SEARCH

SEARCH BY CITATION

Keywords:

  • cell penetrating peptides;
  • drug delivery;
  • cellular translocation;
  • endocytosis;
  • tight junctions;
  • cellular differentiation;
  • Rho-GTPases;
  • inflammatory model;
  • review

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. ACCOMPLISHMENTS AND FAILURES. REVISITING THE PERFORMANCE OF CPPs FOR DRUG DELIVERY
  5. CELLULAR BARRIERS LIMITING CPP TRANSLOCATION
  6. NEW PERSPECTIVES FOR APPLICATION OF CPPs IN DRUG DELIVERY
  7. CONCLUSION
  8. REFERENCES

The cell membrane poses a substantial hurdle to the use of pharmacologically active biomacromolecules that are not per se actively translocated into cells. An appealing approach to deliver such molecules involves tethering or complexing them with so-called cell penetrating peptides (CPPs) that are able to cross the plasma membrane of mammalian cells. The CPP approach is currently a major avenue in engineering delivery systems that are hoped to mediate the non-invasive import of problematic cargos into cells. The large number of different cargo molecules that have been efficiently delivered by CPPs ranges from small molecules to proteins and even liposomes and particles. With respect to the involved mechanism(s) there is increasing evidence for endocytosis as a major route of entry. Moreover, in terms of intracellular trafficking, current data argues for the transport to acidic early endosomal compartments with cytosolic release mediated via retrograde delivery through the Golgi apparatus and the endoplasmic reticulum. The focus of this review is to revisit the performance of cell penetrating peptides for drug delivery. To this aim we cover both accomplishments and failures and report on new prospects of the CPP approach. Besides a selection of successful case histories of CPPs we also review the limitations of CPP mediated translocation. In particular, we comment on the impact of (i) metabolic degradation, (ii) the cell line and cellular differentiation state dependent uptake of CPPs, as well as (iii) the regulation of their endocytic traffic by Rho-family GTPases. Further on, we aim at the identification of promising niches for CPP application in drug delivery. In this context, as inspired by current literature, we focus on three principal areas: (i) the delivery of antineoplastic agents, (ii) the delivery of CPPs as antimicrobials, and (iii) the potential of CPPs to target inflammatory tissues. © 2007 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 97:144–162, 2008

INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. ACCOMPLISHMENTS AND FAILURES. REVISITING THE PERFORMANCE OF CPPs FOR DRUG DELIVERY
  5. CELLULAR BARRIERS LIMITING CPP TRANSLOCATION
  6. NEW PERSPECTIVES FOR APPLICATION OF CPPs IN DRUG DELIVERY
  7. CONCLUSION
  8. REFERENCES

The full therapeutic potential of peptide-, protein-, and nucleic acid-based drugs is frequently compromised by their limited ability to cross the plasma membrane of mammalian cells, resulting in poor cellular access and inadequate therapeutic efficacy.1, 2 Today this hurdle represents a major challenge for the biomedical development and commercial success of many biopharmaceuticals. Over the past decade, however, attractive prospects for a substantial improvement in the cellular delivery of such molecules have been announced that were supposed to result from their physical assembly or chemical ligation to so-called cell penetrating peptides (CPPs), also denoted as protein-transduction domains (PTDs). CPPs represent short peptide sequences of 10 to about 30 amino acids which can cross the plasma membrane of mammalian cells and may thus offer unprecedented opportunities for cellular drug delivery. In fact, in a widely recognized landmark study in mice, the intraperitoneal injection of a fusion protein conjugated to Tat(47–57), an oligocationic CPP derived from human immunodeficiency virus (HIV) Tat protein, was found to let the ligated protein, β-galactosidase, overcome numerous biological barriers, distribute into virtually every organ and even pass the blood brain barrier.3 Nevertheless, the biomedical promise of CPPs is still far from clinical implementation. In fact, whereas most of the scientific motivation in the field of the CPPs derives from the appealing perspectives for their biomedical use, much of the actual research is still very much focused on more fundamental aspects as to their biochemical, biophysical, and cell biological assessment. So far, clinically relevant contributions appear to be rare.

To illustrate the biomedical promise of cell penetrating peptides, we revisit the performance of CPPs for drug delivery, cover both accomplishments and failures and report on new prospects. Besides a selection of successful case histories, we also point towards limitations in CPP mediated translocation. Major obstacles to CPP mediated drug delivery consist in (i) their metabolic degradation, (ii) the cell line and cellular differentiation state dependent uptake of CPPs, as well as (iii) the regulation of their endocytic trafficking as affected by selected Rho-family GTPases. In the final section, we aim at the identification of promising niches for CPP application in drug delivery. In this context, being inspired by recent literature, we focus on three principal areas: (i) the delivery of antineoplastic agents, (ii) the delivery of CPPs as antimicrobials, and (iii) the potential of CPPs to target inflammatory tissue.

Selected CPP Families and Cargo Molecules

Most of the currently recognized CPPs are of oligocationic nature and derived from viral, insect or mammalian proteins endowed with membrane translocation properties. One of the first CPPs, reported as early as 1994, was derived from the third helix of the Antennapedia protein homeodomain of Drosophila.4 Today, this peptide is commonly referred to as penetratin and, together with oligopeptides of the Tat family5 and the chimeric peptide transportan,6 one of the most widely studied CPPs. As for the third α-helix (residues 43–58) of the Antennapedia protein homeodomain the minimal sequence responsible for the cellular translocation of the Tat protein has also been identified and is represented by the predominantly cationic residues 47–57.7 In addition, a wealth of further oligocationic CPPs has been revealed and described in the literature.8, 9 Prominent examples are the peptides of the so-called MPG family,10 antimicrobial-derived CPPs,11–15 pVEC,16, 17 and VP22.18–20 Expectedly, by the genomic approach more and more CPP sequences are likely to be uncovered.

Besides oligocationic CPPs, enhanced translocation of the cellular membrane has also been reported for weakly cationic peptides such as, for example, for a family of peptide sequences derived from the C-terminal domain of human calcitonin (hCT).21–23 Our discovery of this CPP class stemmed from the observation that a C-terminal fragment of hCT was subject to endocytosis when exposed to excised nasal epithelium.24 More recently, sweet arrow peptide (SAP), a linear trimer of moderately cationic, repetitive VRLPPP domains, and conceived as an amphipathic version of a polyproline sequence related to γ-zein, a storage protein of maize, has been identified as CPP.25–27 A selection of names, origins and sequences of representative CPP families is shown in Table 1.

