Targeting the Central Nervous System (CNS): A Review of Rabies

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Review

Targeting the central nervous system (CNS): a review of rabies virus–targeting strategies Mira Oswald, Simon Geissler, and Achim Goepferich Mol. Pharmaceutics, Just Accepted Manuscript • Publication Date (Web): 17 May 2017 Downloaded from http://pubs.acs.org on May 19, 2017

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Molecular Pharmaceutics

Targeting the central nervous system (CNS): a review of rabies virus–targeting strategies Mira Oswald1, Simon Geissler1, Achim Goepferich2* 5

1

Merck KGaA, Chemical & Pharmaceutical Development, Frankfurter Straße 250, 64293 Darmstadt, Germany; phone: +49-6151-72-3144 email: [email protected] 2

10

University of Regensburg, Dept. for Pharmaceutical Technology, Universitätsstraße 31, 94030 Regensburg, Germany; phone: +49-941-943-4842 fax: +49-941-943-4807 email: [email protected]

* Corresponding author

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Abstract 15

20

25

30

The transport of drugs across the blood-brain barrier is challenging. The use of peptide sequences derived from viruses with a central nervous system (CNS) tropism is one elegant option. A prominent example is the rabies virus glycopeptide-29 (RVG-29), which is said to enable a targeted brain delivery. Although the entry mechanism of the rabies virus into the CNS is very well characterized, it is unknown whether RVG-29-functionalized drug delivery systems (DDSs) follow this pathway. RVG-29-functionalized DDSs present themselves with modifications of the RVG-29 peptide sequence and different physicochemical properties compared to the rabies virus. To our surprise, the impact of these changes on the functionality is completely neglected. This review explores virus-related CNS-targeting strategies by comparing RVG-29-functionalized DDSs with regard to their peptide modification, physiochemical properties and their behavior in cell culture studies with a special focus on the original pathway of rabies virus entry into the CNS.

Keywords CNS-targeting strategies, Rabies virus glycopeptide, Blood-brain barrier

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45


Introduction In brain-targeted delivery, short virus peptide sequences with a central nervous system (CNS) tropism have gained great popularity as a potential targeting motif.1-3 Although classical CNS delivery approaches target receptors, which are directly located on the endothelial cells of the

50

blood-brain barrier (BBB),

4, 5

it is unknown whether drug delivery systems (DDSs), which

are functionalized with virus peptide sequences, follow this pathway.2 An interesting fact is that for many viruses, entry into the CNS starts at the peripheral nervous system (PNS).6-10 By entering through the PNS, viruses benefit from the tremendously long nerve tracts that enable their transport from the periphery to the CNS via the spinal cord, circumventing the highly 55

regulated blood-brain barrier.2,

11

The question of whether the uptake mechanism into the

CNS of particles carrying only motifs of virus proteins is identical to that of the whole virus remains unanswered. In particular, short peptide sequences derived from virus proteins, which are responsible for the transport of the virus into the brain, are selected for the functionalization of DDSs.12-14 60

This functionalization causes significant deviations from the physicochemical properties of the viral original particle. The peptide sequences of the virus have to be modified to enable covalent linkage to the DDS. In addition, the DDS itself has certain characteristic physicochemical properties that can drastically change the uptake mechanism of the original sequence. To our surprise, the impact of physicochemical properties of a virus-functionalized

65

DDS in the targeting effect has been completely neglected so far. One prominent strategy for virus-related CNS targeting is the use of the rabies virus. Due to its known CNS tropism,15 rabies virus peptide sequences have a widespread application for the modification of DDSs (see Table 1, 2). Although targeted delivery to the CNS has been shown by in vitro and in vivo analyses (see Table 3), the uptake mechanisms and impact of

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physicochemical properties on the cellular uptake mechanism are not discussed. This is 3 ACS Paragon Plus Environment


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surprising since the pathway of the rabies virus is very well known and therefore well suited for precise analysis of uptake patterns and transport mechanisms of virus-modified DDSs.8, 15, 16

The goal of this review is to elucidate the impact of physicochemical properties on cellular 75

uptake by comparing the properties of the virus with that of RVG-modified DDSs in the literature. We start with a description of the fate of the rabies virus on its way into the CNS, including a detailed description of relevant receptors with regard to the cellular uptake mechanism. We then review the different types of rabies virus glycoproteins available for DDS modification with a special emphasis on the design and the physicochemical properties

80

of DDSs. Finally, we review in vitro and in vivo work with respect to cell uptake and brain tropism. By doing so, we intend to contribute to a better understanding of virus-related CNS targeting strategies.


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1. The rabies virus – a role model for CNS targeting? 85 1.1. The rabies virus The reason for using rabies virus peptide sequences for CNS-targeting strategies can be explained by the pathology of rabies virus infections, which are accompanied by 90

neuroinvasion and neurotropism of viral particles.17 The virus is bullet shaped, with a length of 180 nm and a diameter of 65 nm.18 The virion is surrounded by a lipid layer, which carries the rabies virus external glycoprotein (RVG).16, 17 Rabies virus glycopeptide is responsible for cellular entry and virus fusion.15, 17, 19 Therefore, RVG is a prime candidate for use as a CNStargeting motif. Rabies virus glycopeptide is organized as a trimer and covers the complete

95

surface of the virus. It consists of 500 amino acids (aa) and has three major antigenic sites.17, 20-22

The sequence has three potential N-glycosylation sites (Asn37, Asn204 or 237 and

Asn309), which ensure the proper function and expression of the glycoprotein.23, 24



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1.2. Infection pathway of the rabies virus into the CNS 100 In accordance with the uptake mechanism of most CNS-tropic viruses, the rabies virus uses axonal transport to reach the CNS (Figure 1).2, 9, 10, 25 The pathway of the rabies virus starts in the periphery with the 105

infection of a dermal or muscular wound.

2, 15, 26

The journey to

the CNS starts directly at the neuromuscular junctions (NMJs).7, 14, 15

These are specialized synapses between muscles and motor

neurons that are responsible for the innervation of muscles. Published data suggest that the rabies virus follows an interesting 110

pathway, first entering the postsynaptic muscle membrane and not the presynaptic neurons.27, 28 In accordance with the uptake mechanisms of other viruses, the first interaction with cell membranes is assumed to be mediated by adsorptive interactions triggered by the positively charged surface protein RVG and

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negatively charged gangliosides, lipids and carbohydrates.15, 26, 27, 29-33

Figure 1 Scheme of the long axonal transport of the rabies virus from the periphery to the CNS. The classical pathway suggests a prior infection of peripheral muscle cells ❶. After the binding of the rabies virus to the acetylcholine receptor (nAchR), the rabies virus enters the muscle cell and replicates ❷. From here, the rabies virus spreads into the neuromuscular junction (NMJ). The entry into neurons is achieved by electrostatic interactions (adsorptivemediated) and receptor-mediated interactions ❸. The neuronal cell adhesion molecule (NCAM) and the neurotrophin (p75NTR) receptor are believed to120 open the portals for the rabies virus into the nervous system ❸. The rabies virus travels in a retrograde manner to the cell body of the motor neuron ❹, which is located in the spinal cord. Hereby, it is hypothesized that either the whole virus or the capsid alone travels to the cell body. From here, the neurons are connected to the CNS. Rabies takes advantage of this network and moves from neuron to neuron ❺-❻ until it hits the CNS.

16,

The nicotinic acetylcholine receptor (nAchR) is

responsible for the first entry into the muscle cells ❶. It is hypothesized that this initial entry is used to multiply the rabies virus and, therefore, results in a more efficient infection of the neurons ❷ .15,

34

After the muscle cells are infected with the

rabies virus, it spreads from the muscle into the synapse ❸. The resulting approach enables the virus to have close contact with the cell membrane. The entry into motor neurons can again be 6 ACS Paragon Plus Environment

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mediated by electrostatic interactions (adsorptive-mediated)15, 16, 26, 27, 29-32 and by the binding 125

at specific receptors (receptor-mediated), which are located in the NMJ at the motor neurons

❸ .8, 11, 35 Receptors such as the neuronal cell adhesion molecule (NCAM) and low-affinity nerve growth factor (p75NTR) have been described as potential portals for rabies virus entry into the neuron. 8, 29, 35 However, the fact that NCAM- and p75NTR-deficient mice can still be infected with rabies32, 35 shows that the complexity of the virus entry mechanism is still not 130

completely understood.36 So far, the published data clearly reveal the involvement of nACH, NCAM and p75NT receptors in the pathogenesis of the rabies virus.8,

29, 35

However, the

complete pathway is still not completely identified, including the identification of all relevant receptors needed for the uptake. For example, it is of high interest to determine whether the interaction with presynaptic neurons is dependent on the preceding interaction with the 135

nAchR—which is located at the postsynaptic muscle cell membrane and would therefore require a specific order of receptor interaction36—or whether the rabies virus can directly enter presynaptic neurons.34

1.3. Rabies virus receptors 140

For a solid understanding of rabies virus receptor interactions, this section provides deep insight into the designated receptors, beginning with their structure and occurrence and then discussing the potential receptor binding sites of the rabies virus. The nAchR was the first receptor discovered to be responsible for the entry of the rabies virus into the CNS. Its involvement in the pathogenesis of rabies was revealed after the

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intramuscular injection of mice with the rabies virus. A virus antigen could be detected 1 h after injection by immunofluorescence at the NMJ. This observation directly reflects the pathogenesis of rabies and confirms that the initial infection is transmitted by the bite of an animal.27 Lentz et al. analyze the binding of the rabies virus glycoprotein to acetylcholine 7 ACS Paragon Plus Environment


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receptors by using the α-1-subunit.26 The α-1-subunit receptor belongs to the class of muscle 150

subunits (α1, ß1, δ, ε and ϒ), which is one of three major classes of nAchR subunits. Entry via the muscle subunit describes the classical pathway of the rabies virus with an initial muscle infection in the periphery.34 An interesting question is whether other nAchRs can play a further role in the transport of the rabies virus to the CNS by enabling a synaptic passage between neurons.6, 29 Nicotinic acetylcholine receptors are widely distributed in the nervous

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system and can, therefore, serve as potential door openers for a fast infection of the CNS. Furthermore, the interaction of the rabies virus with the α-subunit could indicate that rabies is also able to interact with other nAchR subtypes. An important note is that the muscular α-1subunit and the neuronal α-7-subunit can both be inhibited by the nAchR antagonist αbungarotoxin.29 Therefore, some authors assume that α7 is also able to interact with the rabies

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virus and, therefore, might also play a role in the uptake of the rabies virus into the CNS.29, 32 Furthermore, the mammalian brain mostly contains the α4ß2 or α7 receptor subtypes. Thus, deeper investigations into α4ß2 and α7 are of high interest.29, 37 After observing that several lymphocyte cell lines could be infected with the challenge virus standard (CVS) and Evelyn Rotnycki Abelseth (ERA) rabies virus strains,38 Thoulouze et al.

