|
Drin G, Rousselle C, Scherrmann JM, Rees AR and Temsamani J
Peptide Delivery to the Brain via Adsorptive-Mediated Endocytosis: Advances With SynB Vectors
AAPS PharmSci
2002;
4
(4)
article 26
( https://www.aapspharmsci.org/scientificjournals/pharmsci/journal/ps040426.htm
).
Peptide Delivery to the Brain via Adsorptive-Mediated Endocytosis: Advances With SynB Vectors
Submitted: June 6, 2002; Accepted: August 20, 2002; Published: October 7, 2002
Guillaume Drin
1
, Christophe Rousselle
1
, Jean-Michel Scherrmann
2
, Anthony R Rees
1
and Jamal Temsamani
1
1
Synt:em, Parc Scientifique Georges Besse; 30000 Nîmes, France
2
Universite Rene Descartes Paris 5 and INSERM U26, Hôpital Fernand Widal, 200 Rue du Faubourg Saint-Denis; 75475 Paris Cedex 10, France
|
Correspondence to:
Jamal Temsamani
Telephone:
Facsimile:
E-mail: jtemsamani@nimes.syntem.com
|
Keywords:
intracellular delivery
peptide vector
blood-brain barrier
multidrug resistance
|
Abstract
Biological membranes normally restrict the passage of hydrophilic molecules.
This impairs the use of a wide variety of drugs for biomedical applications. To
overcome this problem, researchers have developed strategies that involve conjugating
the molecule of interest to one of a number of peptide entities that are efficiently
transported across the cell membranes. In the past decade, a number of different
peptide families with the ability to cross the cell membranes have been identified.
Certain of these families enter the cells by a receptor-independent mechanism, are
short (10-27 amino acid residues), and can deliver successfully various cargoes
across the cell membrane into the cytoplasm or nucleus. Surprisingly, some of these
vectors, the SynB vectors, have also shown the ability to deliver hydrophilic
molecules across the blood-brain barrier, one of the major obstacles to the
development of drugs to combat diseases affecting the CNS.
Introduction
A large number of hydrophilic molecules such as peptides, proteins, and
oligonucleotides are poorly taken up by cells, since they do not efficiently cross
the lipid bilayer of the plasma membrane. This is considered to be a major limitation
for their use as therapeutic agents in biomedical research and in the pharmaceutical
industry. In particular, it has been widely accepted that peptide neuromodulators
fail to significantly affect their target cells within the brain when administered
peripherally. This is likely due to the existence of the blood-brain barrier (BBB), a
complex biological interface that prevents transport of most drugs from the
vasculature into the brain parenchyma. The endothelium of the central nervous system
(CNS) vasculature has a structure somewhat different than that of most other organs.
Between the endothelial cells are tight junctions that block the entry of
water-soluble agents into the internal environment of the brain via aqueous
paracellular pathways.
1
While a wide variety of neuropharmaceutical drugs are presently available, few
possess the physicochemical properties that would render them ideal for the treatment
of CNS disease.
The most important factors determining the extent to which a molecule will be
delivered from the blood into the CNS are lipid solubility, molecular mass, and
charge. Therefore, based simply on lipid solubility and molecular mass, any
peptide-based or oligonucleotide-based neuropharmaceutical will almost certainly be
impeded by the BBB.
To overcome the limited access of drugs to the brain, at least 3 strategies have been developed that achieve BBB penetration
2-5 :
- Neurosurgery-based strategies, which bypass the BBB by means of intraventricular drug infusion, intracerebral infusion, or disruption of the BBB;
- Pharmacology-based strategies, some examples of which employ lipidation, or chemical modification of the drug to improve its ability to diffuse across the BBB; and
- Physiology-based strategies, which take advantage of BBB nutrient carriers or specific receptors, mediating transport via these transporter systems. One example has been to conjugate the therapeutic drug with a protein or a monoclonal antibody that gains access to the brain by either receptor - or adsorptive-mediated transcytosis (eg, transferrin receptor-mediated transfer).
