanocarriers inside cells. The invasive techniques include electroporation, micro-injection, biolistic transfections (with a hand-held gene gun), sonoporation, and relatively recent techniques which rely on the use of microfluidic device-based electroporation [12-14]. However, many of these cause damage to the cell membrane. A relatively simpler, efficient, and non-invasive option for intracellular delivery is the use of drug-loaded nanocarriers. These are described in detail in the subsequent sections which deal with the properties, role, and modifications of nanocarriers for organelle-specific intracellular delivery and targeting. 2.
Nanocarriers for intracellular targeting
Properties and advantages of nanocarriers A host of nanocarriers have emerged as vehicles of choice for drug delivery, especially for cancer therapy. By virtue of their physico-chemical properties including size, shape, surface charge, and the density of targeting ligands on their surface, nanocarriers impart many desirable features for drug delivery. These include, but are not limited to, avoidance of rapid uptake by the mononuclear phagocyte system (MPS) or immediate renal clearance, increased stability of drugs and an improved pharmacokinetic profile, longer circulation times, tuned release of payloads, modification for active targeting to desired locations without off-target effects, and triggered release of payloads by various internal and external stimuli [15,16]. Some important features of nanocarriers are discussed in Sections 2.2 and 2.3. 2.1
Expert Opin. Drug Deliv. (2015) 13(1)
Intracellular delivery of nanocarriers and targeting to subcellular organelles
Mammalian cell Cargo
Cytoplasm
Clathrin
Caveolin Early endosome Caveosome Clathrin coated vesicle Endosomal escape
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Late endosome
Golgi
Lysosome
Mitochondrion
Nucleus
Endoplasmic reticulum
Figure 1. Target sites for intracellular drug delivery: The cytoplasm, nucleus, mitochondria, and the lysosomes constitute the major sites for intracellularly targeted drug delivery.
Role of size, charge, and steric stabilization of nanocarriers
2.2
Size is a critical parameter that dictates nanocarrier disposition in the body. Uptake by the MPS, tissue extravasation, renal clearance, diffusion into tissues, biodistribution, as well as the mechanism of nanoparticle internalization into cells (phagocytosis, macropinocytosis, clathrin, or caveolaemediated endocytosis) are impacted by the nanocarrier size [17,18]. A disorganized endothelium characterized by large fenestrations coupled with a defective lymphatic drainage in tumors allows the extravasation and retention of macromolecules within the tumor interstitium [19-21]. This phenomenon, termed the enhanced permeability and retention (EPR) effect, constitutes the basis of passive targeting [22]. Nanocarriers in the 10 -- 200 nm size range can optimally take advantage of the EPR effect [23,24]. Larger (> 200 nm) and smaller (5 -- 10 nm) particles are removed by the MPS and through renal clearance, respectively. The surface charge of a nanocarrier can affect its association with cells and can be modified to promote active tumor targeting as well as delivery to the cytosol. The presence of a surface charge influences the opsonization of materials, their recognition by cells of the MPS, as well as their overall plasma circulation profile [18]. Generally, positively charged nanoparticles have a higher rate of cell uptake, greater plasma protein adsorption, higher non-specific internalization rate, and a shorter blood circulation half-life compared to neutral or negatively charged formulations [18].
Imparting longevity to nanocarriers in the circulation diminishes their uptake by the MPS and allows enhanced accumulation at the tumor site via the EPR effect. Long circulating nanocarriers can undergo multiple passages through the target from the systemic circulation, which gives them more time to interact with their target. This greatly enhances retention, as well as intracellular delivery of targeted nanocarriers. Hydrophilic, flexible polymeric coatings such as poly(ethylene glycol) (PEG) and other polymers can be grafted or adsorbed on the nanoparticle surface to make them more hydrophilic or to neutralize the surface charge of the particles, thereby slowing down the process of their opsonization [25,26]. Currently, a majority of the nanocarriers are coated with PEG for biocompatibility and for extended circulation time. However, the hydrophilic surface coating of PEG may hinder intracellular uptake of the carriers by cells and endosomal escape, giving rise to a so-called “PEG dilemma” [27]. Limitations on the effectiveness of the passive targeting approach result from the pathophysiological heterogeneity in tumors, variable vascular permeability, and a weak EPR effect, particularly in the central portion of tumors [19]. Clearly, the simple modification of nanocarriers to circulate for extended periods is inadequate for optimized therapeutic outcomes. To overcome these limitations, and to enhance their binding to specific targets after extravasation, active targeting strategies must be employed.