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analyze rabies virus-susceptible and -non-susceptible cell lines with regard to surface molecules, which can enable the uptake of the rabies virus. They discover that all RVsusceptible cell lines express two isoforms of NCAM, 140 and -180, on their surface. Neuronal cell adhesion molecule, also known as CD56, D2CAM, Leu19 or NKH-1, belongs to the immunoglobulin superfamily and is a cell adhesion molecule. Members of this family

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can be found on neurons, astrocytes, myoblasts, myotubes, activated T cells and natural killer (NK) cells.35 Due to their expression in neurons and lymphocytes, other viruses such as the adeno-, coxsackie and herpes simplex virus also use NCAM to enter the CNS.39 Thoulouze et al. find that the recombinant expression of NCAM in NCAM-negative cells enhances their 8 ACS Paragon Plus Environment

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susceptibility to the rabies virus, whereas blocking the NCAM receptor in NCAM-positive 175

cells using antibodies leads to a decrease. In addition, the mortality of NCAM-deficient mice is compared to wild-type animals upon infection with the CVS strain of RV. A delayed mortality is noted for NCAM-deficient mice (mean length of survival 10 days for wild-type and 13.6 days for NCAM-deficient mice). The neutralization of RV infection by soluble NCAM, which is composed of five immunoglobulin(Ig)-like domains and two fibronectin

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domains, proves that the ectodomain is a potential binding site for the rabies virus.35 The p75 neutrophin receptor (NTR) has been identified as another relevant receptor for rabies virus binding. It was discovered by screening a neuroblastoma cell library for soluble RVG.40 The neutrophin receptor, also known as a low-affinity receptor for nerve growth factor, is a type I transmembrane protein of the tumor necrosis factor receptor family.29 It is

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important during early neuronal development and in different pathological conditions such as neurodegeneration and epilepsy.41 In adults, its prevalence is limited to the peripheral and central nervous systems. The ectodomain of the receptor consists of four cysteine-rich domains (CRDs). In contrast to other members of the tumor necrosis factor family, its ligands are organized as dimers.40 Different CRD mutants were used to analyze the interaction

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between RVG and p75NTR.8 These experiments reveal that CRD1 is an RVG binding site because the antigen binds to all mutants except those with a deleted CRD1. Gluska et al. analyze the interaction of p75NTR and the rabies virus with respect to uptake into axons and track the transport machinery by live cell imaging.8 An enhanced green fluorescent protein (EGFP)-tagged rabies virus is applied to dorsal root ganglion (DRG) explants, which are

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cultured in a microfluidic system in the presence of a fluorescent antibody against p75NTR. They find that both the rabies virus and p75NTR are internalized together. In addition, a rabies virus infection of p75NTR knockdown DRG cultures reveals lower infection rates,



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which emphasizes the role of p75NTR as a rabies receptor. Furthermore, p75NTR-dependent uptake causes faster transport of the rabies virus.8


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1.4.

CNS entry

After successful receptor binding on the motor neurons, the rabies virus moves along the axons in a retrograde manner from neuron to neuron by passing the synaptic gap (transsynaptic spread).15 This makes the rabies virus a perfect tool for transneuronal tracing.30, 42 205

The exact mechanism of axonal transport is highly relevant to the potential use of RVG as a CNS-targeting peptide. What matters is whether RVG is only needed for receptor binding at the motor neurons or is a necessary element for the further transport of the rabies virus into the CNS. After receptor binding, the whole virus is endocytosed via clathrin-mediated uptake.43-45 The rabies virus takes advantages of the long axonal transport machinery, which

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enables fast transport to the cell body (3-10 mm per hr).46, 47 This transport has been described for the whole virion as well as for the capsid alone ❹ .15 It has been reported that the rabies virus phosphoprotein, which is part of the rabies capsid, interacts with the dynein light chain LC8;16, 48, 49 this interaction requires uncoating the rabies virus after cell entry. However, it is questionable whether the dynein LC8-phosphoprotein interaction is really needed for the

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axonal transport of rabies virus. As it was shown that recombinant viruses with a deletion of the dynein light chain binding site in the phosphoprotein were still able to reach the CNS.22. This fact favors the assumption that the external rabies virus glycoprotein is not only responsible for entry into the cells, but is also a decisive factor in long-distance transport across axons (intra-neuronal transport).46 For example, RVG pseudotyping of lentiviral

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vectors enable the retrograde transport of the lentiviral vectors to the CNS.50 In addition, the retrograde axonal transport of the whole virion was tracked by using double-labeled rabies viruses with a red fluorescent envelope and a green fluorescent phosphoprotein.51 Finally, the rabies virus reaches the cell body of the motor neuron ❺,, which is located in the spinal cord. These cell bodies are in synaptic contact with motor centers in the brain.8, 15 Thereafter, the

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chain-like connection of neurons via synaptic junctions allows the entry of the rabies virus 11 ACS Paragon Plus Environment


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into the CNS ❺-❻.. Therefore, infection with the rabies virus heavily relies on the ability of the virus to interact with receptors and axonal proteins, enabling synaptic passage and entry into the subsequent neuron and fast axonal transport.6, 16 With respect to the use of RVG as a targeting peptide, RVG’s involvement in long axonal transport is as important as its initial 230

receptor binding. Therefore, a detailed analysis of the contribution of RVG to axonal transport is required. 2. RVG primary sequence and modification: from pathogenesis to use as a CNStargeting motif

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The targeted journey of the rabies virus from the periphery to the brain points directly to a key element of the virus that is responsible for its CNS tropism, the external surface glycoprotein RVG. This glycoprotein contains short peptide sequences, which hold the relevant information for interaction with the above-mentioned receptors. Today, a key strategy in designing CNS-targeted DDSs is the functionalization of a DDS with receptor-active peptide

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sequences. Using this strategy provides a better understanding of the pathogenesis of rabies and concomitantly allows the transport of such systems into the CNS. A major aspect of virus-related targeting strategies is the risk of pathogenicity of the selected peptide sequence. Selected peptide sequences should only contain the relevant information for the uptake mechanism into the CNS but should not induce an immune response.

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By investigating the receptor binding of RVG to the nAchR, Lentz et al. provide us with a better understanding of the significant structural elements in the primary sequence of RVG that are necessary for successful receptor binding. A second aspect of major interest has been the assessment of the physicochemical properties of the peptide sequences. Because the rabies virus takes advantage of electrostatic interactions to bind to the host cell membranes, a

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description of physicochemical properties is important. An interesting and often-neglected fact is that the functionalization of a DDS requires the modification of virus peptide 12 ACS Paragon Plus Environment

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sequences to enable linkage to the DDS. These modifications could impact the sensitivity of RVG to its receptors and its physicochemical properties. We, therefore, shed some light on relevant peptide sequences, their physicochemical properties and the impact of RVG 255

modifications on their functionality. 2.1.

RVG receptor-binding studies

The interaction of RVG with nAchR was somewhat anticipated due to the structural similarities between RVG and curare-mimetic snake venom toxins, which also bind with high 260

affinity to nAchR. The alignment of the primary sequences of the snake venom toxin from

Naja melanoleuca (residues 1-71) and the RVG (residues 151-237) reveals 26 matches. Further, Lentz et al. show that different cholinergic antagonists, neurotoxin peptides and RVG peptides are able to inhibit the binding of nAchR-antagonist α-bungarotoxin to the synthetic peptides of the α-subunit of the Torpedo californica ray and human acetylcholine receptor 265

(residues 173-204). The IC50-values for RVG and neurotoxin peptides are comparable to those of cholinergic agonists and antagonists. The CVS rabies virus strain (residues 175-203) (CVS 29mer, hereafter referred to as RVG-29), a homologous segment of the RVG, is found to be the most effective of the rabies glycoprotein peptides in inhibiting α-bungarotoxin (1.2x10-6M and 2.5x10-6M) and shows its receptor binding capabilities for nAchR.52

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2.2. Physicochemical properties The impact of natural glycosylation on receptor binding affinity is an important aspect to focus on when it comes to the critical review of the physicochemical properties of targeting peptides. It is known that the glycosylation of peptides can improve the uptake of targeting peptides by

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increasing stability and ensuring the right conformational structures for receptor binding and improve the possibility of potentially interacting with glucose transporters.53 The impact of 13 ACS Paragon Plus Environment


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glycosylation on receptor binding has been, for example, shown for encephalin-based glycopeptides, where the receptor binding affinity is highly dependent on the glycosylation grade.54-56 280

Rabies virus glycopeptide carries three potential N-glycosylation sites, which are said to enable RVG’s proper expression on cell surfaces and its function. The question is whether RVG-29 requires further glycosylation for proper receptor binding. It is reported that nonglycosylated glycoprotein shows a decrease in surface expression and antigenicity.57 However, an impact of missing glycosylation on the uptake has not been described. In

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addition, RVG-29 is obtained from residues 173-204 of the primary RVG sequence, which carry no glycosylation sites.52 The physicochemical properties of RVG play an important role in the cell uptake behavior of the rabies virus. RVG-29 has good water solubility with an isoelectric point at pH 9.7, causing a positive net charge under physiological conditions. The positive charge of RVG-29 at pH

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7.4 explains the adsorptive interaction of the rabies virus in addition to the receptor-mediated uptake.27,

29, 32, 35

Because heparan sulphate proteoglycans lead to a negative charge of the

plasma membrane surface, the uptake of positively charged substances is believed to be driven by electrostatic interactions.33, 58

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2.3. Immunogenicity of RVG-29 Due to the viral origin of the RVG-29 peptide, its potential immunogenicity should be taken into account prior to its application as a targeting peptide. To our surprise, none of the presented RVG-29-functionalized DDSs address this potential risk of pathogenicity. RVG is said to be the major viral antigen. However, the primary sequence of RVG-29, which is in

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accordance with position 175-203 of the RVG primary sequence, is not part of the described antigenic sites of RVG. Two major antigenic sites are described. Antigenic site II is located 14 ACS Paragon Plus Environment

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between positions 34 and 42 and positions 198 and 200. Antigenic site III, which is associated with virulence, is located between positions 330 and 340 of the primary sequence.16, 20, 59-61 2.4. RVG-peptide modifications 305 For CNS-targeted delivery, DDSs have frequently been functionalized with the RVG-29 peptide. The anchoring of RVG on a DDS is one of the most critical steps during formulation. The functionalization requires modifications of RVG-29 to enable covalent coupling or adsorption of the peptide to the DDS. The different modifications of the RVG-29 peptide and 310

resulting changes in physicochemical properties are explained in detail hereafter. Table 1 lists the RVG-29 derivatives that have been used to date.