Table 1. Selection of Representative CPP Families, Model Cargoes and Related References
NameOriginSequenceReferencesCargo/Reference
Tat family
 Tat (48-60)HIV-1 proteinGRKKRRQRRRPPQQ3, 52–55β-galactosidase3
 OligoarginineTat derivativeRn56–59Cyclosporin A60
Penetratin family
 pAntpAntennapedia homeodomainRQIKIWFQNRRMKWKK4, 61–63Polo-box30
 pIslIsl-1 homeodomainRVIRVWFQNKRCKDKK64–66Biotin/avidin65
Chimeric CPPs
 TransportanGalanin-mastoparanGWTLNSAGYLLGKINLKALAALAKKIL6, 67GalR-1 (antisense)68
 MPG peptides  10, 69siRNA
  Pβgp41-SV40GALFLGFLGAAGSTMGAWSQPKKKRKV69, 70Fluorophore
  Pαgp41-SV40GALFLAFLAAALSLMGLWSQPKKKRKV71, 72Fluorophore
Pep-1Trp-rich motif-SV40KETWWETWWTEWSQPKKKRKV72, 73Proteins73
Antimicrobial-derived CPPs
 Buforin 2Toad stomachTRSSRAGLQWPVGRVHRLLRK11, 74GFP74
 Bac715-24Bactenecin familyPRPLPFPRPG12, 14NeutrAvidin75
 SynB(1)Protegrin 1RGGRLSYSRRRFSTSTGR15, 76Doxorubicin76
Other CPPs
 pVECMurine VE-cadherinLLIILRRRIRKQAHAHSK17, 77Fluorophore17
 VP22Viral protein (HSV-1)DAATATRGRSAASRPTE−18, 19GFP18
  −RPRAPARSASRPRRPVE  
hCT-derived peptides  78 
 hCT(9-32)Human calcitoninLGTYTQDFNKFHTFPQTAIGVGAP22–24,79GFP22
 hCT(9-32)-brHuman calcitonin, SV40LGTYTQDFNKFHTFPQTAIGVGAP21, 80GFP21
  AFGVGPDEVKRKKKP-NH2  
SAPModified maizeVRLPPP-VRLPPP-VRLPPP25–27Fluorophore26
ProtaminSalmon roeMixture of protamin 1–481Fluorophore81

The large number of cargo molecules that have been efficiently delivered by the CPP approach includes biologics like peptides,28–30 proteins,31–34 antisense oligonucleotides,35–38 siRNA,39, 40 plasmid DNA,41–44 as well as model drugs.45, 46 Even particulate systems such as liposomes,47 and microparticles48–50 could be delivered. A more detailed overview over various cargos delivered by CPPs was recently presented by Jarver and Langel.51 A representative selection of model cargoes and related references is part of Table 1.

Mechanisms of Internalization

The exact mechanisms underlying the internalization of CPPs across the cellular membrane still await complete understanding. During the pioneering phase, the cellular translocation of penetratin and Tat peptides was frequently (but not exclusively) assigned to a passive, temperature-independent process4, 5, 57 that appeared to be insensitive to endocytosis inhibitors.5, 61 These observations were thought to be consistent with various theoretical models for a CPP-induced physical perturbation of the lipid membrane leading to a direct translocation of the plasma membrane into the cytosol without prior compartmentalization into subcellular vesicles.4, 5, 61 More recently, however, the related hypotheses involving direct translocation have been widely challenged following several reports on artifactual results that were caused, for example, by cell fixation protocols prior to confocal laser scanning microscopy (CLSM) of cells incubated with fluorescence labeled CPPs. Another source of misinterpretation was found to derive from experimental difficulties to distinguish cell surface-associated CPP from CPP internalized in cytoplasmic compartments.82 Unequivocal discrimination between associated and internalized fluorescence, however, is a prerequisite to interpret both CLSM and fluorescence-activated cell sorting (FACS) analyses with fluorescence labeled CPPs.80 In contrast to initial reports, more recent work demonstrated clearly an involvement of endocytosis in the internalization of a Tat-derived peptide82 and penetratin.83 Prior to endocytosis, both peptides were shown to first interact electrostatically with the extracellular matrix (ECM) of the cell surface through binding to negatively charged glycosaminoglycans.84 In a yet early stage, we proposed an endocytic mechanism for a hCT derived CPP24 possibly owing to a β-sheet induced aggregation of the peptide on the cell surface. In fact, to date endocytic uptake is the accepted pathway for many of the currently known CPPs, though still with some exceptions.61, 72, 85, 86 Endocytosis may involve several pathways.80 So far, several classical studies focused on uptake via clathrin-coated pits.87, 88 Meanwhile, however, technologies and reagents are available that can cast light on non-classical, clathrin-independent pathways such as macropinocytosis and endocytosis via lipid rafts.89–93 A number of excellent reviews report on pathways of cellular CPP internalisation.9, 92, 94 Below we present an updated overview of the field.

Intracellular Trafficking Routes of CPPs

Very little information is available regarding the downstream fate of CPPs after endocytic capture at the plasma membrane. Thus, towards future therapeutic implementation, the mechanisms of CPP internalization and the pursued intracellular trafficking routes need to be studied in more detail. Increasing evidence for endocytic uptake mechanisms of CPPs caused concerns that the vectors might be trapped in endosomes, rendering them unable to release the therapeutic cargo. Wadia et al.93 suggested the rate-limiting step of drug delivery by CPPs to be the escape from the macropinosomal vesicle. Having entered cells by lipid raft macropinocytosis, most of the studied Tat(48–57)-Cre CPP remained trapped in macropinosomes, even after 24 h, indicating that escape from macropinosomes is an inefficient process. Chloroquine, an ion-transporting ATPase inhibitor that disrupts endosomes by preventing their acidification, enhanced Tat-Cre release from macropinosomes but was associated with extremely high cytotoxicity. To circumvent these deficiencies, the authors developed a transducible, pH-sensitive fusogenic dTat-HA2 peptide that markedly enhanced the release of Tat-Cre from macropinosomes.93 HA2 is a well-characterized, pH sensitive fusogenic peptide that destabilized lipid membranes at low pH.93 Zaro et al.85, 86 recently presented experimental evidence that in HeLa cells membrane transduction of oligoarginine occurs separately from macropinocytosis. Their experimental method was said to allow for separate measurements of cytosolic versus vesicular uptake.