2
However, problems have been encountered with many of these approaches: for
example, increasing the lipophilicity of a peptide also decreases its solubility in
serum, whereas direct injections into the brain could result in toxic manifestations.
Thus, pharmacological disruption of the BBB may not always be desirable as a general
approach, even though the barrier can be opened during periods of CNS trauma,
allowing for acute delivery of drugs (as in, for example, brain cancer or stroke).
Our view is, therefore, that new, noninvasive methods of administration are urgently
needed.
During the past decade, several peptides have been described—such as
penetratin,
6,7 a basic segment of the transcription-activating factor (Tat),
6,7 and SynB vectors
5 —that allow the intracellular delivery of polar, biologically active
compounds in vitro and in vivo. As reported in
Table 1, the peptides, belonging to various families, are heterogeneous in
size (10-27 amino acids) and sequence. However, all these peptides possess multiple
positive charges, and some of them share common features, such as important
theoretical hydrophobicity and helical moment (reflecting the peptide
amphipathicity), the ability to interact with a lipid membrane and to adopt a
significant secondary structure upon binding to lipids (
Table 2). Since these peptides penetrate into cells by a receptor-independent
nonendocytotic process (see later), attempts have been made to demonstrate that
interaction of some of them with the lipid matrix of the plasma membrane could play a
key role in their cell uptake (for a detailed overview, see Langel
7
). Although the detailed mechanism by which these peptides enter cells is
poorly understood, the facility with which they cross the membrane into the
cytoplasm even when carrying hydrophilic molecules has provided a new and powerful
tool in biomedical research.
This review will address the use and mechanism of transport of cell-penetrating
peptides for the delivery of molecules into cells and across complex physiological
barriers, with a focus on their potential use for the transport of drugs across the
BBB.
Cell-Penetrating Peptides
SynB vectors
SynB vectors are a new family of vectors derived from the antimicrobial peptide protegrin 1 (PG-1), an 18-amino-acid peptide originally isolated from porcine leukocytes.
22 The peptide has a b-hairpin structure in which 2 antiparallel strands linked by a β-turn are stabilized by 2 disulfide bridges.
24 As previously reported, the PG-1 peptide interacts with, and forms pores in, the lipid matrix of bacterial membranes.
24-25 Since it has been shown that the pore formation capability of PG-1 depends on its cyclization,24 researchers designed various linear analogues of PG-1 that lack the cysteine residues. These linear peptides (SynB vectors) are able to interact with the cell surface and cross the plasma membrane without their membrane-disrupting activity (Table 2). Furthermore, the internalization of these peptide vectors into cells does not appear to depend on a chiral receptor, since the D-enantio form penetrates as efficiently as the parent peptide (L-form), and retro-inverso sequences exhibit identical penetrating activity. These linear protegrin analogues were the starting point for developing a new potent strategy for drug delivery into complex biological membranes such as the BBB (see later). Penetratin
It has been shown that the homeodomain of the Antennapedia protein. (AntpHD), a
Drosophila
homeoprotein, is internalized by cells in culture and is conveyed to the nucleus, where it binds specifically to its DNA cognate site.26,27 The sequence responsible for this translocation has been mapped to a region comprising the third helix of AntpHD.9 Furthermore, it was established that a short peptide segment, pAntp
43-58
(penetratin), corresponding to the helix itself is able to penetrate into primary neuronal cultures.9 An original feature of penetratin is that it can be recovered within the cells after incubation at 4°C. Additional studies showed that analogues of penetratin corresponding to its enantio- (43-58 all D) or retro-inverso form (58-43) penetrated as efficiently as the parent peptide,28,29 suggesting that pAntp