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Table 1: Peptide properties of the RVG-29 peptide and its modifications estimated by the Innovagen peptide calculator. Mod. = modification, N = number of residues, MW = molecular weight, pI = isoelectric point, dR = D-arginine, RVG-29 = rabies virus glycopetide 29mer, C = cysteine, G = glycine, R = Arginine, H = Histidine, GST = glutathione Net charge RVG-29 peptide sequence+ Linker

Mod.

N

MW [g/mol]

pI [pH]

Ref. at pH7

RVG-29

29

3267

9.7

2

62-65

RVG-29+C

30

3370

8.75

1.9

4, 66-69

RVG-29+GGGGC

34

3542

8.75

1.9

70, 71

RVG-29+GGGG9dR

41

4844

12.25

11

72-77

42

5221

12.25

11.3

78

47

5666

12.25

11.5

79

RVG-29-Protamine

54

6458

12.17

14

80

RVG-29+9D/LR

41

4844

12.25

11

81

RVG-29-+ Biotin

29

3267

9.7

2

82, 83

RVG-29+HHHH rRrRrRrRr Recombinant fusion protein GSTRVG-29-9R-His

C-Terminal

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2.4.1. Addition of positively charged peptides 320 RVG-29 peptides have frequently been conjugated to nucleic acids. In this strategy, the RVG29 peptide acts as a navigator, leading the complex to the CNS. Because nucleic acids carry a negative charge, they can be used to form electrostatic complexes with positively charged peptides. 325

Therefore, by adding positively charged residues to the RVG-29 sequence, the resulting peptide forms complexes with nucleic acids. An example is the complex formed by attaching the cell-penetrating peptide (CPP) nona-arginine to the C-terminal of the RVG-29 peptide sequence.72, 73 The addition of nona-arginine leads to an increase of the isoelectric point and a positive net charge. The construct is positively charged even at neutral pH values. Nucleic

330

acids can be condensed and complexed by such molecules. A short sequence of glycine or histidine serves as a linker between RVG-29 and nona-arginine, minimizing undesired interactions. The RVG-29 part of the whole molecule is still available for the receptor interaction.71-73, 75, 78, 79, 84 The addition of the positively charged nonapeptide not only favors the adsorption of nucleic acids, but also facilitates the cell uptake of the DDS.81

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The increased uptake of nona-D-arginine RVG-29 compared to nona-L-arginine RVG-29 is shown by Zeller et al..81 The use of racemic arginine leads to a higher resistance against proteases and shows reduced toxicity.81, 85 Therefore, racemic arginine is promising for use as a CPP for brain delivery. In addition to short oligonucleotides, Ye et al. aim to formulate DNA complexes with RVG-

340

29. They use the protamine RVG-29 instead of the nona-arginine peptide, as nona-arginine RVG-29 is not able to bind their DNA.80


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2.4.2. Maleimide-thiol coupling 345

In addition to modifying RVG-29 with positively charged peptides, further modifications have been undertaken by coupling the RVG-29 peptide directly to a DDS. One of the bestknown coupling reactions is maleimide-thiol coupling. Maleimides react with thiols to form stable thio-ether bonds.86 One reason for the popularity of this coupling reaction is that peptides can easily be functionalized with cysteines to introduce a thiol group.87 An

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interesting fact is that the original RVG-29 sequence already contains a cysteine residue, which is located in the middle of the sequence and amenable to maleimide-thiol coupling.62, 63 A coupling reaction with this native cysteine can, however, impact the binding affinity of the RVG-29 peptide to the described receptors. Evidence of this cysteine’s importance for the receptor binding is given by Lentz et al.

355

The native cysteine belongs to the matches, which have been identified via the alignment of the RVG sequence and snake neurotoxin peptide sequence.52 Therefore, this cysteine can be involved in the receptor binding. Further, Lentz et al.’s results imply a reduced receptor interaction for RVG peptide sequences without cysteines in their primary sequence. A good example is the RVG peptide CVS 10mer, which consists of the 190-199 residues of the CVS

360

strain and contains no cysteine. Compared to RVG-29 (CVS 29mer), which contains a cysteine in the middle of the sequence, CVS 10mer shows a decreased binding to synthetic peptides of the Torpedo and human acetylcholine receptor α-subunit with IC50 values of 7.2x10-5M, compared to 1.2x10-6M for RVG-29.52 To avoid such a loss of receptor affinity, one elegant approach is to modify the peptide with an additional cysteine on the C-terminal

365

site.66-68,

70, 88-90

A disadvantage of this strategy is the risk of obtaining undesirable side

products. Furthermore, if the central cysteine is not protected, the maleimide group is able to react with both cysteines. In addition, the presence of the two cysteines may facilitate disulfide bond formation that decreases the number of free thiol groups. 17 ACS Paragon Plus Environment


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Page 18 of 57

2.4.3. Biotin/(strept)avidin coupling 370 Biotin/(strept)avidin coupling is used to attach RVG-29 to albumin nanoparticles83 and poly(lactic-co-glycolid) (PLGA) nanoparticles.82 Streptavidin mediates the linkage between RVG-29 and the nanoparticles by carrying an additional biotin on the C-terminal. Since the interaction between streptavidin and biotin is one of the strongest non-covalent interactions, 375

with a dissociation constant of 1.3x10-15M, it is very well suited as a conjugation technique.91

2.4.4. Control peptides

To verify the receptor-mediated uptake of an RVG-29-functionalized DDS, a comparison of 380

these delivery systems with negative controls is mandatory. In the literature, two different examples, the RV-Matrix peptide and the scrambled RVG peptide, have been used. Their length and physicochemical properties are similar to those of the RVG-29 peptide. However, they are supposed to have no CNS-targeting effect. While the RV-Matrix peptide is obtained from the rabies matrix virus protein,72, 74-77, 80 the scrambled RVG peptide is synthesized by

385

changing the amino acid sequence.73


Page 19 of 57

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

390

395

400

405


Table 2 gives detailed information about published RVG-29-targeted DDSs. By screening all DDSs for formulation type, RVG-29 modification and modification type, use of negative controls and cargo, the table provides an overall view and comparison of the common DDSs. Furthermore, the table provides information about physicochemical characterizations including conjugation yield, size, surface charge and stability analysis. + GGGG9dR = RVG-29 + glycine linker + nine arginine residues; +C = RVG-29 with additional cysteine on C-terminal; +GGGGC = RVG29 + glycine linker with additional cysteine on C-terminal; +HHHHrRrRrRrRr = RVG-29 + histidine linker + nine arginine residues; AFM = atomic force microscopy; Asp = Aspartate; BCA = bicinchoninic acid; BPEI = bioreducible polyethylenimine; CD = cyclodextrins; CPT = Camptothecin; DIR = 1,2´-Dioctadecyl-3,3,3´,3´-Tetrematheylindotricarbocyanine Iodide; DLS = dynamic light scattering; DNA = deoxyribonucleic acid; DSPE = 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine; DTT = Dithiothreitol; EDC = 1-Ethyl-3-(3dimethylaminopropyl)carbodiimide; FBS = fetal bovine serum; FRET = Förster resonance energy transfer; Glu = Glutamate; GSH = gluthation; GTA = methoxy PEG and trimethylammonium groups; ITZ = Itraconazole; miRNA = microRNA; mPEG-Mal = Monofunctional PEG Maleimide; n.t. = not tested; N/P ratio = nitrogen/phosphate ratio; NC = nanocarrier; NR = nanorod; NHS-PEG-Mal/Mal-PEG-SCM = N-hydroxy-succinimid-polyethylene glycol-maleimide; NMR = nuclear magnetic resonance; NR = Niele red; PAH = polyasparthydrazide; PAM-ABP = poly(cystaminebisacrylamidediaminohexane) grafted with 9–11 residues of the amino acid arginine with a molecular weight of ∼4.45 × 10 3 Da/mole; PAMAM = polyamidoamine; PBS = phosphate-buffered saline; PCR = polymerase chain reaction; pDNA = plasmid DNA; PEG = Polyethylenglycole; PLGA = poly(lactic-co-glycolic acid); RPMI = Roswell Park Memorial Institute; RVG-Mat = RVG-Mat (MNLLRKIVKNRRDEDTQKSSPASAPLDDG); RV-Mat = rabies virus matrix protein; SCM= succinimidyl carboxymethyl; Scrambled RVG-(WESYRTRAIPKCSPGTDPMINPFTRGNGN); SDS-PAGE = sodium dodecyl sulfate polyacrylamide gel electrophoresis; siRNA = small interfering RNA; SMPT = 4-succinimidyloxycarbonyl-α-methyl-α-[2-pyridyldithio]toluene; SNALPs = stable nucleic acid lipid particles; SSPEI = disulfide crosslinked bioreducible polyethylenimine; TMC = trimethylated chitosan; w/o = without; w = with



RVG peptide sequence

No.

1

RVG-29

Neg. cont rol

-

Conjugation DDS

Cargo

Linker

Size

Surface charge

Agarose gel electrophoresis

yield

RVG-29-conjugated

pDNA

Fluorescence activity of tryptophan: RVG29:BPEI-SH (molar ratio 1:11,8)

RVG-29

-

RVG-29-conjugated SSPEI

miRNA

n.t.

Ref.