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3.
Approaches for intracellular delivery
The most commonly employed ligands for intracellular delivery include cell-penetrating peptides (CPPs) and proteins, which can translocate through cell membranes [28], nuclear localization signals (NLS) [29], mitochondrial localization signal [30], ER signal peptide [31], and other non-peptide ligands [32,33]. Among vehicles, pH-sensitive nanocarriers as well as cationic lipids and polymers, along with many other nanosystems, have been investigated for intracellular delivery. This section discusses the major intracellular delivery approaches with a greater focus on CPPs as one of the most widely used agents to promote intracellular delivery. Active targeting of nanocarriers for intracellular delivery using ligands
3.1
Active targeting utilizes various affinity ligands to modify nanocarriers, which enables them to bind molecules that are selectively or differentially expressed on the tumor cells or on the membranes of various intracellular organelles [16]. Still, EPR-mediated passive targeting remains the first step of the active uptake process. These targeting ligands include antibodies and their fragments, peptides, proteins, sugar moieties, aptamers, and small molecules like folate. To ensure delivery of nanocarriers inside cells and subsequently to their subcellular targets, they must first traverse the plasma membrane. Nanocarriers may be internalized directly via receptormediated endocytosis (RME) following their interaction with the receptors on the plasma membrane, or indirectly by associating with the lipid bilayer [34]. RME may be by either clathrin- or caveolae-mediated routes. Molecules may also be internalized via pinocytosis or phagocytosis. However, for nanocarriers, endocytosis is the major route of entry into the intracellular space. The intracellular trafficking pathway of nanocarriers is controlled by the particular endocytic pathway that it follows, which in turn depends on the ligand involved as well as the cell type [34,35]. For example, transferrin (Tf), the iron-binding protein, binds to transferrin receptors and internalizes via clathrin-mediated endocytosis, whereas viruses such as those belonging to the polyomavirus family utilize caveolae-mediated endocytosis as their entry mechanism [36,37]. Nanocarriers accumulate at their initial target (tumor) by passive targeting via the EPR effect or by specific ligandmediated active targeting. However, their subsequent intracellular delivery can be mediated by various internalizable ligands (e.g., folate, transferrin) [16]. Specific examples for various ligand-targeted nanocarriers have been discussed in section 4. Modification of nanocarriers with cellpenetrating peptides for intracellular delivery
3.2
A popular approach for surmounting the cellular barrier and enabling intracellular delivery of various therapeutic and diagnostic molecules is the use of cell-penetrating proteins 4
or peptides to modify them [16,38]. CPPs can penetrate the cell membranes by a phenomenon known as “proteintransduction” and enhance the delivery of the CPP-modified cargoes inside the cell [38]. The CPPs contain domains of less than 20 amino acids, termed protein transduction domains, which are highly rich in basic amino-acid residues [39]. This property of translocation through the plasma membrane has been found for a number of other peptides including transportan [40], VP22, a herpes virus protein [41], model amphipathic peptide (MAP) [42], synthetic polyarginines [33], DNA-binding peptides like human c-Fos (139--164), c-Jun (252--279), and RNA-binding proteins like HIV-1 Rev (34--50), BMV Gag (7--25), and FHV coat proteins (35--49) [43]. CPPs can be classified based either on their origin or their sequence characteristics [44]. On the basis of their physicochemical properties, CPPs may be divided into cationic, amphipathic, and hydrophobic CPPs [45]. Cationic CPPs such as trans-activating transcriptional transactivator (TAT) [46-48], polyarginines [33], and protamine are rich in arginine [49]. Amphipathic CPPs include peptides such as model amphipathic peptide (MAP) and transportan which are rich in lysine and arginine-rich peptides like RW9 and RL9 derived from penetratin [50]. The third class of CPPs includes those peptides that have apolar residues, including prenylated peptides [51], pepducins [52], and the signal sequences from integrin b3 [53]. A variety of mechanisms for CPP accumulation in the cytoplasm have been proposed. However, no single pathway has been accepted as a unifying mechanism for CPP-mediated uptake. This is due to differences in uptake because of peptide properties such as molecule length, their concentration, sequence characteristics, conformation, and charge delocalization, as well as cargo characteristics like size and charge [54,55]. CPPs or CPP-cargo conjugates may enter the cell using one or multiple mechanisms and may localize in different subcellular compartments. CPPs utilize both energy-dependent and energy-independent mechanisms for translocation through cell membranes. CPPs alone, or when attached to small molecules, are usually internalized via electrostatic interaction and hydrogen bonding which bring about their direct transduction through the lipid bilayer and do not require energy [56,57]. CPPs conjugated to large cargoes typically utilize the energy-dependent endocytic process of macropinocytosis, which a key mechanism for their uptake [58]. Other routes of entry include clathrin-mediated endocytosis and lipid raft-mediated caveolae endocytosis [59-61]. For any of these mechanisms of uptake, it is essential that the CPPs interact with the negatively charged cell-surface residues for successful transduction [1]. CPPs can deliver a variety of cargoes including drugs, antibodies, proteins, nucleic acids (DNA, siRNA, oligonucleotides), fluorochromes, quantum dots, nanocarriers, and contrast agents for magnetic resonance imaging (MRI), both in vitro and in vivo [38,62,63].