43-58
translocates through cell membranes without binding to a stereospecific receptor and by a nonendocytotic pathway. Moreover, the uptake of peptide is not saturable and appears to be independent of cell type since it has been shown to enter into various cell lines, such as lymphocytes or endothelial cells.8,30 The unusual cellular import of the pAntp
43-58
peptide could depend on its capacity to interact with the lipid matrix of plasma membrane,18,19 although the exact mechanism is still unresolved. Tat peptide
As found for the homeoproteins, the transcription factor Tat, involved in the replication cycle of human immunodeficiency virus (HIV), was demonstrated to penetrate into cells.31 In addition, a 35-amino-acid peptide corresponding to fragment 37-72 of the HIV Tat protein has been shown to promote the intracellular delivery of covalently bound proteins.32 Vivès et al13 showed that several fragments (Tat37-60, Tat43-60, Tat48-60) derived from the most basic region of the protein penetrate into HeLa cells at 37°C as well as 4°C. In addition, it was observed that neither endocytosis nor potocytosis seemed to be involved in this process.13,33 One of the shortest peptides, Tat48-60, which contains a nuclear localization signal (NLS), was defined as the minimal translocating fragment, although the deletion of 3 nonbasic residues within this sequence did not affect its cell-penetrating ability.13 This observation underlines the role of basic residues in the translocating ability of Tat-derived peptides. In addition, it has been reported recently that arginine-rich peptides deriving from DNA- or RNA-binding domain are also imported into eukaryotic cells.33 Other cell-penetrating peptides
A number of other cell-penetrating peptides that derive not from natural proteins but from the engineering of various short peptides have been described, such as, for example, transportan,10,20 the model amphipathic peptide (MAP),16,17 and various signal sequence-based peptides,11,12 and homoarginine vectors34 (see Tables 1 and 2). Transportan is a 27-amino-acid chimeric peptide composed of the neuropeptide galanin and mastoparan-X linked by a central lysine. The cell uptake of this peptide is rapid, occurs at 4°C, and is unaffected by the presence of inhibitors of endocytosis.10 Likewise, it has been reported that MAP and some of the other sequence-based peptides seemingly enter into cells via a nonendocytotic pathway.11,12,16 The sequence-based peptides resulted from the fusion of a hydrophobic peptide (eg, a signal sequence, a fusion peptide) with the NLS motif, whereas the MAP peptide is a complete canonical amphipathic helix. As observed with pAntp
43-58
, the unconventional internalization of these peptides seems to be neither saturable nor dependent on the cell type8,10 and could be related to their lipid-binding capacity.11,12,17,21 As far as the homoarginine peptides are concerned, they have also been described as entering into cells by an energy-dependent nonendocytic process.34 Intracellular Delivery
One of the first applications of these peptide vectors was the design of cell-permeable constructs that are able to enhance the cell uptake of cargo molecules. For instance, it has been demonstrated that phosphopeptides linked in tandem with pAntp
43-58
can stimulate a mitogenic response by activating a regulatory protein involved upstream of this process.35 Similarly, it has been shown that such constructs can specifically inhibit ligand-dependent transduction pathways in various cell lines.36-39 Several studies have also focused on the use of the pAntp
43-58
peptide to promote the delivery of fragments of proteins able to inhibit cyclin-dependent kinase (Cdk), involved in the regulation of the cell cycle.40-42 These cell-penetrating constructs have been proposed to have an anticancer potential since it was shown that they block, in vitro, the phosphorylation activity of Cdk and arrest cell proliferation.