NHSPEG5k-Mal

≥ 4 wt ratio size decreased to 200 nm

Neutral surface charge at ≥ 4

NHSPEG5k-Mal

Polymer/miR-124a wt of 6.6 : 290.5 nm

6.6 wt ratio (polymer/miRNA) change to positive charged values

0.8-6.6 wt ratio (polymer/miRNA) form stable complexes

6.6 wt ratio polymer/miRNA complexes were incubated with 10%FBS remained stable over 4h

N/P-ratio from 96/1 to 12/1: 9.0 ± 2.5 mV to 4.6 ± 1.3 mV

Stable complexes formed at N/P ratio of ≥ 48/1 and ≥ 24/1

Incubation with 50% serum for 72h

GPC analysis

2

Serum stability

BPEI-SS-PEG-RVG29 was able to form polyelectrolyte complexes with pDNA at wt ratio of 0.75

BPEI

Stable in 10% serum in RPMI at 37° C over 48h; 63

Stable up to 50% serum RPMI in size and surface charge

62

N/P-ratio from 96/1 to 12/1: 308 ± 16 nm to 134 ± 19 nm 1

3

Electrostatic interaction

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

Page 20 of 57

RVG-29 +C

RVG-29-peptide linked siRNA/TMC-PEG

siRNA

H-NMR 26% (by weight)of RVG-29peptide linked to TMCPEG

Mal-PEGSCM

siRNA loading prior to RVG-29linkage: 135 ± 7 nm

66

siRNA loading after RVG-29linkage: 207 ± 2 nm Possible coupling products RVG-29 linked C-terminal or with Glu and Asp residues pDNA RVG-29

4

+GGGGC

PAM-ABP: poly (amido amine) PAMAM dendrimer grafted with 9-11 arginine residues

1,2 and 3 molecules of RVG-29 conjugate with each PAM-ABP molecule at peptide:polyplex conjugation ratios of 2,4 and 8 detection of thiol group by Ellman´s assay

EDC-NHS via Carboxygro up at Argininresidues of PAM-ABP

Size decrease in dependence of carrier:DNA ratio (520) from 188 ± 9 to 152 ± 9 nm for the Bare-nanocarriers, other NC similar results in size decrease

Increase of surface charge in dependence of carrier:DNA ratio (520) from 11.4 ± 1.8 to 21.1 ± 3.8 mV

RVG-29-PAM-ABP (ratio 2:1) complete condensed at a 3-fold ratio of carrier:DNA, peptide:polymer ratios of 4:1 and 8:1

Environment-Sensitive DNA release: 5 mM DTT (reducing agent) 70% of DNA was released by 2 hours

20

ACS Paragon Plus Environment

70

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1 2 3 4 No. 5 6 7 8 9 10 5 11 12 13 14 15 16 17 6 18 19 20 21 7 22 23 24 25 26 27 8 28 29 30 31 32 33 34 410 35 36 37 38 39 40 41 42 43 44 45 46 47 48



Neg. cont rol

Conjugation DDS

Cargo

siRNA RVG-29 +C

Linker

Size

Surface charge

COOH-PEG-g-PAHy-GTA/si RNA (100:1): 221 ± 9.35 nm

COOH-PEG-g-PAHyGTA/si RNA (100:1): 2.78 ± 1.82 nm

yield

Polyion complex RVG-29 peptide tagged PEGylated polyasparthydrazi de derivatives

Fluorescence intensity of tryptophan: molar ratio of conjugate RVG-29 to PEG 0.32:1 SCM-PEGMal

RVG-29-PEG-g-PAHyGTA/siRNA (100:1): 248 ± 10.32 nm

RVG-29-PEG-gPAHy-GTA/siRNA (100:1):


>50:1 COOH-PEG-gPAHy-GTA/siRNA with PEG 1/3/5% were able to complex siRNA 3% PEG micelles were chosen for further studies

Serum stability

Ref.

50% FBS at 37° C siRNA was protected more than 24h

67

9.81 ± 3.74 nm PAMAM-PEGRVG-29/

RVG-29 +C

DNA Nanoparticles

RVG 29 -

Dendigraft-PolyLysine-RVG-29FRET

Plasmid pEGFP-N2

NMR

NHS-PEGMal

-

UV-absorbance

Sulfo-LCSMPT

+C

150 ± 16.7 nm

n.t.

Complete encapsulation at ratio of 10:1 of PAMAM to DNA

n.t.

68

n.t.

n.t.

Incubation in serum, GSH at 37° C, Activated caspase 3 at 37° C for 2h

4

DLS: ~5 nm AFM:30 nm

Nano-device

RVG 29 +C

Modified βcyclodextrins: cationic amphiphilic CD and neutral PEGylated derivative

1

-

H NMR: estimated by number of maleimides in the intermediated species 4 RVG-29 peptides conjugate per cylcodextrin molecule

SMPT

Coformulation of CD1 {7} with CD2 {25} and RVG-29 CD1:CD2:CD4 Increase in size > 200 nm, PDI < 0.4

Charge decrease from 35 – 20 mV

CD1:CD2:CD4 (molar ratio 1:1.5:0.5) complexed with siRNA mass ratio (1:10)

Aggregation in 50% OptiMEM of CD:siRNA (mass ratio 10) nanocomplexes no size increase for complexes with higher molar content of CD2

Incubated with 50% FBS protection up to 24h

21


89


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

No.


DDS

RV-Mat

siRNA-RVG29-9dR

siRNA

Scrambled RVG

siRNA-RVG29-9dR

siRNA

+GGGG9dR RVG-29 10 +GGGG9dR

Surface charge


Serum stability

Ref.

n.t.

none

n.t.

n.t.

RVG-29-9dR was able to bind siRNA in dose dependent manner

stable over 8h in 50% FBS

72

n.t.

none

n.t.

n.t.

n.t.

n.t.

73

n.t.

Incubation with 90% mouse serum at 37° C addition of 100 pmol prevent degradation for at least 4hours

n.t.

n.t.

LiposomesiRNA-peptide complexes

siRNA

n.t.

none

siRNA:liposomes: RVG-29-9r (100:10/100/1000:1000 pmol 178 ± 20 nm

RV-Mat

RVG--29siRNA

siRNA

n.t.

none

n.t.

n.t.

RVG-29 +GGGG9dR

Size

yield

RV-Mat +GGGG9dR

12

Linker

Cargo

siRNA:liposomes: RVG-29-9r 100:10/100/1000:1000 pmol 6.25±0.75/9.78±0.98/ 19.89±4.46 mV (with increasing concentration of liposome

RVG-29 11

Conjugation

Neg. control

RVG-29 9

Page 22 of 57

75

22


76

Page 23 of 57

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

No.



RVG-29 13 GGGG+9dR

14

+HHHH

Conjugation

Neg. cont rol

DDS

RVMat

n.t.

-

-

RVG-299rR/pDNA

pDNA

Linker

Size

Surface charge


Serum stability

Ref.

n.t.

none

n.t.

n.t.

n.t.

n.t.

74

n.t.

none

N/P ratio: ≥ 3 80 nm (Size of naked pDNA was 308 ± 24 nm)

N/P ratio: ≥ 2 27 mV

Complete encapsulation of pDNA at N/P ratio: ≥ 3

Stability proved after Incubation with 10% serum for 20 min by gelelectrophoresis

Cargo yield

rRrRrRrRr

15

Recombinant fusion protein GST--RVG-299R-His

-

RVG-29-9R6His/pDNA

pDNA

SDS-PAGE

none

N/P ratio: ≥ 2 118-172 nm (Size of naked pDNA was 323 nm)

N/P ratio: ≥ 2 14.2 mV

16

RVG-29Protamine

RVMat

RVG-29protamine-DNA complexes

pDNA

n.t.

none

n.t.

n.t.

Complete encapsulation at N/P ration: ≥ 2, (shielding of surface charge with mPEG-Mal)

Complete binding at a w/w ratio DNA/RVG29-Protamine of 1:10

78

Stability proved after incubation with 10/30/50 and 90% mouse serum in 0.01 M PBS for 4 and 8h at 37° C

80

n.t.

23


79



RVG29+9D/LR

Neg. control

Conjugation DDS

Cargo

Linker

Size

Surface charge

RVG-29-9DR-siRNA: 208.6 ± 20.4nm

RVG-29-9DR-siRNA: 24.2 ± 0.3 mV

RVG-29-9LR-siRNA = 203.5 ± 3.5 nm

RVG-29-9LR-siRNA = 28.8 ± 0.7 mV

yield

RVG-29-9DRsiRNA -

siRNA

n.t.

none

And RVG-299LR-siRNA


Serum stability

Complete binding at Peptide-siRNA complexes (100 pmol; 10:1 peptide:siRNA)

Both complexes stable for at least 3hours exposed to RNase A; after incubation with 50% human AB serum only RVG-29-9DR-siRNA complex remained stable over 24h in contrast to RVG-29-9LR-siRNA (3h)

n.t.

β-Galactosidase release: loaded nanocarrier in dialysis membrane (300 kDa) in PBS with 10%FBS 100 rpm at 37° C

n.t.

Incubation in PBS + 2% Tween-80 at 37° C

n.t.

Incubation at 37° C in presence of RNase (0.01 or 0.1 mg/ml) or serum (10%FBS)

Ref.

81

At 25° C:

RVG-29 +GGGGC

RVG-29 peptide conjugated pluronic-based nano-carrier

Bare-NC: 2.1 ± 1.3 1

βGalactos idase

H NMR and Fluorescence intensity of tryptophan : 81% (1.8 wt% of the nanocarrier)

NHSPEG-Mal (MW 2.1 K)

Size was measured at 4/25/37° C: At 37° C < 70 nm

W and w/o chitosan

Chito-NC: 12.8 ± 2.4 RVG-29-Bare-NC:1.9 ± 2.4

88

RVG-29-ChitoNC:11.3 ± 0.6

Cysteine-modification

1 2 3 4 5 No 6 . 7 8 9 10 11 17 12 13 14 15 16 17 18 18 19 20 21 22 23 24 25 26 27 28 19 29 30 31 32 33 34 35 20 36 37 38 39 415 40 41 42 43 44 45 46 47 48

Page 24 of 57

+Biotin

+GGGG9dr

-

RVMAT-9r

PLGA nanoparticles

SNALPs

Camptot hecin

siRNA

AvidinBiotinLinkage

n.t.

BCA Protein Assay: 12.2 ± 1.9nmol RV-Mat-9r/µmol 15.8 ± 0.2 nmol RVG-29-9r

DSPEPEG-Mal

RVG-29-PLGA-DiR : 188 ± 44 nm

RVG-29-PLGA-DiR: 0.36 ± 1.76 mV

RVG-29-PLGA-NR:

RVG-29-PLGA-NR:

162 ± 64 nm

-0.88 ± 2.98 mV

RVG-29-PLGA-CPT:

RVG-29-PLGA-CPT:

204 ± 45 nm

-2.50 ± 1.19

RVG-29-PLGA:

RVG-29-PLGA:

253 ± 69 nm

-2.71 ± 1.33

RV-Mat-9r liposomes : 190.0 ± 22.9 nm (PDI: < 0.3) n.t. RVG-29-9r liposomes: 195.8 ± 4.5 nm (PDI: < 0.2)

24


82

77

Page 25 of 57

RVG peptid e sequen ce

Neg. cont rol

21

+C

-

RVG-29 peptide modulated liposomes

Protaminecondensed siRNA

22

RVG-29

-

RVG-29 targeted exosomes

RVG-29

-

24

25

Biotinylated RVG-29

23

Exosomes

No.