Fischer et al.95 suggested that CPPs do not necessarily end up for good in endosomes but, after a certain endosomal sojourn, move on to other organelles within the cell. Their data points towards a shift into acidic early endosomal compartments followed by a retrograde delivery through the Golgi apparatus and the endoplasmic reticulum, and a final release into the cytosol (Fig. 1). The authors also reported about similarities of the oligocationic CPPs Tat and penetratin, respectively, with toxins that were known to be transported by means of the retrograde pathway.95 These toxins, such as ricin and Shigella toxin, reached the cytosol of eukaryotic cells after binding to the cell surface, endocytosis by different mechanisms and retrograde transport to the Golgi apparatus and the endoplasmic reticulum.96–98

thumbnail image

Figure 1. Scheme of the potential retrograde pathway of CPPs as reported by Fischer et al.95

Download figure to PowerPoint

To improve insight into the intracellular trafficking pathways of two recently developed CPPs, SAP, and hCT(9–32)-br, a branched hCT derived CPP, we performed CLSM based colocalization studies with the early endosomal antigen 1 (EEA1), a protein associated with early endosomes.80 The resulting overlays revealed that early endosomes were involved in the uptake and initial intracellular trafficking of the two oligocationic peptides. However, after an incubation time of 3 h, most of the internalized CF-SAP or CF-hCT-(9–32)-br were no longer present in early endosomes but moved to other compartments within the cytosol.80 When we tested whether the peptides may have entered the endoplasmic reticulum or the Golgi network, none of the related experiments yielded unequivocal results, neither in favor nor against the hypothesis.80 More recently, Jones et al.99 presented direct evidence for the localization of Tat and octaarginine (R8), after endocytic uptake, in late endosomes and lysosomes. In two leukemia cell lines with different endocytic profiles, and in epithelial cells, a significant fraction of internalized Tat and R8 entered lysosomal compartments within 60 min of internalization. No effects on the cellular distribution of the peptides with agents that disrupt the morphology of the Golgi and the endoplasmic reticulum could be observed. These findings shift evidence from the retrograde pathway of CPPs involving cytosolic release, to their entry into lysosomal compartments which is likely to be followed by CPP degradation. Obviously, this would negatively affect the integrity of the CPPs and, most likely, could compromise the delivery of their cargoes to the respective intracellular targets.

ACCOMPLISHMENTS AND FAILURES. REVISITING THE PERFORMANCE OF CPPs FOR DRUG DELIVERY

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. ACCOMPLISHMENTS AND FAILURES. REVISITING THE PERFORMANCE OF CPPs FOR DRUG DELIVERY
  5. CELLULAR BARRIERS LIMITING CPP TRANSLOCATION
  6. NEW PERSPECTIVES FOR APPLICATION OF CPPs IN DRUG DELIVERY
  7. CONCLUSION
  8. REFERENCES

Successful Delivery by the CPP Approach

The following section covers selected examples of successful drug (or model drug) delivery by the CPP approach. In particular, we focus on distinct in vivo studies, whereas a broader outline is given in the review of Dietz and Bdeltahr.8 A pioneering piece of work on CPP mediated cellular delivery of heterologous proteins was authored by Fawell et al.53 The authors covalently linked Tat peptides to β-galactosidase, horseradish peroxidase, RNase A, and domain III of Pseudomonas exotoxin A. Cellular uptake was monitored colorimetrically or by the level of cytotoxicity. The Tat chimeras were effective on all cell types tested, and stainings showed uptake into virtually every cell in each experiment. In mice, treatment with Tat-β-galactosidase chimeras resulted in delivery to various tissues, with high levels in heart, liver and spleen, low-to-moderate levels in lung and skeletal muscle, and little or no activity in the kidney and in the brain,53 the latter two observations being in some contrast to the data of Schwarze et al.3

In rats, constructs of CPPs with antisense peptide nucleic acid (PNA) have been shown to regulate galanin receptor levels and modify pain transmission.38 Intrathecal administration of a CPP-PNA construct, consisting of penetratin coupled to an antisense PNA targeting the galanin receptor, was shown to modify galanin-mediated pain and reduced the levels of 125I-galanin binding in the dorsal horn of the spinal cord, although the presence of the penetratin-PNA construct within the cell was not demonstrated.38

Another in vivo study demonstrated the cellular delivery of the caveolin-1 scaffolding domain, resulting in the inhibition of nitric oxide synthesis and the reduction of inflammation.100 A chimeric peptide consisting of the caveolin-1 scaffolding domain peptide attached to penetratin was shown to be successfully taken up into blood vessel endothelial tissue from isolated mouse aorta rings, resulting in an inhibition of acetylcholine-induced vasodilatation and nitric oxide production.100 When administered systemically in mice, the chimeric peptide showed suppression of acute inflammation and a vascular leak. The suppression was as efficient as glucocorticoid or endothelial nitric oxide synthetase inhibitors.100

New advances in the transport of doxorubicin through the blood-brain barrier (BBB) by a CPP mediated strategy were shown by Rousselle et al.13 The capacity of doxorubicin to cross the BBB was studied using an in situ rat brain perfusion technique and also by iv injection in mice. When doxorubicin was coupled to either D-penetratin or SynB1, its uptake was increased by a factor of 6, as compared to free doxorubicin. Moreover, using a capillary depletion method, coupling of doxorubicin to either one of the two CPPs led to a 20-fold increase in the amount of doxorubicin transported into brain parenchyma, demonstrating that the two CPPs enhance the delivery of doxorubicin across the BBB.13

Advantages and versatility of protein transduction over viral transgene delivery were demonstrated by Van Der Noen et al. when they compared the in vivo transduction of Tat-β-galactosidase in rat salivary gland cells with retroviral gene delivery.101 The authors found that in contrast to viral transduction, having a limited capacity to infect non-dividing cells, all cell types were susceptible to Tat-mediated protein transduction. Moreover, the authors were able to achieve equal cellular concentrations of Tat-β-galactosidase in 100% of the cells whereas with viral delivery a transduction efficiency of only 30–50% was achieved.101