Peptide vectors are also able to translocate large proteins into cells. For example, proteins linked to the Tat-translocating domain are able to enter into a wide variety of human and murine cell lines.43-45
Similarly, peptide vectors have been used successfully to deliver oligonucleotides.46-48 For example, it has been reported that the AntpHD-derived peptide and transportan were able to transport into melanoma cells a 21-mer Peptide Nucleic Acid (PNA) that was unable to cross the plasma membrane by itself. Once in the cytoplasm, the oligonucleotide blocked the expression of the galanin type I receptor by interacting with the messenger RNA (mRNA) coding for this protein. In this instance, the oligonucleotide was linked to the peptide vector via a disulfide bridge. It is likely that this bridge was cleaved within the cell, thereby releasing the oligonucleotide for interaction with its target RNA in the cytoplasm or pre-mRNA in the nucleus.47
Brain Delivery
Enhancement of brain uptake
As described earlier, the BBB poses a formidable obstacle to drug therapy for the CNS. The fact that a peptide vector is internalized inside the cell does not guarantee that it will cross the BBB. The BBB is more complex than a simple cell layer; it comprises a specialized endothelium (compared with that of other blood vessels) associated with pericytes and astrocyte foot processes, which together elaborate a cellular barrier containing an efficient system of tight junctions. Despite this complexity, we have demonstrated that SynB peptide vectors can enhance the delivery of many different types of drugs across the BBB.
In one study, we assessed the efficacy of SynB vectors to enhance the brain uptake of the anticancer agent doxorubicin. Doxorubicin was conjugated to SynB vectors via a chemical linker (succinate), and its ability to cross the BBB was studied using in situ cerebral perfusion in rats and mice.14,15 This "ectorization" of doxorubicin to SynB vectors significantly enhanced its brain uptake in all the grey areas assessed—without compromising BBB integrity. The amount of vectorized doxorubicin that was delivered to the brain parenchyma (shown after applying the capillary depletion method) was about 20- to 50-fold higher than for free doxorubicin, depending on the vector used. Interestingly, we also observed that SynB vectorized doxorubicin bypasses the P-glycoprotein (P-gp) that has been shown to be present in the luminal membrane of the BBB endothelial cells.49 This 170-kDa ATP-dependent efflux pump, because of its unidirectional orientation from brain to blood, restricts the brain entrance, or increases the brain clearance, of a broad number of therapeutic compounds, including cytotoxic drugs.50,51 Other experiments, carried out in vitro using resistant cells, have confirmed that vectorized doxorubicin bypasses the P-gp and enhances its potency in those cells.49
As a comparison, we have also conjugated doxorubicin to D-penetratin and assessed its brain uptake by in situ brain perfusion. Although a 5- to 7-fold enhancement in brain uptake was observed, the brain vascular volumes were 2-fold larger than those observed with the SynB vectors, suggesting an opening of the tight junctions.14 Bolton et al have shown that pAntp
43-58
(penetratin L-form) does not pass the BBB after an intravenous injection.52
The ability of SynB vectors to enhance the brain uptake of doxorubicin was also assessed after intravenous injection of vectorized doxorubicin into mice. The tissue and plasma distribution of doxorubicin were dramatically modified when the drug was vectorized. The brain concentrations were higher for vectorized doxorubicin than for free doxorubicin.14 Interestingly, vectorized doxorubicin shows significantly lower levels in the heart, strongly suggesting that cardiotoxicity—the main side effect of doxorubicin—could be reduced using this strategy.
To assess the broader potential of this approach, we have investigated the transport of other small molecules, peptides, and even proteins. We have shown that conjugation of the antibiotic benzylpenicillin (B-Pc) to SynB vectors improves its penetration across the BBB in a significant manner and that the vectorized B-Pc is distributed in all grey areas.53 Antibiotics must cross the BBB to be suitable for use in the treatment of severe cerebral infections such as bacterial meningitis.
The use of SynB vectors has also been successfully applied to brain delivery of druglike peptide molecules. In a pharmacological application focused on pain management, the brain uptake of an enkephalin analogue was enhanced several hundredfold after vectorization. Importantly, systemic administration of the vectorized enkephalin led to a dramatic enhancement of its analgesic effect in mice compared to that of free enkephalin (Rousselle et al, unpublished results, Jan 2002). In a further application, we have shown that attachment of SynB vectors to the protein streptavidin (via a SynB vector-biotin construct) results in enhanced uptake of streptavidin into the brain (Rousselle et al, unpublished results, Feb 2002 ).