RVG-29

Nanoro ds

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48


+C

Linker

Size

Surface charge


Serum stability

Ref.

n.t.

DSPEPEG-Mal

91 ± 6nm

-21.8 ± 1.7

n.t.

n.t.

90

siRNA

n.t.

-

n.t.

n.t.

n.t.

n.t.

65


siRNA

Quantitative PCR

Lamp2b fused to RVG-29 peptide

~80 nm

n.t.

n.t.

n.t.

64

-

RVG-29-anchored nanoparticle

Itraconazole

n.t.

Streptavidi n-biotin

RVG-29-ITZ-NPs: 89.3 ± 1.9 nm

-33.1 ± 0.9 mV

n.t.

Drug release in 10% FBS

83

-

RVG-29-PEGgold NR@SiO2

-

20 ± 2.5 µg RVG-29/ mg AuNRs@SiO2NH2

MALPEG5kNHS

Length: 117.7 ± 7.3 Width: 50.3 ± 3.1 nm Aspect ratio: 2,34

+14.2 ± 2.5 mV

n.t.

n.t.

69

+Biotin

DDS

Cargo

Conjugation yield

25



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 57

3. RVG-29-functionalized DDS 420 RVG-29 was used as a CNS-targeting motif in a wide variety of DDSs. These DDSs differ from each other in their different RVG-29 peptide modifications, the type of DDS functionalization and the physicochemical properties of the resulting DDS. Table 2 gives a summary of the published RVG-29 targeted DDSs, providing detailed information on their 425

formulation, design and characterization. The main characteristics of the materials with respect to composition and therapeutic potential are discussed below.

3.1. 430

Nucleic acid-RVG-29 polymer complexes

RVG-29 peptide has been used for the non-viral delivery of genes to the CNS using cationic polymers for nucleic acid complexation. Complexes with nucleic acids are formed by electrostatic interactions of positively charged polymers and negatively charged nucleic acids, leading to the formation of so-called polyplexes. Disulfide-branched polyethylenimine (PEI)62, 63, trimethylated chitosan66, poly(cystaminebisacrylamidediaminohexane) grafted with

435

9-11 residues of arginine (PAM-ABP)70, polyasparthydrazide67, polyamidoamine68, poly-Llysines4 and β-modified cyclodextrins89 have all been used as polycationic material for complexation with nucleic acids. The attachment of the peptide to the polymer is carried out via the use of heterobifunctional linkers such as N-hydroxy-succinimid-polyethylene glycolmaleimide

440

(NHS-PEG-Mal)62,

63,

66-68

,

(4-succinimidyloxycarbonyl-α-methyl-α-[2-

pyridyldithio]toluene) (Sulfo-LC-SMPT)4 and 3-(Maleimido)propionic acid N-succinimidyl ester89. Amine-reactive NHS-ester enables coupling to the polymer, whereas a maleimide or pyridine-2-thione functionality is used for the covalent coupling of the RVG-29 sequence. 26 ACS Paragon Plus Environment

Page 27 of 57

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


In addition to the original RVG-29 sequence,62, cysteine4, 445

66, 67, 89

63

RVG-29 sequences with an additional

have been used for the formation of nucleic acid-RVG-29 polymer

complexes. The impact of the use of the native cysteine for the coupling reaction has been discussed above (see 2.3.1). Another strategy is employed by Beloor et al., who use 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)/NHS as coupling agent to activate the carboxy group of the C-terminal of the RVG-29 peptide and bind it to the arginine residues on a polymer.70 However, it cannot

450

be excluded that the free carboxy groups of glutamate and aspartate in the RVG-29 sequence can react with the polymer as well. This can result in a variety of orientations of the RVG-29 peptide that can directly impact the receptor binding affinity, as each type of reaction product results in a different presentation of the RVG-29 peptide to the receptor. Introducing polyethylengylcole (PEG) chains as spacers between a DDS and RVG-29

455

reactive groups is a frequently used option. Furthermore, the PEGylation of DDSs can increase their biocompatibility and extend their circulation time in the bloodstream.92 The presentation of the RVG-29 peptide on the distal end of a PEG chain might facilitate binding, in contrast to functionalized DDSs without spacers. This has been shown for antiHER2 antibody fragment (Fab´)–targeted liposomes.93, 94

460

The quantitative analysis of the final amount of RVG-29 peptide is estimated by different analytical approaches. Son et al. use the fluorescence of tryptophan in the RVG-29 sequence and measure the increase in molecular weight of RVG-29-functionalized polymers by GPC analysis.63 Successful coupling can also be shown in 1H-NMR analysis by the disappearance of the maleimide peak. The amount of RVG-29 is calculated by integrating the 1H-NMR

465

spectra.

66, 89

Another approach is the quantification of RVG-29 by using Ellman’s assay to

detect free thiol groups of the cysteine in the sequence.63



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 57

Polyplexes of nucleic acids and RVG-29 polymers are characterized by measuring size, surface charge and gel retardation, which depends on the RVG-29 polymer–to–nucleic acid ratio. By increasing the electrostatic interactions between nucleic acid and polymer, the size 470

of complexes decreases. Agarose gel electrophoresis is used to identify the required ratio of siRNA or DNA (P) to RVG-29-polymer conjugates (N) or to analyze the serum stability of complexes. 3.2. Nucleic acid–RVG-29-9R/protamine complexes

475

Similar to RVG-29-polymer complexes, RVG-29-nona-arginine or protamine conjugates are formed by electrostatic interactions between positively charged poly-arginine or protamine sequences and negatively charged nucleic acids. The described nona-arginine sequences consist either of D-arginine72, 74-76, 81, L-arginine81 or alternating D- and L-arginine residues78. Ye et al. use protamine as a complexing component.80 The poly-arginine and protamine

480

conjugates can be directly synthesized without the use of additional linker chemistry and are added to the C-terminal of the RVG-29 peptide. Like in the polymer complexes, the formation is characterized by the analysis of size, surface charge and gel retardation. Again, the positive charge is a common property for all complexes. 3.3. RVG-29-functionalized nanoparticles

485 Nanoparticles have some advantages over polyplexes with respect to their stability. The encapsulation protects drug molecules such as nucleic acids against degradation, and, furthermore, PEGylated nanoparticles show prolonged blood circulation time.84, 88, 95 In contrast to polyplexes, functionalization with RVG-29 takes place after the formulation of 490

nanoparticles. The use of chitosan-conjugated pluronic-based micelles as a transport system with additional RVG-29 peptide modification to enable receptor-mediated uptake is one example of RVG-29-modified nanoparticles.88, 96 To form these micelles, RVG-29 peptide is 28 ACS Paragon Plus Environment

Page 29 of 57

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


conjugated at the C-terminal to an NHS-PEG-Mal linker on a polymeric micelle. The conjugation yield is estimated by 1H-NMR analysis and the fluorescence of tryptophan in the 495

RVG-29 peptide sequence. The sizes of the different polymeric micelles are determined at different temperatures to show the thermosensitive effect of the pluronic. Surface charge is analyzed at 25° C and shows the impact of the chitosan modification of the polymeric micelles. Bare polymeric micelles have a neutral charge and show no change in surface charge after RVG-29 modification and encapsulation with β-Galactosidase. However,

500

additional modification with chitosan causes a shift to positive values of the polymeric micelles (~12 mV). β-Galactosidase release is estimated at 10% FBS at 37° C. RVG-29 peptides are presented on the distal end of PEG chains with free N-terminals. The additional chitosan modification allows the binding via receptor and electrostatic interactions. Cook et al. encapsulate camptothecin in RVG-29-functionalized PLGA particles.

505

Functionalization is achieved by an avidin-biotin linkage, presenting the RVG-29 peptide with a free N-terminal.82 Besides nanoparticles, RVG-29-functionalized liposomal formulations are described as well. Tao et al. present RVG-29-functionalized liposomes as a DDS for the delivery of siRNA. The liposomes were composed of 1,2-dioctadecanoyl-sn-glycero-3-phosphoethanolamine (DSPE),

510

cholesterol

(CHO)

and

1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-

[maleimide(polyethylene glycol)-2000] (DSPE-PEG-Mal).90 Due to the thiol reactivity of the maleimide group, coupling reactions could take place either with the cysteine located in the middle or on the C-terminal of the RVG-29 peptide. Furthermore, liposomes containing positively charged phospholipids are used for the delivery 515

of nucleic acids. They are also referred to as stable nucleic acid lipid particles.77 Conceicão et al. use 1,2-dioleoyl-3-dimethylammonium-propane (DODAP) as the positively charged lipid in their formulation. The liposomes are further functionalized with RVG-29-nona-arginine 29 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 30 of 57

and conjugated by the reaction of DSPE-PEG-Mal with the cysteine located in the middle of the RVG-29 sequence. Another interesting approach is shown by Pulford et al. This group 520

encapsulates siRNA-RVG-29-nona-arginine complexes into positively charged liposomes to enhance the stability of the siRNA complex.76 In contrast to the other DDSs, in this study, the RVG-29-peptide is not present on the outer surface of the DDS, but rather hidden by a positively charged lipid bilayer of liposome. Lee et al. analyze rabies virus–mimetic silica-coated gold nanorods. By doing so, they are the

525

only ones who take the shape of the rabies virus into consideration in a DDS design. They use non-spherical gold nanoparticles with a SiO2 shell. Functionalization is performed with MALPEG5k-NHS and RVG-29 with an additional cysteine. Conjugation yield is determined by HPLC analysis.69 3.4. RVG-29-functionalized exosomes

530 Exosomes are natural nano-vesicles secreted by numerous cell types.65,64,

97

As a result, they

make an excellent DDS, mimicking natural cell vesicles. Due to their cell-like membrane structure, exosomes are less likely to elicit a foreign body response, thus causing less-adverse effects.98 In the literature, two exosome formulations are presented as promising a BBB535

targeted DDS for gene delivery.64, 65 In these studies, researchers use self-derived dendritic cells for exosome production. An advantage of exosomes is that they contain a number of membrane proteins, which are exclusively expressed on the exosomal surface.99 Lamp2b is one such exosomal membrane protein, which can be used to functionalize exosomes by fusing the RVG-29 peptide to the extra-exosomal N terminus. The RVG-29-Lamb2b functionalized

540

exosomes are analyzed with regard to their size using nanoparticle tracking analysis and electron microscopy. The successful expression of RVG-29 Lamp2b is assessed with quantitative PCR. The loading of exosomes is achieved with electroporation.64,

65


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Table 3: Summary of performed in vitro experiments on RVG-29-targeted DDSs. The table gives an overview of the cellular uptake studies of the RVG-29-functionalized DDS. Each DDS is listed with its physicochemical properties, the cargos and cells used and the studies performed on the cellular uptake.

