Nose-to-brain peptide delivery – the potential of nanotechnology

Eleni Samaridou1 and Maria José Alonso

1,2*

1Center for Research in Molecular Medicine and Chronic Diseases (CIMUS), Av. Barcelona s/n,

Campus Vida, Universidade de Santiago de Compostela, 15782 Santiago de Compostela, Spain.

2Department of Pharmacy and Pharmaceutical Technology, School of Pharmacy, Universidade de

Santiago de Compostela, 15782 Santiago de Compostela, Spain.

*Corresponding Author: Prof. María José Alonso.

Department of Pharmacy and Pharmaceutical Technology, Center for Research in Molecular Medicine

and Chronic Diseases, IDIS research Institute, Universidade de Santiago de Compostela, 15782,

Santiago de Compostela, Spain.

E-mail address: mariaj.alonso@usc.es (M.J. Alonso)

GRAPHICAL ABSTRACT

Olfactory Epithelium

Nose-to-Brain Peptide Delivery

Simple Protein/Peptide solution Co-administration with permeation enhancers

Polymer-based nanosized drug delivery systems

Insulin Erythropoietin

BFGF

Chitosan

Penetratin

PLA, PLGA, Gelatin

or Chitosan NPsPEGylated NPs

Chitosan/P80

coated NPs

Surface functionalized

NPs

Protamine

Protection from proteolytic degradation

Improved uptake by the olfactory mucosa

Facilitated access to the CNS based

either on passive or active targeting

Extended half-life times and higher

concentrations in the CNS

The contribution of nanotechnology

Lipid-based nanosized drug delivery systems

NanoemulsionsNanostructured

lipid carriers (NCLs)Liposomes Cubosomes

Abstract

Nose-to-Brain (N-to-B) delivery offers to protein and peptide drugs the possibility to reach the brain

in a non-invasive way. This article is a comprehensive review of the state-of-the-art of this emerging

peptide delivery route, as well as of the challenges associated to it. Emphasis is given on the

potential of nanosized drug delivery carriers to enhance the direct N-to-B transport of protein or

peptide drugs. In particular, polymer- and lipid- based nanocarriers are comparatively analyzed in

terms of the influence of their physicochemical characteristics and composition on their in vivo fate

and efficacy. The use of biorecognitive ligands and permeation enhancers in order to enhance their

brain targeting efficiency is also discussed. The article concludes highlighting the early stage of this

research field and its still unveiled potential. The final message is that more explicatory PK/PD

studies are required in order to achieve the translation from preclinical to the clinical development

phase.

Keywords:

Nose-to-brain delivery; intranasal drug administration; therapeutic peptides; protein drugs; olfactory;

nanomedicine; nanoparticles; polymer-based nanocarriers; lipid-based nanocarriers.

1. Introduction

Neurological disorders, such as Alzheimer’s disease, Parkinson’s disease, Multiple sclerosis etc., but

also diseases like obesity, behavior disorders and sexual dysfunction have been directly associated to

different modalities of brain dysfunction. These debilitating diseases are nowadays continuously

growing, affecting more and more people worldwide. In addition to the social burden and the

individual suffering that they cause, the treatment of these diseases is also associated with very high

costs.1 Up to date, most of the drugs intended to treat CNS disorders and other brain related diseases

are administered systemically and, for this, a prerequisite is that they are able to cross the blood–

brain barrier (BBB).2,3

Unfortunately, there is a significant number of drugs, notable peptide and

protein drugs which could potentially have a powerful effect in the CNS provided that they could

overcome the BBB or acquire other routes of access to the brain.

Nose-to-brain (N-to-B) delivery may represent a non-invasive method that enables the delivery of

complex drugs to the CNS, while avoiding the BBB. This route is based on the principle that drugs

can access the CNS following a “shortcut” from the nose directly to the brain along the trigeminal or

olfactory nerves, located at the upper part of the nasal cavity. The increasing numbers of peptide and

protein drugs which may be of interest to treat chronic CNS diseases and the recent identification of

important brain functions have stimulated research in the nose-to-brain delivery field. Within this

field, the level of evidence of the value of nanotechnology for the direct CNS targeted peptide

delivery is still limited, however the knowledge generated over the last decade about this specific

topic has raised some expectancies.4,5

Based on this background information, the main objective of this article is to focus on the potential

of nanotechnology-mediated peptide delivery to the brain via the nose. More precisely, this review

will provide the reader with a view of the challenges associated to this modality of administration,

followed by the current status of the nose-to-brain peptide transport, and it will end with a critical

analysis of the value of nanotechnology as compared to that of penetration enhancers for helping

peptide drugs to reach brain targets.

2. Challenges and barriers to N-to-B peptide delivery

In early 1937, Faber reported for the first time the possibility of a direct passage from the nose to the

brain, after administering a dye in the nostrils of rabbits.6 Still, it was only in the late 90s, that the

growing interest in the field of brain delivery motivated the scientific community to start exploring

this alternative route.7–13

Mechanistic studies in animal models have proven that N-to-B drug

transport takes place either by extracellular or transcellular transport mechanisms along the olfactory

epithelium or via the trigeminal nerve, after administration of the drug into the nasal cavity (Fig.