More recently, Cao et al.102 created a Bcl-xL fusion protein, denoted as PTD-HA-Bcl-xL, containing the protein transduction domain derived from the human immunodeficiency TAT protein. Bcl-xL is a well-characterized anti-apoptotic protein that may enhance cell survival. The Bcl-xL fusion protein was shown to be highly efficient in transducing primary neurons in cultures and potently inhibited staurosporin-induced neuronal apoptosis. Furthermore, intraperitoneal injection of PTD-HA-Bcl-xL into mice resulted in robust protein transduction in neurons of various brain regions and decreased cerebral infarction in a dose-dependent manner.102

Finally, in a highly recognized study in mice, Schwarze et al.3 demonstrated the intraperitoneal delivery of a fusion protein, Tat(47–57)-β-galactosidase (Tat-β-gal), to result in an uptake into almost every organ or tissue, as detected by significant local β-gal activities. Although substantially less intense as compared to organs close to the peritoneum, even the blood-brain barrier could be overcome showing β-gal activity in sagittal brain sections of the mice. Importantly, Tat-β-gal could not disrupt the blood-brain barrier as found after a treatment with protamine as positive control. As late as in 2000, Schwarze et al.55 claimed that such protein-transduction domains (PTDs) can efficiently deliver most protein cargoes into virtually the entire organism.

Limitations of CPP Mediated Translocation

Nevertheless, in contrast to the promise of these reports, the literature also provides evidence that the hypothesis of CPPs as unrestricted delivery tools should be challenged. In fact, the alleged universality of CPPs as a delivery tool has recently come under scrutiny. For instance, Koppelhus et al.103 investigated the translocation of Tat and penetratin in five different cell cultures, namely HeLa (cervical carcinoma), SK-BR-3 (breast carcinoma), IMR-90 (fetal lung fibroblast), H9 (lymphoid) and U937 (monocytic) cells. The two CPPs demonstrated either poor or no uptake in the investigated five cell lines. This observation was explained to potentially result from either a very low CPP translocation efficiency, or from quick cleavage of fluorescence labeled CPPs in the cells. Moreover, the authors demonstrated that Tat and penetratin showed distinct differences in their uptake patterns relative to the tested cell line. Therefore, they concluded that the internalization of the CPPs could be limited to certain cell types and depend on cell-specific membrane components or lipid composition.103 Likewise, CLSM studies by Kramer and Wunderli-Allenspach104 showed that Tat(44–57) carrying a fluorescent tag, Tat(44–57)-fluorescein, did not enter Madine Darby canine kidney cells (MDCK) with intact plasma membranes but accumulated at their basal side. Only cells permeable for ethidium homodimer-1 (EthD-1), a marker for plasma membrane impairment, showed uptake and intracellular accumulation of Tat(44–57). A similar phenomenon was found by Violini et al.105 The authors demonstrated a complete lack of intracellular accumulation of fluorescein-conjugated Tat(48–57) peptide in confluent, tight junction (TJ) forming epithelial MDCK and CaCo-2 monolayers. However, membranes of epithelial cells that are known not to form TJs such as HeLa cells could be efficiently translocated. Previous studies in our group revealed the efficiency of CPP uptake to depend strongly on the investigated cell line.23 Fully polarized and organized MDCK cells that efficiently mimic epithelial barriers demonstrated only slight translocation of linear human calcitonin derived peptides, Tat(47–57) and penetratin(43–58) as compared to more “leaky” HeLa cells.23

To evaluate the efficiency of CPP mediated delivery into the cytosol, Falnes et al.106 fused Tat peptide to the diphtheria toxin A-fragment (dtA), an extremely potent inhibitor of protein synthesis. The Tat–dtA fusion protein was found to avidly bind to the cell surface but failed to show detectable cytotoxicity of the toxin which would exclude uptake. Equally, fusion proteins of Tat basic domain and VP22 as CPPs and diphtheria toxin A-fragment (dtA) showed association with cells in the absence of a cytotoxic effect, indicating inefficient transport of dtA into cells by Tat or VP22.106 The authors used a much lower concentration as commonly applied for the cellular delivery of biologically active molecules by CPPs and argued that, since relatively high CPP concentrations were required to elicit biological effects, CPPs may not yet represent sufficiently established and efficient vehicles for the intracellular delivery of macromolecules. Also, the authors questioned relevant intrinsic membrane penetrating activities of CPPs and claimed that while CPPs may elicit massive association with the cell surface, possibly through interaction with cell-surface heparans, only a very small fraction of the cell-associated molecules may be able to cross the cell membrane. Poor efficiency of Tat-mediated cell transduction has also been reported by other groups. Zhang et al.,107 for example, recently demonstrated that a retro-inverso form of Tat(49–57) CPP, denoted as RI-Tat-9, did not enter confluent MDCK and Caco-2 cells but only translocated into non-epithelial T lymphatic MT2 and HeLa cells. Therefore, a cell type-specific barrier was suggested to control the uptake of RI-Tat-9 by HeLa, MT2, MDCK, and Caco-2 cells. Furthermore, the authors hypothesized that oral and dermal delivery by the used CPPs would be inefficient owing to the observed low permeability of epithelia and the repulsion of free RI-Tat-9 molecules by molecules bound at the monolayer surface.107

Several studies reported on the limited in vivo applicability of the CPP approach. Caron et al.108 showed that the direct delivery of a Tat-eGFP fusion protein to muscle tissue, using subcutaneous or intra-arterial injections, led to only few positive fibers in the muscle periphery or surrounding the blood vessels. Muscles injected with Tat-eGFP showed intense labeling of the ECM, suggesting that Tat binds to components of the ECM surrounding myofibers which could interfere with the intracellular transduction process.108

Further evidence for a permeability barrier was found with radiolabeled [99mTc]Tat peptide that was directly instilled into the urinary bladder of rats but failed to show any distribution into the body.105 In fact, Violini et al.105 demonstrated that [99mTc]Tat was retained by the epithelium of the bladder, an uroepithelial tissue of similar embryonic origin as MDCK cells. Furthermore, Niesner et al.109 showed efficient in vitro translocation of D- or L-amino acid Tat(49–57) into target cells when conjugated to fluorophores and/or antibody fragments, suggesting receptor-independent cell entry mechanisms. However, a concomitant in vivo study in mice revealed that the conjugation of an engineered single chain antibody fragment specific for the ED-B domain of fibronectin, a marker typical for tumor neovasculature, with the Tat peptides resulted in a severely reduced tumor targeting performance as compared to the unconjugated antibody. Apparently (but not surprisingly), by conjugation to Tat, the antibody fragment completely lost its intrinsic targeting capacity, caused by a indiscriminate binding of the cationic peptide domain to the negatively charged extracellular matrix of the endothelial cells lining the vasculature. The authors, therefore, suggested CPPs to lack an in vitroin vivo correlation for drug delivery.109 Leifert et al.110 came to similar results and concluded from an in vivo study with Tat(47–57) and VP22 in mice or in live MC57 cells, respectively, that these peptides would neither enhance translocation into cells nor exhibit enhanced immunogenicity.