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Intracellular delivery of nanocarriers and targeting to subcellular organelles
Recently, TATp-modified liposomes (TATp-L) were shown to deliver DNA into dendritic cell (DC) cultures [46]. TATp increased the uptake and internalization of liposomes in DC and enhanced the in vitro transfection efficiency as was evidenced from EGFP and bovine herpes virus type 1 (BoHV-1) glycoprotein D (gD) expression. Although DCs treated with plain-L or TATp-L loaded with pCIgDA (plasmid for eukaryotic expression of BoHV-1) both expressed gD, the transfection with TATp-L resulted in a two-fold higher protein expression than that of plain-L. The de novo synthesized gD was immunologically active only when DCs were transfected with TATp-L, and surpassed the threshold needed to trigger the secretion of the interleukin-6 (IL-6) cytokine [46]. Another CPP, octa-arginine (R8) was used to modify the surface of PEGylated doxorubicin (DOX)-loaded liposomes, commercially available as Doxil or Lipodox using a polyethylene glycol-dioleoyl phosphatidylethanolamine (PEG-DOPE) amphiphilic co-polymer [33,64]. The R8-modified liposomes enhanced the anti-cancer activity, improved intracellular delivery, and endosomal escape of DOX relative to the unmodified liposomes, resulting in a higher DOX delivery to the cytoplasm and its eventual accumulation in the nucleus. The higher accumulation of DOX led to greater apoptosis and cytotoxicity in vitro, and translated into effective tumor growth suppression in BALB/c mice bearing subcutaneous (s.c) 4T1 tumors [64]. Similar results with R8-modified Lipodox were also seen both in vitro in A549 (non-small cell lung cancer) cells and in vivo in A549 tumor xenograft-bearing nude mice [33]. CPPs have also been used for the intracellular delivery of imaging and contrast agents or both simultaneously [63]. Liu et al. reported the synthesis of water-soluble, biocompatible, carboxyl-, and PEG-functionalized indium phosphide (InP)/zinc sulphide (ZnS) quantum dots (QDs), designated QInP, and evaluated their uptake into cells using four CPPs [65]. The CPPs evaluated included synthetic nonaarginine (SR9), R9 modified with polyhistidine (HR9), a CPP composed of nona-arginine and the penetration accelerating peptide sequence (Pas) (PR9), and a CPP composed of an INF7 fusion peptide and nona-arginine (IR9). All the CPPs interacted non-covalently with QInP in vitro to form stable CPP/QInP complexes. These complexes efficiently delivered QInP into human non-small cell lung cancer (A549) cells. Moreover, the complexes were non-toxic when 500 nM of CPP was complexed with QInP at a molecular ratio of 30, and QInP did not cause significant cytotoxicity when the concentrations were £ 1 µM [65]. Table 1 lists additional examples of nanocarriers modified with CPPs for intracellular delivery of drugs or biologicals. CPPs can target diverse cell types in heterogenous patient populations due to their ability to facilitate non-specific and receptor-independent cellular uptake of various carriers, and thereby overcome limitations of many active targeting agents. Moreover, they can guide cellular internalization through less degradative pathways than RME to minimize the breakdown
of labile therapeutic cargoes [66]. However, the non-specificity of CPPs is a major deterrent to their systemic application and hinders their utility to target specific sites in vivo, thus increasing the risk of toxicity and off-target effects. Other limitations of CPPs include their susceptibility to proteolytic cleavage under physiological conditions and charge-mediated blood clearance, both of which result in a shorter duration of action due to rapid renal clearance [45,48]. A number of approaches have been suggested to overcome challenges associated with the systemic administration of CPPs. These typically involve control of the presentation of CPPs only at a target site in