Similar results have also been seen with the Tat peptide. Schwarze et al,54 after fusion of this peptide to β-galactosidase, assessed the tissue distribution after intraperitoneal injection in mice. They observed strong β-Gal activity in all the tissues analyzed, including liver, kidney, heart muscle, lung, and brain. This suggested that the protein had crossed the BBB and, in addition, had passed into most other biological tissues. Mechanism of brain uptake
We have shown by in situ perfusion studies, a technique allowing a first-pass exposure, that the internalization of Dox-SynB is a saturable process.15 The measured Km, which was in the range of 4 to 9µM, compares well with the values observed for substrates reported to be taken up by adsorptive-mediated endocytosis. Furthermore, no difference in brain uptake was seen between doxorubicin linked to L-SynB or D-SynB vectors, indicating that a stereospecific receptor is not a requirement for its brain transport. In addition, we have reported that the passage of peptides can be inhibited in a competitive manner by polycationic molecules such as poly(L)lysine or protamine, which act as endocytosis inhibitors. These observations suggest that the crossing of BBB by SynB vectors is via an energy-dependent adsorptive-mediated endocytosis mechanism.15
It is known that at physiological pH values the luminal surface of the brain endothelium presents an overall negative charge (due in part to significant sialylation) and thus creates an environment more selective to positively charged substances.55,56 The SynB peptides are positively charged. This net positive charge is likely to play a key role in the adsorptive-mediated endocytosis process, wherein electrostatic interactions of the peptide vector with the surface of endothelial cells may mediate surface binding and subsequent internalization of the peptide vectors into the brain capillaries.
For the transcytosis of peptides through the BBB, 3 steps have been proposed: (1) binding and internalization at the luminal side of endothelial cell membrane, (2) diffusion through the cytoplasm of endothelial cells, and (3) externalization at the basolateral side of endothelial cells.57 The main components of the basal membrane are type IV collagen, fibronectin, laminins, chondroitin, and heparan sulfate from glycosaminoglycans. The most abundant component, type IV collagen, polymerizes with laminin and fibronectin proteins via protein-binding domains such as integrin and lectin receptors.58 These components not only provide a mechanical supporting structure for the capillary wall, they are also important as a negatively charged barrier arising from the chondroitin and heparan sulfate residues, in addition to the anionic properties of the luminal and abluminal membranes of the endothelial cells.59 Our results suggest that adsorptive-mediated endocytosis occurs at least at the luminal side of brain capillaries. The similarity in behavior observed for the peptide vectors studied suggests that the externalization at the abluminal side of endothelial cells may also be via a receptor-independent mechanism. However, since endocytosis inhibitors have been tested at only the luminal side of the endothelial cells, it is possible that a different mechanism may be involved in the externalization step.
Conclusion
Biological membranes pose a formidable obstacle to drug therapy where the drugs
are large or hydrophilic. As new drugs for neurological and other disorders are
discovered, new delivery techniques will have to be developed in concert to overcome
this transport obstacle. As rapid advances in cell and molecular biology lead to a
proliferation of potent molecules that cannot be effectively delivered to cells and
brain by conventional means, continuing refinement of the new delivery methods will
be essential to realizing the potential of these molecular drugs. The use of peptide
vectors as a delivery system represents a novel and promising approach. The recent
results obtained both in vitro and in vivo demonstrate that these vectors can
successfully transport drugs across the membranes of many different cell types, and
furthermore can traverse the more demanding BBB. The knowledge already acquired about
the various peptide sequences will result in the design of more efficient peptide
vectors. The optimization of these vectors will also be aided by a greater
understanding of the transport mechanisms operating at the cell membranes and the
BBB.
Acknowledgements
We are thankful to our colleagues at Synt:em and at INSERM U26 for helpful
discussions.
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