Xia et al.111 tested how the Tat(47–57) motif affected uptake and biodistribution of the lysosomal enzyme β-glucuronidase when expressed from recombinant viral vectors. The continuous in vivo production of Tat-β-glucuronidase from adenovirally transduced hepatocytes only led to a moderately increased uptake of the enzyme in certain tissues.111 Lee and Pardridge112 examined pharmacokinetics and organ uptake of Tat(48–58) and Tat-protein conjugates in rats. They analyzed the effects that the Tat peptide exerted on the plasma AUC of the model protein streptavidin as well as the extent to which changes in the plasma AUC influence its uptake into other organs. The study revealed rapid clearance of the Tat-biotin conjugate in rats and no enhancement of bioavailability of the protein when bound to the conjugate.

Tseng et al.113 investigated the cellular translocation of liposomes by penetratin and Tat(47–60). The authors demonstrated that both Tat and penetratin enhanced the translocation efficiency of liposomes in proportion to the number of CPP units ligated to the liposomal surface. However, the improvement in the uptake of liposomal doxorubicin was not reflected by increased cytotoxicity in vitro or tumor control in vivo, demonstrating that merely adding CPPs to liposomes loaded with a cytotoxic drug was inadequate to improve its antitumor activity.

Further evidence for boundaries to the CPP approach was provided by several in vivo studies showing that Tat conjugates did not change the common distribution pattern of macromolecular cargoes that was heavily skewed towards the organs of the mononuclear phagocytic system (MPS) or the organs of waste elimination.53, 112, 114 It needs to be pointed out that these reports do not dispute the cellular entry of CPPs per se or the validity of CPP-assisted cargo delivery that is based on the cargo's functionality. More likely, differences in the experimental settings including the use of in vitro or in vivo experiments, the used cell lines, cellular differentiation states, enzymatic activity and metabolic stability of the CPPs and the type of CPPs itself may be of great importance for the efficiencies of CPPs in translocation studies. In other words, the concept of CPPs as a universal tool in drug delivery needs to be abandoned.

CELLULAR BARRIERS LIMITING CPP TRANSLOCATION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. ACCOMPLISHMENTS AND FAILURES. REVISITING THE PERFORMANCE OF CPPs FOR DRUG DELIVERY
  5. CELLULAR BARRIERS LIMITING CPP TRANSLOCATION
  6. NEW PERSPECTIVES FOR APPLICATION OF CPPs IN DRUG DELIVERY
  7. CONCLUSION
  8. REFERENCES

Metabolic Degradation

A major obstacle to CPP mediated drug delivery is thought to consist in the often rapid metabolic clearance of the peptides when in contact or passing the enzymatic barriers of epithelia and endothelia. Koppelhus et al.,103 for example, reported that the observed poor intracellular uptake of CPPs might results from quick degradation of the fluorescence labeled CPPs in the cells. Until today, however, despite its general relevance, information on the momentous subject of enzymatic stability and degradation of CPPs is rare and only few studies have so far investigated the cellular metabolism of CPPs. Elmquist et al.17 studied the metabolic degradation of pVEC in murine brain endothelial cell lysate. pVEC was found to be rapidly degraded when incubated with this model. By contrast, when replacing all L-amino acid residues of its sequence by their non-natural D-counterparts, pVEC was no longer subject to any metabolic degradation.16 Another related study focused on the metabolic stability of transportan, a transportan analogue, and penetratin when in contact with a Caco-2 human colon cancer cell line. The stability of the peptides was shown to be in the order of transportan > TP10 > penetratin.115 At least ten degradation products were found for both transporter and TP10, and the identified cleavage products were shown not to penetrate cell membranes.116 In a previous study we analyzed the metabolic degradation kinetics and the cleavage patterns of selected CPPs, namely human calcitonin (hCT) derived peptides, Tat(47–57) and penetratin(43–58) by incubation in three epithelial models, MDCK, Calu-3, and TR 146 cell layers.117 The proteolytic activities among the different epithelial models and the CPPs were highly variable, whereas the individual patterns between the three models for metabolic degradation of each peptide were quite similar or even congruent.117 The stability of Tat was superior to that of hCT-derived CPPs, such as hCT(9–32), and penetratin. We further found that the metabolic stability of hCT-derived CPPs could be largely increased—possibly by steric hindrance of endopeptidases in the mid section of the peptide—through conjugation of the oligocationic simian virus 40 (SV40) large T antigen to the side group of the Lys in position 18, resulting in a novel branched hCT-derived CPP, hCT(9–32)-br.21, 80, 118 In the same work we envisaged a potential relation between the metabolic stability of the investigated CPPs and their translocation capacity.118 To this end we focused on (i) metabolic degradation kinetics and cleavage patterns, and on the (ii) cellular uptake of four oligocationic CPPs, namely SAP, hCT(9–32)-br, [Pα] and [Pβ], when in contact with three epithelial cell cultures, namely subconfluent HeLa, confluent MDCK and confluent Calu-3 cells. SAP and hCT(9–32)-br demonstrated marked metabolic stability under all experimental conditions. In contrast, [Pα] and [Pβ] showed drastic enzymatic degradation, and decomposed quickly even when incubated in serum free medium. Moreover, we identified the specific cleavage sites for each of the four peptides. Emphasizing the relevance of the chosen cellular models, we found the uptake efficiencies of all four CPPs to be strongly cell line dependent. No direct relation between metabolic stability and translocation efficiency was observed, indicating that metabolic stability is no prerequisite for efficient cellular translocation. Nevertheless, for individual CPPs structural modifications that improve metabolic stability are expected to contribute to enhanced translocation. Holm et al.119 reported about uptake and metabolic stability of CPPs in two yeast species, Saccharomyces cervisiae and Candida albicans. The intracellular degradation of the three investigated CPPs, namely pVEC, penetratin and (KFF)3K, varied from complete stability to complete degradation. Moreover, the authors showed that intracellular degradation into membrane impermeable products could significantly contribute to the fluorescence signal. pVEC displayed the highest internalizing capacity. Considering its stability in both yeast species, it was considered as an attractive candidate for further studies.