vivo. These have been summarized very well in a recent review by MacEwan and Chilkoti [66]. To achieve control over CPP presentation, “smart” or stimuli-responsive systems have been developed based on the physiological or microenvironmental features peculiar of the targeted tissue or cell type [44,66]. The triggers for CPP activation may be intrinsic to the tumor environment (low pH or over-expression of enzymes like proteases) or extrinsic such as local application of heat or light. For the controlled presentation of CPPs, five approaches have been investigated including: i) controlled display of CPPs by removal of “stealth” polymers [48], ii) triggered display of CPPs by actuation of molecular tethers [67], iii) use of ionizable residues to manipulate CPP charge [68], iv) presentation of CPPs by dissociation from ionic inhibitors [69], and (v) modulation of the interfacial density of critical CPP residues by temperature-triggered micelle assembly [66,70].
Cationic molecules Other approaches for promoting intracellular delivery make use of cationic molecules, which interact with the negatively charged plasma membrane of the cells. These include amine containing polymers, cationic lipids (e.g., DC-Cholesterol, DOTAP), dendrimers, and polypeptides. For example, polyethyleneimine (PEI) is widely used for its ability to induce membrane permeability, and allow the nanoparticle entry into cells. It finds applications in gene delivery because it binds and condenses nucleic acids very efficiently. However, cationic molecules can cause concerns about cytotoxicity and poor stability in biological buffers. To circumvent this, they are often modified or shielded with PEG. PEG-modified amines, for example, exhibit stability in biological buffers, penetrate cell membranes, can disrupt endosomes, and are able to access the cytosolic space [34]. The endosomedisrupting ability of amine-containing polymers results from the “proton-sponge effect.” The amine groups of these polymers are able to absorb protons generated by the ATPase, causing osmotic pressure build-up within endosomes. This leads to the destabilization and rupture of endosomes releasing their contents into the cytosol [71,72]. Another mechanism for the membrane permeability by cationic molecules is their ability to form nanoscale holes in the plasma membrane [73]. 3.3
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Table 1. CPPs for intracellular delivery of nanocarriers. CPP (sequence)
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CKRRMKWKK (derived from penetratin) RRRRRRGGRRRRG derived from protamine CGRRMKWKK
Peptide for ocular delivery (POD) CGGG[ARKKAAKA]4 or HIV-Tat (CGGGGYGRKKRRQRRR) R8 (octaarginine) R9 (nonaarginine)
TAT
ACPP (Suc-e8-(Aop)-PLGC(Me)AGr9-c-NH2)
Nanocarrier
Thermo-sensitive liposomes Lipid nanoparticles
Cargo
Method of CPP activation/ exposure
Ref.
Doxorubicin
Mild hyperthermia
[143]
–
[144]
Liposomes
siLuc2 (against luciferase), and siGFP c-myc siRNA
[145]
PLGA nanoparticles (NPs)
Fluorbiprofen
NIR triggered two-photon absorption and cleavage of photoresponsive group from inactive CPP --
Graphene oxide sheets Phenyl boronic acid targeted mesoporous silica NPs Micellar NPs
pEGFP Doxorubicin
-Cleavage of MMP-2 responsive peptide PVGLIGK(Cy5)G
[147] [148]
Plk-1 siRNA
[149]
Dendrigraft poly-L-lysine (DGL-G3) NPs
pEGFP
Shell cross-linked knedellike NPs
Cleavage of MMP-2 responsive peptide PLGLAG Low tumor extracellular pH and MMP-2 triggered activation of CPP –
Peptide nucleic acid for iNOS mRNA and alexa fluor 633 for imaging Doxorubicin Cathepsin B triggered activation of TAT Gadolinium-DOTA and/or MMP 2/9 triggered release of Cy5 the ACPP and cargo
Mesoporous silica coated QD NPs PAMAM generation 5 dendrimer
[146]
[150]
[151]
[152] [63]
Aop: 5-amino-3-oxapentanoyl; ACPP: Activable cell penetrating peptide; C(Me): S-methylcysteine; c-NH2: C-terminal amide; lower case letters: D-amino acids; Suc: Succinyl.