31



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

No .

DDS

Cargo

Cell line

Physicochemical characterization

1

RVG-29-conjugated BPEI

Firefly luciferase coding sequence region in pcDNA3.1 (+) vector

Neuro2a (nAchRpositive)

RVG-29: unmodified sequence

HeLa (nAchR negative)

Size: ~200 nm

Cy5.5-labeled miR-124a oligomer

Neuro2a (nAchRpositive)

CMV promoter driven Gaussia luciferase vector

HeLa (nAchR negative

PPIL2 siRNA Carboxyfluorescein -labeled siRNA

Neuro2a (nAchR-positive)

2

3

RVG-29-conjugated BPEI

RVG-29-peptide linked siRNA/TMC-PEG

HeLa (nAchR-negative) 5

6

Polyion complex RVG-29 peptide tagged PEGylated polyasparthydrazide derivatives

PAMAM-PEG-RVG29/DNA

PPIL2 siRNA Negative control siRNA FAM-labeled siRNA Cy5-labeled siRNA pEGFP-N2 and pGL2-control vector

Neuro2a (nAchR-positive)

Charge: 0 RVG-29: unmodified sequence Size: 290.5 nm (Polymer/miR.124a wt of 6.6) Charge:+ RVG-29: +C Size: siRNA/TMC-PEGRVG-29 :~ 200 nm Charge: + RVG-29: +C

HeLa (nAchR-negative)

Size: RVG-29functionalized micelles: ~ 250 nm

BCECs

Charge: + RVG-29: +C

Page 32 of 57 Cellular uptake

Ref.

Confocal microscopy: Competitive inhibition assay using free RVG-29: Negative control: BPEI-SS-PEG/pDNA polyplex

63

Fluorescence intensity: Detection of miR-124a release from RVG-29-SSPEI/miR-124a-complex: Negative control: RVG-29-SSPEI +scramble miRNA

FACS: Negative control: siRNA/TMC-mPEG complexes CLSM: In vitro gene silencing: Expression of BACE1 was 57% for unmodified complexes and 50% for RVG-29-functionalized complexes FACS/CLSM: Negative control: COOH-PEG-g-PAHy-GTA/FAM-siRNA micelles In vitro gene silencing: Reduced expression of BACE1 detected by Western Plot analysis

62

66

67

Cellular uptake by fluorescent microscopy: Negative control: PAMAM-PEG/DNA

Size: ~150 nm Charge: n.t.

Cell uptake mechanism: ↓ uptake at 4° C, prior incubation with free RVG-29 or GABA, endocytosic pathway inhibitor filipin,

68

Transport studies 7

Dendigraft-polylysineRVG-29-FRET

Cy5 labeled nine amino acid peptide

SH-SY5Y

RVG-29: +C Size: ~5 nm

In vitro caspase-3 activation model: 4

Charge: n.t.

32


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No .

DDS

Cargo

Cell line


8

RVG-29-functionalized βCyclodextrins

Fluorescein-tagged siRNA

U87 HeLa

RVG-29: +C Size: RVG-29functionalized cyclodextrin formulation (R3): ~250 nm

GAPDH siRNA

Cellular uptake

Ref.

Cellular uptake in U87: Negative control: free siRNA, cationic CD siRNA complex, PEGylated CD Control: Lipofectamine + siRNA Competitive inhibition assay: Preincubation with RVG-29

89

Charge: +

9

10

11

12

siRNA-RVG-29-9dR

siRNA-RVG-29-9dR

Liposome-siRNA-peptide complexes

RVG-29-9R-p137

Biotinylated RVG29 or RVG-Mat Anti GFP siRNA

FITC-siRNA

PRP (cellular prion protein) siRNA

p137-RVG-29-9r

Neuro2a (nAchR-positive) BHK21 292T HeLa CHO (nAchR-negative)

RVG-29: GGGG+9dR

Raw 264.7 N9 Primary splenic macrophages of wild type and AchR knockout mice Neuro2a (nAchR-positive) HEK293 HeLa 4T1 (nAchR-negative)

RVG-29: GGGG+9dR

SH-SY5Y HEK293 U373

RVG-29: GGGG+9dR

GAPDH knockdown analysis: Negative control: naked siRNA, cationic cyclodextrin w/o siRNA Positive control: Lipofectamine+siRNA Cellular uptake of RVG-29-peptide: Competitive inhibition of RVG-29 by α-bungarotoxin in dose-dependent manner

Size: n.t. Charge: n.t.

Size: n.t.

Cellular uptake of RVG-29-9R/siRNA complexes: Negative control: RV-Mat-9R/siRNA complexes Positive control: Lipofectamine

72

GFP-silencing: nAchR-expression: Cellular uptake of RVG-29-FITC on primary macrophages: Negative control: scrambled RVG-29-FITC 73

Charge: n.t.

RVG-29: GGGG+9dR Size: ~180 nm

Cellular uptake of RVG-29-9dR/FITC-siRNA complexes: Gene silencing in Raw 264.7 cells: Uptake to Neuro2a (MFI): Competitive inhibition of RVG-29-9r with liposome in Neuro2a: Excess of unlabeled RVG-29-9r or RVM-9r

76

Charge: + Cell specificity (MFI):

Size: n.t.

PrP siRNA-RVG-29-9r LSPC suppress expression: α3/α5nAchR-expression in SH-SY5Y, HEK293, U373: Cellular uptake in α3/α5nAchR –positive U373 cell line Negative control: naked siRNA, RVMat9R-p137 and RVG-299R-pXef

75

Charge: n.t. Protection of SH-SY5Y from rotenone-induced cell death: Negative control: RVG-299R- scrambled p137, RVMat9R-p137

33



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

No .

DDS

Cargo

Cell line


14

RVG-29-9rR/pDNA

pEGFP

Neuro2a (nAchR-positive) HeLa (nAchR-negative)

RVG-29: HHHHrRrRrRrRr Size: ~80 nm

Cellular uptake Cellular uptake of RVG-29-9rR/pDNA in Neuro2a: Negative control: 9rR/pEGFP-N1, naked plasmid Inhibition of endocytosis at 4° C Internalization indicated by LysoTracker

Page 34 of 57

Ref.

78

Charge: +

15

17

RVG-29-9R-6His/PDNA

RVG-29+9D/LR-siRNA

pRNATU6.3/Hygro encoding green fluorescent protein

GFP

Neuro2a (nAchR-postive) HeLa BV-2 BHK-21 (nAch-negative) Neuro2a (nAchR-postive)

RVG-29: +9R-6His Size: < 200 nm

Brain targeted SNALPS

Si-EGFP FAM labeled siRNA siMutAtax3

Neuro2a (nAchR-positive) HT-22 HeLa (nAchR-negative)

Specificity of cell recognition: Positive control: Lipofectamine

79

Charge: + RVG-29: +9R

Cellular uptake :

Size: ~200 nm

Gene silencing (Increase of GFP-negative cells %): Positive control: Lipofectamine Negative control: 9DR/LR-siRNA

Charge: +

20

Luciferase expression assay: Positive control: Lipofectamine Neuro2a cells-based ELISA:

RVG-29: GGGG+9R

81

Cellular trafficking: Cellular uptake: Negative control: Non-targeted SNALPs, RV-MAT-9R targeted liposomes

Size: < 200 nm siRNA delivery in Neuro2a cells: 77

Charge: n.t. Uptake inhibition: Uptake was reduced at 4° C and inhibited by free RVG-29 peptide

21

RVG-29-peptide modulated liposomes

FAM-labeled siRNA EGFPsiRNA+Protamine

BMM U87

RVG-29: +C Size: ~90 nm

Gene silencing: Cellular uptake in BMM cells: GFP expression silencing: Negative control: naked GFP-siRNA, RVG-29-liposome

90

Charge: -

34


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 545 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

Nu m be r 22


DDS

Cargo

Cell line


Cellular uptake


siRNA alphasynuclein

SH-SY5Y

Gene silencing:

GAPDH siRNA BACE1 siRNA

C2C12 (muscle) Neuro2a

RVG-29: unmodified sequence Size: n.t. Charge: n.t. RVG-29: unmodified sequence

23

Ref.

65

Gene silencing: Positive control: Lipofectamine Negative control: naked siRNA, unmodified exosomes

64

Cellular uptake on bEND3:

83

Cellular uptake Compared to spherical gold-NPs Inhibited by α-bungarotoxin

69

Size: ~80 nm Charge: n.t. 24

RVG-29 anchored nanoparticle

Itraconazole

25 RVG-PEG-gold-NRs @SiO2

bEND3 Neuro2a (nAchR-positive)

HeLa (nAchR-negative)

RVG-29+C Length: 117.7 ± 7.3 nm Width: 50.3 ± 3.1 nm Aspect ratio: 2,34 Charge: +

35



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Page 36 of 57

4. In vitro testing of RVG-29-functionalized DDS In vitro testing of RVG-targeted DDSs should provide an answer of whether functionalization 550

with RVG enables receptor-mediated uptake in relevant cell lines. By using RVG-29 as a CNStargeting peptide, the functionalized DDS should follow the pathway of the rabies virus. Therefore, the cell lines and in vitro experimental designs used should reflect the pathway of the rabies virus. The uptake mechanism of the rabies virus has been described as a combination of receptor- and adsorptive-mediated uptake. According to the classical pathway of the rabies virus,

555

muscle cells and neurons are of particular relevance and could be, therefore, used for in vitro testing of RVG-targeted DDSs. 4.1. Interaction of RVG-modified particles with primary cells and cell lines

The in vitro effect of RVG-functionalized DDSs on cells has been investigated in several studies 560

(Table 3). Figure 2 gives an overview of the cell lines used, including their origin and receptor expression. The uptake of RVG-functionalized DDSs by neuronal cell lines, such as Neuro2a expressing nAchR (nAchR-positive), compared to non-neuronal cell lines, such as HeLa, BHK21, 292T, CHO, HEK293, 4T1, BV-2 and C2C12, which do not express nACH receptors (nAchR-negative),35, 62-64, 66, 67, 72, 76-79, 81 has been investigated by a number of groups. Neuro2a

565

cells are derived from mouse neuroblastoma. An important question concerns the involvement of the neuronal nAchR subunits. In the classical rabies pathway, only the interaction of RVG with the nAchR α-1-subunit, which is typically expressed in muscle cells, has been described.27 The uptake of RVG-targeted DDSs in Neuro2a cells has allowed researchers to draw conclusions about the receptors relevant to the uptake of the rabies virus and RVG-29-targeted DDSs, as

570

Neuro2a cells express several neuronal nAchR subunits and NCAM and p75NT receptors.