In conclusion, metabolic stability of CPPs is an important biopharmaceutical factor for their cellular bioavailability. For the development of optimized CPP sequences, a balance between two features is required: On the one hand, for successful therapeutic application of CPPs, CPPs need to be stable enough to carry their cargo to the target before they are metabolically cleaved. On the other hand, in the interest of therapeutic efficacy, CPPs ligated to a therapeutic agent must be cleaved off the conjugate. Subsequently, CPPs need to be cleared from the tissue before they can accumulate and reach toxic levels.

Cell Line and Differentiation State Dependent Uptake

Increasing numbers of contributions to the field report about a cell line and differentiation state dependence of the translocation efficiency of CPPs.23, 79, 105, 107, 120 In particular, massive differences in CPP translocation between “leaky” and “non-leaky” cell culture models were demonstrated.23, 79, 105, 107, 120 “Leaky” cell culture models, such as HeLa cells, lack the ability to form TJs and, therefore, grow to quite leaky and highly permeable monolayers.107 On the other hand, when grown to “non-leaky” epithelial type cell cultures, as represented by confluent MDCK monolayers, cells are connected by junctional complexes encircling the apex of each cell. TJs constitute the most apical element of the junctional complex which also includes adherens junctions, desmosomes and gap junctions. TJs form an efficient barrier to the paracellular diffusion of molecules from the lumen to the tissue parenchyma (gate function) and restrict the diffusion and exchange of lipids and proteins between the apical and basolateral domains of the plasma membrane (fence function).121–125

The relevance of TJ formation for the translocation of CPPs has been reported on several occasions. For instance, Violini et al.105 found a plasma membrane-mediated permeation barrier to Tat(48–57) related cationic peptides in selected well-differentiated epithelial cells. L- and D-stereoisomers of Tat(48–57) peptide conjugates labelled with 99mTc were quantitatively analyzed in confluent monolayers of MDCK renal epithelia and CaCo-2 colonic carcinoma cells forming TJs, and compared to HeLa and KB 3-1 cells repesenting epithelial cell lines that do not form TJs in monolayer culture. In confluent MDCK and CaCo-2 monolayers, the transepithelial permeability of the investigated vectors was comparable to that of inulin as a macromolecular marker of very low paracellular permeability, but much less than that of propanolol, a highly permeable marker compound for transcellular permeability. Additionally, confluent MDCK and CaCo-2 cells showed a complete lack of intracellular accumulation of fluorescein-conjugated Tat peptide. By contrast, in “leaky” HeLa and KB 3-1 cell cultures, baseline cytoplasmic and nucleolar accumulation was readily observed. These data suggest a cell-type specific, tight junction-dependent barrier for the investigated CPPs.

Similar findings were obtained in a prior study of our group.23 Using linear human calcitonin derived peptides, Tat(47–57) and penetratin(43–58), we found the efficiency of CPP uptake to depend strongly on the investigated cell line. For all peptides, HeLa cells demonstrated a greater uptake potential as compared to confluent MDCK cells.23 Further evidence of a cell type-specific barrier was recently given by the study of Zhang et al.107 showing the uptake of the retro-inverso form of Tat(49–57), namely RI-Tat-9, to depend on the cell-type specific barrier properties of HeLa, MT2, MDCK and Caco-2 cell cultures. Again, significantly limited CPP uptake was observed in cell cultures with TJs.

In another study we investigated in detail the impact of cellular differentiation upon efficiency and mechanism of the translocation of selected CPPs.120 We observed that progressive cellular differentiation of the MDCK cells, such as the formation of TJs, correlated well with an endocytic slow-down and a marked drop in the cellular uptake of the CPPs. The observed endocytic slow-down was not specific for the investigated CPPs, namely SAP and hCT(9–32)-br. Instead, its mechanism was compound unspecific as it was generally observed for several markers of endocytosis. Both CPPs were readily taken up by HeLa cells through lipid raft-mediated endocytosis followed by endosomal escape.80 However, for the translocation of SAP and hCT(9–32)-br in proliferating MDCK cells, we observed the involvement of both lipid raft-mediated as well as clathrin-dependent endocytosis. After reaching confluence, translocation of the investigated CPPs into MDCK monolayers dropped markedly. In fact, when confluent, MDCK monolayers widely lost their capacity for translocation via lipid rafts. The large differences in the translocation efficiencies of the CPPs between “leaky” HeLa and “non-leaky” MDCK cell cultures on the one hand, as well as between “leaky” MDCK cells shortly after seeding, and “non-leaky,” well-differentiated MDCK monolayers on the other, demonstrate the crucial impact of confluence upon CPP uptake. Epithelial type cell cultures, such as confluent MDCK cells, feature important elements of polarized epithelia, and represent a more stringent and realistic model to test the potential for therapeutic applications of CPPs as compared to “leaky” cell cultures without tight junctional complexes such as HeLa, KB 3-1, Bowes Human Melanoma or MC57 fibrosarcoma cells.95, 105, 126–128

Regulation of Endocytic Activity by Rho-GTPases

Within the context of increasing evidence for endocytosis as an underlying mechanism for the cellular translocation of many CPPs, a more detailed understanding of the cell biology involved in this process is valuable. As documented in literature, members of the Rho-family of small GTPases are intimately involved in the regulation of endocytic activity.129–131 Rho-GTPases are ubiquitously expressed across eukaryotes where they act as molecular switches, cycling between an active GTP-bound state and an inactive GDP-bound state.130, 132, 133 Rho GTPases are activated in response to extracellular cues, allowing the potential for dynamic regulation of membrane-trafficking processes in response to the extracellular environment.134 Activation enables Rho GTPases to interact with a multitude of effectors that relay upstream signals to cytoskeletal and other components, eliciting rearrangements of the actin cytoskeleton and diverse other responses. Rho GTPases regulate a variety of cellular events such as actin polymerization, cell morphology and polarity, cell growth control, transcription, and membrane trafficking events such as endocytosis.130, 131, 133, 135–138 Apparently, Rho-GTPases mediate the signaling interface between endocytic traffic and actin cytoskeleton as increasing connections between endocytic traffic and the actin cytoskeleton are revealed.139 In Swiss 3T3 fibroblasts, RhoA controls the assembly of actin stress fibers and focal adhesion, Rac1 promotes the formation of lamellipodia and Cdc42 regulates filopodium formation.138, 140, 141 Cross-talk between Rho proteins has been observed; in particular, Cdc42 is a strong activator of Rac, such that filopodial extensions are usually seen associated with lamellipodial protrusions.138, 141, 142 On the other hand, opposing activites of Rho versus Rac and Cdc42 GTPases on several cellular functions, such as actin reorganization and endocytosis could be observed.130, 143, 144