Targeting nanocarriers to intracellular organelles 4.
Agents need to be delivered to specific locations within the cell to elicit their therapeutic effects. For example, genes and anti-sense therapy preparations, siRNA, pro-apoptotic drugs, and lysosomal enzymes need to be delivered to the nucleus, cytoplasm, mitochondria, and the lysosomal compartment, respectively, to exert their therapeutic actions [1]. To ensure the delivery of therapeutics to these intracellular organelles, nanocarriers can be modified with suitable ligands to direct them to specific compartments and avoid chemical modification of the therapeutic molecule itself. This section includes discussion of targeted delivery of therapeutic, diagnostic, or imaging payloads to specific intracellular compartments including the cytosol, nucleus, mitochondria, and lysosomes. Delivery of nanocarriers to cytoplasm A number of therapeutics require delivery into the cytosol either because their receptors are present in the cytosol or because their target is an intracellular organelle accessible through cytosolic transport [74]. Nanocarriers, which deliver a variety of cargoes from small molecules to biologics 4.1
6
(DNA, siRNA, proteins, and antibodies) have been developed and employed for cytosolic delivery. Liposomes are the oldest and by far, the most widely investigated nanocarriers. Various strategies have been employed to utilize liposomes for cytosolic delivery of therapeutics. One of the strategies includes the development of “fusogenic” or pHsensitive liposomes [75,76]. Fusogenic lipids undergo a phase transition under acidic conditions, which facilitates their interaction and fusion or destabilization of the endosomal membranes, resulting in the release of the encapsulated cargo into the cytoplasm [74]. Depending on the mechanism for triggering the pH-sensitivity, various strategies may be utilized for the formulation of pH-sensitive liposomes. Typically, combinations of DOPE with compounds containing an acidic group (e.g., carboxylic groups in oleic acid, methacrylic acid, cholesteryl hemisuccinate (CHEMS), and N-isopropylacrylamide) that can stabilize PE at a neutral pH are utilized [77]. Fusogenic properties may also be imparted to liposomes by the incorporation of synthetic fusogenic peptides or polymers in the lipid bilayers [78,79]. Serum or plasma proteins are known to hamper formulations by destabilizing them, causing the premature leakage of their contents and also reduce their pH-sensitivity, thus rendering them inappropriate as carriers for in vivo delivery to the cytoplasm [79]. Long-circulating,
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Intracellular delivery of nanocarriers and targeting to subcellular organelles
Table 2. Nanocarriers for cytoplasmic delivery of payloads. Nanocarrier Liposomes
Liposomes
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Micelles (mixed micelles) Doxil
Core-shell colloids
Multi-layered polyion complex (PIC)
Composition
Payload(s)
(DPPC/Chol or DPPC/DOPE) liposomes modified with fusogenic polymers PEG-PMMICholC6 or PMMI-co-PDMAEMA DOPE/CHEMS/DOPE-PEG1K liposomes modified with R8 DSPE-PEG2000/DSPE-3400--2C5/ PHIS-PEG2000 DOX-loaded liposomes modified with MCF-7 cell specific chimeric phage fusion coat protein (Phage-Doxil) Proton sponge-based core made of PEGDMA crosslinked PDEAEMA and shell of PAEMA
PEG-SS-PAsp(DET)/silica-coated PAsp(DET)/siRNA PIC
Mechanism of cytoplasmic delivery
Ref.
Mitoxantrone
pH-sensitivity and fusogenic ability
[79]
Bleomycin
Fusogenic ability and CPP mediated cytoplasmic delivery pH-sensitivity and fusogenic ability of PHIS pH-dependent membrane fusion property of phage-Doxil
[153]
Paclitaxel Doxorubicin
Streptavidin coated QDs (605-SA-QDs) Luminescent nanocrystals for in vitro bioimaging siRNA against VEGF or luciferase
[154] [155]
[81] Proton-sponge core driven volume expansion (~50-fold) and accompanying increase in zeta potential at pH