36

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Therefore, this cell line is ideal for investigating the cell uptake of an RVG-functionalized DDS by receptor-mediated effects.35, 100 However, Neuro2a cells are derived from neuroblastoma and, therefore, cannot be used for modeling the rabies virus pathway.101 Although the rabies virus does not belong to the group of viruses which enter the CNS via the 575

BBB,6, 15, 17, 102 the uptake of RVG-modified nanoparticles has also been assessed using brain capillary endothelial cells (BCECs)68 and the mouse brain endothelial cell line bEnd.383, 103. The uptake of RVG-modified nanoparticles in these cell lines sheds new light on the uptake characteristics of RVG-functionalized DDSs. These studies assume that RVG-functionalized DDSs also target non-neuronal cells at the BBB. The bEnd.3 is an immortalized mouse

580

endothelial cell line, which expresses several typical BBB transporters and receptors, such as glucose and l amino acid transporters and p-glykoprotein receptors, and important tight junction proteins.104 Due to its BBB functionality, this cell line is widely used for testing interactions of DDSs with the BBB.5, 90, 105 The observed uptake of RVG-modified DDSs in these cells can be a result of the involvement of further unknown rabies receptors but can also be due to electrostatic

585

interactions between the RVG-functionalized DDS and the endothelial cell membrane. This is also underlined by the results of Tian et al., who analyze several poly(2-(diisopropylamino)ethyl methacrylate) (PDPA)-based polymersomes functionalized with either Angiopep-2 or RVG-29. Although RVG-29-functionalized polymersomes show uptake in bEnd.3 cells, RVG-29functionalized polymersomes show no internalization in the later-used 3D in vitro model

590

composed of brain endothelial cells cocultured with a mouse astrocyte cell line and pericytes.103 Further experiments are required to create a complete picture of the pathway of an RVG-targeted DDS. In vitro testing on human glioblastoma cell line U87 aims at the application of RVGtargeted DDSs for the treatment of cancer.75,

89, 90

The use of the SH-SY5Y cell line directly


37


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Page 38 of 57

addresses the application of RVG-modified DDSs in Parkinson’s disease.4, 595

65, 75, 106

The SH-

SY5Y is a neuronal tumor cell line.107 Interestingly, it expresses several neuronal nAchR subunits: the α3, α5, β2 and β4 and the α7-subunit.75,

108, 109

The α7-subunit is the prime

candidate for the receptor-binding of an RVG-modified DDS among the nAchR subunits. Like the α-1-subunit, which is used in the receptor-binding studies of RVG, the α-7-subunit is also able to bind the antagonist α-bungarotoxin. This fact demonstrates structural similarities between 600

the two subunits, which might also enable the binding of the rabies virus and RVG-modified DDSs on the neuronal α-7-subunit. With regard to pathological conditions of the brain, microglia cells play an important role as an immune barrier.5,

58, 73

. Kim et al. investigate the RVG-

targeting effect on macrophages (Raw 264.7), the glial cell line (N9) and primary macrophages obtained from wild-type mice, all expressing the α7-subunit of nACHR on their surface. The 605

group uses the wild-type primary macrophages to assess the binding of the RVG-29 peptide. The results assume that the FITC-labeled RVG-29 is also able to bind at the primary macrophages via the α7-subunit 73.


38

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610

615


Figure 2 Overview of the cells used for in vitro testing of an RVG-functionalized DDS, including their location and receptor expression. Neuronal cell lines directly mirror the classical pathway of the rabies virus along the axonal nervous system. This includes neuronal cells such as the Neuro2a and the SH-SY5Y cell lines, which are said to express several identified rabies receptors, such as nAcHR, NCAM and p75NTR, and the potential rabies receptor GABA. Microglia cells such as N9 play a role in the immune response of the brain and are also expressed with nAchRs. The use of brain endothelial cells directly targets a potential uptake of an RVG-functionalized DDS across the BBB. The mouse endothelial cell line bEnd.3 and BCECs are used. In contrast to the nAchR-expressing cells, no rabies virus receptors have been identified. This is the same for the glioblastoma cell line U-87, which shows the application of RVG-targeted DDSs in the field of cancer therapy.

4.2. Receptor-mediated uptake

The first step of the analysis of an RVG-modified DDS should be the proof of the selective 620

uptake of the targeting peptide RVG-29 itself. This is impressively shown by the work of Kumar et al., who prove the selective uptake of biotinylated RVG-29 peptide compared to the RV-Mat peptide, which is used as a negative control, on Neuro2a cells.72 The preliminary investigation of the uptake of RVG-29 peptide alone is also performed by Kim et al. They show that an FITClabeled RVG-29 peptide is taken up by primary macrophages of wild-type mice. In contrast,

625

primary macrophages of nAchR-knockout mice show no uptake of the RVG-29 peptide.73


39


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Page 40 of 57

The mechanism of receptor-mediated uptake of the RVG-modified DDS is analyzed by cellular uptake studies conducted at 4° C and compared to 37° C68, 78 or by competitive inhibition with free RVG-29 peptide63, 72, 89,76 agonists or antagonists. This is, for instance, shown by Liu et al., who analyze the cellular uptake of RVG-modified nanoparticles in BCECs. The results suggest a 630

receptor-mediated uptake via the GABA receptor, which is indicated by a reduced uptake after the pre-incubation with free GABA. The prior incubation with potential inhibitors of the nACHR (acetylcholine, mecanylamine and nicotine) cause no reduced uptake of RVG-modified nanoparticles.68 The suggested pathway is of high interest as the involvement of GABA in the pathogenesis of rabies has not been described so far. Furthermore, the potential involvement of

635

GABA receptors is also shown for the RVG peptide RDP. The 39 amino acid–long peptide sequence is partly derived from the 330-357 amino acid sequence of RVG and shows a reduced uptake in SH-SY5Y cells in the presence of free GABA.110 Therefore, further investigations with respect to a potential binding of RVG-29 at GABA receptors are required. In addition to the inhibition studies, BCECs have been incubated with different endocytosis

640

inhibitors to investigate the endocytosis mechanism. Furthermore, the nACHR receptor specificity for Neuro2a has been proven by the reduced uptake of RVG-29-peptide after incubation with α-bungarotoxin.64, 72


40

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4.3. Do RVG-29-functionalized DDSs follow the classical rabies virus pathway? 645 The use of RVG-29 as a CNS-targeting peptide is a promising strategy, underlined by its wide application as a targeting peptide for CNS-targeting strategies.29 Although the uptake of the presented RVG-29-functionalized DDS into the CNS is proven by in vivo testing, the data lack an analysis of the precise way the DDS functions from the injection site to the CNS. 650

The higher uptake of RVG-targeted DDSs in neuronal cells compared to non-neuronal cells clearly reveals an uptake mechanism that is similar to that of the rabies virus with respect to receptor binding. Unfortunately, the selected cell lines do not mirror the pathway of the rabies virus. The fact that an RVG-functionalized DDS also shows cellular uptake in brain endothelial cells suggests that the uptake mechanism of these DDSs differs from that of the rabies virus.

655

Two major aspects have to be taken into account. First, the presentation of the RVG peptide on the surface in the DDS and, second, the overall surface net charge. The orientation of an RVG molecule on the surface is highly dependent on the type of DDS and the selection of the RVG-29 modification. While RVG is organized in trimeric spikes at the surface of the rabies virus,17, 69 it is questionable whether RVG-29-functionalized DDSs need a similar arrangement of RVG-29 on

660

their surface to enable receptor binding. This point is only addressed by Lee et al., who analyze the impact of increasing surface density of RVG-29 on the uptake. However, it is questionable whether the higher uptake with an increasing surface density of RVG-29 is a result of active receptor binding or only due to an increase in the positive surface charge of the whole construct. To our surprise, neither the different ways of RVG-29 orientation nor the amount of RVG-29 on

665

the surface of the presented DDS seem to have an impact on cellular uptake. Therefore, it is arguable that RVG-functionalized DDSs are endocytosed via highly selective receptor interactions. Lentz et al.’s results clearly show that receptor binding affinity at the α-1-subunit is


41


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Page 42 of 57

highly dependent on the primary sequence of the RVG peptide.27 These facts emphasize an uptake of an RVG-functionalized DDS, which is mediated via the electrostatic and hydrophobic 670

interactions between the RVG-peptide coupled onto the surface of the DDS and cell membranes.29 Another important fact, which is raised by Lee et al., is the shape of RVG-29functionalized DDSs. They design their DDS according to the physicochemical properties of the rabies virus.69 The bullet-shaped gold nanorods show a higher uptake in Neuro2a cells than spherical gold nanoparticles. This clearly emphasizes that shape has an impact on the uptake of

675

RVG-29-functionalized DDSs. Furthermore, it raises the question of whether RVG-29functionalized DDSs with a clear difference in shape from the rabies virus are generally able to be taken up by the cell and transported along the long axonal neurons. This can only be proven by tracking an RVG-29-functionalized DDS after intravenous application. This is only shown by Lee et al., who prove the uptake of their RVG gold nanorods in ex vivo images of the spinal

680

cord.56 The presented data are not sufficient to find an answer to the question posed. So far, a clearly identified gap in the in vitro models used is the missing analysis of an active receptor binding at the rabies virus receptors. Several factors such as shape, RVG-29 surface density and peptide stability have been stated as influences on receptor binding. The authors clearly encourage

685

analyzing the impact of the stated facts for a deeper understanding of CNS-targeting strategies. The second identified gap is that the current in vivo data only prove targeted delivery into the CNS but are not able to verify whether RVG-29-functionalized DDSs exactly follow the pathway of the rabies virus. However, an important fact is that the pathway of the rabies virus has still not been completely identified. For example, it is still not known whether the rabies

690

virus requires a specific order of receptor interaction or whether all stated receptors can be used