In confluent MDCK cells, RhoA activation has been found to stimulate apical and basolateral endocytosis whereas Rac1 activation led to a drop in endocytosis.143, 144 Moreover, confluent MDCK cells have been found to feature elevated Rac1 and Cdc42 activity but decreased RhoA activity compared to subconfluent cultures.145 During the formation of epithelia, Cdc42 and Rac-1 seem to have a prominent role in the generation of intimate cell-to-cell contact.146, 147 Overexpression of constitutively active Rac1 or Cdc42 increased E-cadherin localization and actin accumulation at cell–cell junctions, whereas these events were inhibited by dominant negative mutants of Rac1 and Cdc42.146–149 Kazmierczak et al.150, 151 suggested that incompletely polarized MDCK cells possess a pathway for the internalization of Pseudomonas aeruginosa which is then downregulated during the acquisition of polarity. This pathway is sensitive to actin depolymerising agents and requires activity of a Rho-A small GTPase but not of Rac-1 or Cdc42. Incompletely polarized MDCK cell monolayers (day 1) efficiently internalized apically applied P. aeruginosa via a pathway that required actin polymerization and activation of Rho-family GTPases, and was accompanied by an increase in activated RhoA. In contrast, the entry of P. aeruginosa into highly polarized MDCK monolayers (day 3) was 10- to 100-fold less efficient and insensitive to inhibitors of actin polymerization or of Rho-family activation. RhoA was not activated. Instead, Cdc42-GTP levels increased significantly.150, 151

With respect to the cellular uptake of selected CPPs, recent findings in our group indicate an involvement of Rho-family GTPases in the differentiation restricted regulation of endocytic traffic.120 In fact, the rates of CPP endocytosis are likely to be related with active form Rho-A, whereas active form Rac-1 relates with endocytic slow-down, cell density and cellular differentiation. The results reflect the above mentioned inverse roles of Rho-A and Rac-1 in the regulation of endocytosis. To our knowledge, this is the first study to cast light on the cell line and differentiation restricted translocation of CPPs into epithelial cell models and its underlying cellular mechanism.

NEW PERSPECTIVES FOR APPLICATION OF CPPs IN DRUG DELIVERY

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. ACCOMPLISHMENTS AND FAILURES. REVISITING THE PERFORMANCE OF CPPs FOR DRUG DELIVERY
  5. CELLULAR BARRIERS LIMITING CPP TRANSLOCATION
  6. NEW PERSPECTIVES FOR APPLICATION OF CPPs IN DRUG DELIVERY
  7. CONCLUSION
  8. REFERENCES

As a consequence of the described limitations of CPP mediated translocation, for example, the dependence on the state of cellular differentiation, we aim in this final section at the identification of promising niches for CPP application in drug delivery. Ideally, these approaches should either overcome or bypass the given limitations of CPP mediated drug delivery described above and, thus, offer a realistic avenue toward the usage of CPPs in a therapeutic context. As inspired by current literature we will focus on three principal areas: (i) the delivery of antineoplastic agents, (ii) the delivery of CPPs as antimicrobials, and (iii) the potential of CPPs to target inflammatory tissues.

Delivery of Antineoplastic Agents Through CPP Mediated Translocation

The rationale of antineoplastic therapy, that is to specifically eradicate proliferating tumor tissue while leaving healthy tissue unharmed, is notoriously difficult to accomplish. Traditional chemotherapy is often poorly specific, making the search for more targeted treatments quite attractive. The reconstitution of tumor-suppression following the mutation or deletion of tumor-suppressor proteins, such as p53, is often considered a prime goal for effective antineoplastic treatment.92 Recently, various studies revealed the potential of the HIV-1 Tat protein transduction domain to modulate the cell biology of living organisms by the direct cellular delivery of proteins and peptides.92, 152, 153 In the first example of using CPP mediated transduction to deliver a p53 peptide in vitro, Selivanova et al.154 linked a C-terminal p53 peptide, which was previously shown to activate wild type p53 and several DNA-contact mutant forms of p53,155 to the Antennapedia transduction domain, and found that the conjugate induced p53-dependent apoptosis in several tumor cell lines. Normal cells, being wild-type for p53, were resistant to the p53 peptide.154 Recently, Snyder et al.156 used Tat mediated protein transduction to deliver structurally modified p53 C-terminal fragments, resistant to degradation, into ovarian tumor bearing mice. Although the Tat-p53C′ peptide was able to enter all cells, p53-specific genes were only activated within cancer cells but not in normal cells. The potential of this approach was further illustrated by in vivo experiments where intraperitoneal injection of Tat-p53C′ for 12 days caused a significant reduction in tumor growth and a sixfold extension in lifespan, with some mice remaining free of the disease for more than 200 days.156

Another strategy for the delivery of an antineoplastic agent using CPP mediated transduction is the regulation of the cell cycle. In order to reconstitute tumor suppressor function, a fusion protein consisting of p27 and the Tat transduction domain was tested in a cell culture of human Jurkat cells.157 Transduced Tat-p27 was found to bind to Cdk2 in the cells, and loss of Cdk2 kinase activity compared to control cells was demonstrated. Moreover, treatment of cells with Tat-p27 resulted in a substantial, dose-dependent, G1 phase cell cycle arrest.157 Another study from Gius et al.158 demonstrated that covalent linkage of the p16 peptide to the Tat transduction domain was sufficient to control its intracellular accumulation, subsequent inhibition of cyclin D:Cdk4/6 activity, and G1 arrest in synchronized human keratinocytes.158 The observations showed that both, transducible peptides and proteins, were capable to target active cyclin:Cdk complexes. Since cell cycle control genes are deregulated in the vast majority of human tumors, the targeting of these genes and/or their products is now under investigation as potential antineoplastic therapy.159 In an excellent review on various strategies for the application of Tat mediated protein transduction, Wadia and Dowdy92 focused on technologies to deliver peptides and proteins in the treatment of cancer.

Delivery of CPPs as Antimicrobials

The functions of CPPs have been extensively studied in cell cultures of various mammalian cells, but there are only few reports on their ability to overcome the membranes of bacteria and yeast. Given the fact that many CPPs are cationic and often amphipathic, similar to membrane active antimicrobial peptides, Nekhotiaeva et al.160 examined CPPs as to their antimicrobial potency. Two CPPs, TP10 and pVEC, were found to pass the membranes of bacteria and fungi. The observed uptake route involved rapid surface accumulation within minutes followed by microbial entry. TP10 inhibited the growth of Candida albicans and Staphylococcus aureus, and pVEC inhibited Mycobacterium smegmatis growth at low doses, below the levels that harmed human HeLa cells. Therefore, although TP10 and pVEC entered all cell types tested, they preferentially damaged microbes. Indeed, this effect was sufficient to clear HeLa cell cultures from S. aureus infection. Also, when exposed to TP10 the cytoplasmic conversion of the marker dye SYTOX Green demonstrated a rapid and lethal permeabilization of the plasma membrane of S. aureus. Furthermore, pVEC permeabilized M. smegmatis, but not HeLa cells. Therefore, TP10 and pVEC may enter both mammalian and microbial cells, but preferentially permeabilized the microbial rather than the mammalian plasma membrane.160

Very recently, the uptake of three CPPs, namely pVEC, penetratin and (KFF)3K, into two yeast species, Saccharomyces cervisiae and Candida albicans was studied by Holm et al.119 By comparing the capacity of the investigated CPPs to traverse the yeast cell envelope the authors could show that the cellular uptake of the peptides varied widely. pVec and (KFF)3K displayed the highest translocation into yeast, although being partly degraded. Considering the stability of pVEC in both yeast species, it was suggested as attractive candidate for further studies.119 Nevertheless, to date the therapeutic value of CPP based antimicrobials has yet to be established.

Potential of CPPs to Target Inflammatory Tissue

In a recent study in our laboratory we observed that a marked drop in the cellular uptake of CPPs into MDCK cells related directly with a general slow-down in their endocytic activity, and inversely with the cellular differentiation of the MDCK cells.120 Our findings were further corroborated in an inflammatory epithelial model which was induced by pretreatments of confluent MDCK monolayers with a cocktail of IFN-γ and TNF-α. Inflammatory epithelial conditions such as inflammatory bowel diseases, for example, Crohn's disease and ulcerative colitis,161–164 or inflammatory airway diseases associated with cystic fibrosis165 or asthma bronchiale,166 typically feature increased cytokine production, and significant barrier dysfunction. Correspondingly, we found the translocation efficiencies of selected CPPs in the IFN-γ/TNF-α induced inflammatory MDCK model to be significantly enhanced as compared to untreated MDCK monolayers as negative control. In fact, in a cytokine concentration dependent manner, we found the endocytosis rates of CPPs to be boosted by up to 90%. These findings led us to propose that—under inflammatory conditions—the redistribution of TJ proteins associated with lipid raft microdomains167, 168 reopens a lipid raft-mediated pathway for CPPs in confluent MDCK cells. The results appear to be of particular interest in the context of a CPP mediated delivery of anti-inflammatory drugs.

The potential of this approach was previously demonstrated by the CPP mediated delivery of cyclosporin A for the treatment of cutaneous inflammation,60 or caveolin-1 scaffolding domain against vasodilatation and nitric oxide production.100 In fact, Bucci et al.100 demonstrated that the CPP mediated in vivo delivery of the caveolin-1 scaffolding domain inhibited nitric oxide synthesis and reduced inflammation.100 A chimeric peptide consisting of the caveolin-1 scaffolding domain peptide ligated to penetratin was successfully taken up into blood vessel endothelial tissue from isolated mouse aorta rings, resulting in an inhibition of acetylcholine-induced vasodilatation and nitric oxide production.100 Moreover, when used systemically in mice, the chimeric peptide showed suppression of acute inflammation and vascular leak. The suppression was as efficient as glucocorticoid or endothelial nitric oxide synthetase inhibitors.100

A further example for an anti-inflammatory application of CPPs was recently presented by Rothbard et al.60 The authors studied the topical delivery of cyclosporine A in the form of a conjugate with an arginine heptamer through a pH-sensitive linker, yielding R7-CsA. In contrast to unmodified cyclosporine A, which failed to penetrate skin, topically applied R7-CsA was efficiently transported into mouse and human skin. R7-CsA reached dermal T lymphocytes and inhibited cutaneous inflammation. The data provides a novel approach to the topical treatment of inflammatory skin disorders.60

CONCLUSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. ACCOMPLISHMENTS AND FAILURES. REVISITING THE PERFORMANCE OF CPPs FOR DRUG DELIVERY
  5. CELLULAR BARRIERS LIMITING CPP TRANSLOCATION
  6. NEW PERSPECTIVES FOR APPLICATION OF CPPs IN DRUG DELIVERY
  7. CONCLUSION
  8. REFERENCES

Over more than a decade, a broad variety of CPPs has been evaluated for their capacity to support the cellular delivery of therapeutics that normally do not cross the plasma membrane. Although a number of landmark studies in the field claimed a practically unrestricted cellular access of CPPs and CPP associated cargos, crucial limitations to these shuttles have been pointed out more recently. In this review, we attempted to review distinct aspects of CPP mediated cellular delivery. Besides the wealth of data in the literature that has demonstrated what has been achieved in the field, we also report in more detail about cellular barriers to CPP uptake and the resulting hurdles for the use of CPPs in drug delivery. In particular, we emphasize the massive impact of (i) metabolic degradation, (ii) cell line and cellular differentiation dependent effects, as well as (iii) the role of Rho GTPase signaling upon CPP translocation. Despite critical aspects, which need to be considered with regard to future therapeutic applications of CPPs, we also discuss a number of potential niches for CPP mediated drug delivery, including the cellular delivery of antineoplastic drugs, or the delivery of antimicrobials and anti-inflammatory medications.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. ACCOMPLISHMENTS AND FAILURES. REVISITING THE PERFORMANCE OF CPPs FOR DRUG DELIVERY
  5. CELLULAR BARRIERS LIMITING CPP TRANSLOCATION
  6. NEW PERSPECTIVES FOR APPLICATION OF CPPs IN DRUG DELIVERY
  7. CONCLUSION
  8. REFERENCES