42

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for initial entry into the CNS. In addition, it cannot be ruled out that further receptors are involved in the uptake process.15, 29

5. In vivo testing of RVG-functionalized DDSs

695 The question of whether RVG-modified DDSs show uptake into the CNS is finally answered by an in vivo analysis of RVG-modified DDSs and is shown for most of the presented DDSs. The targeted uptake into the CNS is shown for RVG-29-functionalized DDSs by comparing them to the uptake of the DDSs’ used cargo78, the unmodified DDSs62, 700

63, 66, 67, 90

or the used DDSs

functionalized with scrambled versions of RVG, instead of the CNS-leading RVG-29 sequence72, 76

. In addition to proving CNS-targeting using fluorescent cargoes, active cargoes are used to

detect luciferase activity68, 78 , caspase-3 activation4 and the expression or silencing of genes64, 65, 72, 76

. As the performed in vivo experiments mainly focus on the final delivery of the RVG-29-

functionalized DDS into the brain rather than showing the complex fate of RVG-29705

functionalized DDSs, it is still questionable whether RVG-29 targeting results in delivery via the long axonal transport. Therefore, a focused analysis of the pathway of RVG-29-functionalized DDSs is highly required, as this is the only possibility for unveiling the targeting abilities of the RVG-29 peptide. In that regard, the authors want to highlight the work of Kumar et al., Cook et al. and Lee et al.,

710

whose contradictory works underline the importance of a deeper investigation of the CNStargeting effect of RVG-29 (see Chapter 7). While Kumar et al. are one of the first to show the potential of RVG-29 as a CNS-targeting motif, Cook et al. argue for the CNS-targeting potential of RVG-29. Kumar et al. show a targeted delivery of RVG-29-9R-FITC-siRNA to the brain after 10 hr, which is not seen for the negative control RVG-Mat-9R-FITC-siRNA and FITC-siRNA


43


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715

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alone. The microscopic examinations of the different brain sections reveal the detection of RVG29-9R-FITC-siRNA in the cortex, thalamus and the striatum. Furthermore, the RVG-29 functionalization has an application in mice infected with Japanese encephalitis virus (JEV). Mice treated with the antiviral siRNA/RVG-29-9R complex show 80% survival. In contrast, mice treated with siRNA alone, luciferase siRNA or siRNA complexed with RV-MAT-9R die

720

within 10 days.72 In contrast, Cook et al.’s results challenge the CNS-targeting potential of RVG29.82 They quantify the delivery of the fluorescence marker DiR, encapsulated in RVG-29functionalized or biotin-functionalized nanoparticles, to specific CNS regions. These results provide only minimal evidence of a targeted uptake via the spinal cord into the CNS. A higher uptake of RVG-functionalized nanoparticles is shown only in the cortex region. This observation

725

leads to an assumption about the participation of GABAB receptors in the entry mechanism of the rabies virus, as these receptors are mainly located in this region. Cook et al. further refer to the results of Liu et al., who show that the uptake of RVG-modified nanoparticles is inhibited by GABA in brain endothelial cells.68 This is an interesting hint, as the GABA receptor is not described as a rabies receptor and should, therefore, be taken into account for a deeper analysis

730

of receptor participation (see 4.2). Furthermore, Lee et al. demonstrate an uptake via the spinal cord for bullet-shaped RVG-29-gold-nanorods-SiO2 compared to spherical RVG-29-gold nanoparticles.69 These results emphasize the ex vivo analysis of spinal cords for the proof of a transport of RVG-29-functionalized DDSs along the long axonal transport. Furthermore, they highly encourage an examination of whether RVG-29-functionalized DDSs are able to mimic the

735

rabies virus.


44

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6. Future Outlook The use of the RVG-29 peptide for targeted delivery into the brain is a promising strategy. A proof of targeted delivery of RVG-29-functionalized DDSs should be done using a point-to-point 740

comparison of a functionalized DDS with the original virus. While there is a significant amount of knowledge about the original virus with respect to physicochemical properties (size, shape) and uptake mechanism, a detailed characterization of RVG-29-functionalized DDSs is missing. This section focuses on identified gaps, which should be filled in order to prove the CNStargeting effect of RVG-29 and RVG-29-functionalized DDSs.

745

For in vitro testing, the key question is whether RVG-29-functionalized DDSs follow exactly the same route as the rabies virus to the CNS. Most cell lines are selected only due to their receptor expression of rabies virus receptors but do not mirror the pathway of the rabies virus. A precise selection of cell lines based on the pathway of the rabies virus is highly needed to assess whether RVG-29-functionalized DDSs follow exactly the same route. Therefore, peripheral

750

muscle cells, an in vitro model of the NMJ and long axonal neurons are of utmost interest for in vitro testing of RVG-29-functionalized DDSs. The use of brain endothelial cells addresses a potential direct uptake of RVG-29-functionalized DDSs. A deep analysis of the cellular uptake mechanism is advisable to assess which mechanism and which receptors are involved in the cellular uptake of RVG-29-functionalized DDSs at the BBB.

755

Another important fact to focus on is the potential impact of other subtypes of the nAchR on the uptake mechanism of the rabies virus and RVG-29-functionalized DDSs. Lentz et al. show that RVG-29 is able to interact with the α-1-subunit, which can be found in muscle cells.26, 27, 31 This interaction explains the initial entry of the rabies virus in muscle cells; however, as RVG-29 can be inhibited by prior incubation with α-bungarotoxin,72 the uptake of RVG-29 should be


45


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analyzed with other α-bungarotoxin-sensitive nAchR subtypes such as α7 or α4β2. These subtypes are widely distributed in the mammalian brain29, 37, 111 and, therefore, important targets with respect to a targeted delivery of the rabies virus and RVG-29-functionalized DDSs to the CNS. To our surprise, none of the presented RVG-29-functionalized DDSs address the uptake

765

machinery of the rabies virus. A final delivery is not only dependent on the initial receptor binding, but also heavily depends on the interaction with axonal proteins. The work of Gluska et al. nicely shows how the hijacking of RVG-29-functionalized DDSs can be analyzed. They use live cell tracking to analyze the entry of the rabies virus at the NMJ and the retrograde transport along the axon.8

770

The missing direct comparison of RVG-29-functionalized DDSs and the original viral particle has been identified as a major drawback to the physicochemical characterization of the presented DDSs. The authors emphasize the work of Lee et al., who design their DDS by mimicking the rabies virus.69 By doing so, they identify concrete values for size, shape and surface density of RVG-29 on the DDS. Their role model is a bullet-shaped particle with a length

775

of 180 nm and width of 65 nm17 with around 400 trimeric spikes of RVG, which covers the surface of the rabies virus.15, 69, 112 Therefore, the size, shape and surface density of RVG-29functionalized DDSs should be compared to the properties of the original viral particles. In particular, the impact of different shapes, densities and presentations of RVG-29 on the surface of the DDS should be addressed to understand the big picture. This can be further underlined by

780

the results of Lee et al. They show a higher uptake for bullet-shaped nanorods than for spherical nanoparticles in vitro and in vivo. In addition, they show an increased uptake with increasing surface density of RVG-29 on the surface of the nanorods (2-20 µg/mg gold-NRs).69


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The use of RVG-29 as a CNS-targeting peptide requires several modifications to enable the coupling of the peptide to the DDS. The impact of these modifications on cellular uptake is 785

barely discussed. A prior analysis of the cellular uptake of the RVG-29 modification compared to RVG-29 is required by the authors to understand the impact of such modifications on cellular uptake. With regard to in vivo analysis, the authors strongly suggest including the analysis of the spinal cord as proof of an uptake in accordance with the original pathway of the rabies virus, as shown

790

by Cook et al. and Lee et al..69, 82 In addition further possible pathways to the CNS need to be assessed. So far, there is no evidence for transport across the BBB based on the pathway of the rabies virus. However, as the majority of the presented RVG-29-functionalized DDSs clearly show a targeted uptake into the CNS, the question of how they enter it is of utmost importance and should be answered prior to further application of RVG-29 as a CNS-targeting peptide.

795

7. Summary and conclusion The rabies virus carries a decisive factor for entry into the CNS, controlled by the external surface glycoprotein RVG. Although the complete infection pathway of the rabies virus is still not understood, nAchR, NCAM-1, p75NTR and interactions with carbohydrates, gangliosides

800

and lipids hold a stake in its entry into the CNS. It is assumed that the virus uses a dual-entry strategy of receptor and electrostatic interaction. This is further emphasized by the identification of a 29mer peptide derived from RVG binding to an α-1-subunit of nAchR and the fact that this peptide has an overall positive charge under physiological conditions, enabling electrostatic interactions with cell membranes. For the functionalization of DDSs, the RVG-29 sequence

805

undergoes modifications. The impact of these modifications with regard to the uptake mechanism still needs to be investigated. The functionalization with RVG-29 is applied to


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several DDSs, such as those used for the delivery of nucleic acids with polymer or polyarginine complexes, nanoparticles and exosomes. The targeting effect is proven by an increased uptake of targeted DDSs in neuronal versus non-neuronal cells. Although the classical pathway of the 810

rabies virus is along the long axonal nervous system, the uptake of RVG-functionalized DDSs in brain endothelial cells and macrophages emphasizes a versatile uptake strategy for these DDSs. The functionalization of DDSs with the RVG-29 sequence results in changes of the primary sequence of RVG-29 and in different physicochemical properties compared to RVG-29. Interestingly, the uptake into the used cells was independent of all changes, which could be due

815

to the significance of positive charges of the molecules for the cell uptake. Nevertheless, to identify the uptake mechanism of a targeted DDS, impact factors such as the modification of a peptide and physicochemical properties should be taken into account. The results assume a dual approach of receptor binding and electrostatic interactions, which cannot be reduced to a unique key-lock principle. Although potential CNS targeting has been shown in

820

in vivo experiments, they have not been able to record the pathway of RVG-targeted DDSs. In particular, the contradictory results of Kumar et al., Lee et al. and Cook et al. (see Chapter 5) show that it is important to record the track of RVG-29-functionalized DDSs and take the structure modifications of RVG-29 and the DDS’s own physicochemical properties into account.

825

Acknowledgements This work has received support from the EU/EFPIA Innovative Medicines Initiative Joint Undertaking COMPACT grant no. 115363. The author wants to thank Robert Hennig for the valuable discussions. Abigail Brooks is gratefully acknowledged for her support in text revision.


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835 3. 4.

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Figure 1


1


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Figure 2


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