1).14–19

Despite this increasing interest, the mechanisms underlying this direct N-to-B pathway are

not fully elucidated yet.

Pathways related to the transport of peptides from the nose directly to the brain

The olfactory region is located at the top part of the nasal cavity under the cribiform plate in close

proximity to the olfactory bulb, interlocking the nose with the brain (Fig. 1). More specifically, the

olfactory epithelium consists of three types of cells, namely the basal epithelial cells, sustentacular

cells, and the olfactory neurons with their cilia extending towards the nasal cavity (Fig. 2).16,20

More

detailed information about nasal physiology can be found in previous reviews.16,18,19,21

Figure 1. Olfactory and Trigeminal nerve position in the nasal cavity

Trigeminal

Nerve

Olfactory

Nerve

Brain

Nose

Nasal

Epithelium

Olfactory

Epithelium

After administration of the drug into the nasal cavity, N-to-B drug transport may occur through the

olfactory epithelium, either (i) by axonal transport after internalization into the neurons, (ii) by

paracellular transport across the spaces between cells and, notably across the channels next to the

olfactory nerves, or (iii) by transcellular transport across the basal epithelial cells (Fig. 2).14–17,22,23

The paracellular pathway is considered to be the dominant transport mechanism based on animal

studies, and it allows a more rapid drug transport (usually <30 minutes) than the others, which can

last from a few hours up to days.16,24

This could be explained by the slow regeneration of the

olfactory neurons (every ~1 month) and the coexistence of mature and newly formed neurons,

resulting in the absence of tight junctions in some parts of the olfactory epithelium.24

This leakiness,

in combination with the bulk flow of the cerebrospinal fluid (CSF) into the brain, enables the

transport of the intranasally administered drugs to the CNS.16

However, the predominance of one

specific transport mechanism vs. the others depends on the properties of the drug or the delivery

system used.25

Depending on the pathway, the drug may reach the olfactory bulb by intraneuronal

uptake and, from there, it may go into the brain regions connected to the olfactory tract (i.e., the

piriform cortex, hypothalamus, amygdala) and finally disseminate through the CNS, and/or it may

diffuse directly from the CSF into the whole CNS (Fig. 2).26

Figure 2. Schematic representation of the olfactory pathway and possible uptake mechanisms

involved in the transport of peptides from the nose directly to the brain. (CSF: cerebrospinal fluid;

DDS: drug delivery system)

Olfactory Pathway

Thalamus

Hippocampus

Hypothalamus

Piriform cortex

Olfactory

Neuron

Drug

or DDS

Olfactory

Bulb

Cribriform

Plate

Mucus

Layer

Olfactory

Epithelium

Intra-

neuronal

Trans-

cellular

Para-

cellular

CSF

Extra-

neuronal

Uptake Mechanisms

Amygdala

Based on studies in different animal models, some authors have proven that direct N-to-B drug

delivery can also take place along the less explored trigeminal nerve, parts of which extend from the

brainstem through the nasal respiratory epithelium, and provide thus a direct passage to the caudal

and the rostral parts of the brain (Fig. 3).24,25,27–30

Still, the contribution of the trigeminal pathway is

not fully understood and is considered to be less relevant than the olfactory track.31

Figure 3. Schematic representation of the Trigeminal nerve pathway. (CSF: cerebrospinal fluid)

Challenges encountered in the transport of peptides from the nose directly to the brain

Despite the potential of this patient-friendly drug delivery route to the CNS, there are significant

challenges associated to this modality of administration. Nose-to-brain transport is significantly

affected by the surface and structural properties of the administered biomolecules (e.g., size and

lipophilicity, degree of ionization). Proteins, because of their larger size (>1000 Da) and

hydrophilicity, are transported in a far less extent than smaller lipophilic molecules. Another

important factor is the presence of metabolic enzymes (cytochrome P450, esterases and transferases)

in the mammalian olfactory mucosa.15,32–35

On the other hand, from an anatomical point of view, the

localization of the olfactory epithelium in the roof of the nasal cavity makes it difficult for drugs to

gain access to the targeted region.36,37

To address these pitfalls and enhance the bioavailability of the

protein molecules, different approaches have been suggested, such as the use of permeation

enhancers, cell penetrating molecules, mucoadhesives or nano-based drug delivery systems.4,5,38–48

The last ones have the additional advantage of protecting the therapeutic load, while improving its

interaction with the olfactory region. However, a limitation of this route, is related to the low

volumes that can be administered (maximal dosing volume in humans is 0.4 mL), which implies the

need of designing nanocarriers with a high drug loading capacity.49

Lastly, it is worth mentioning

Trigeminal Pathway

Brain

StemTrigeminal

Nerve

CSFBulk Flow

Nasal

Epithelium

that so far, the mechanistic studies have been mainly performed in animals, whereas the studies in

humans have focused on the evaluation of the drugs therapeutic effects, or, in some exceptional

situations the measurement of the drug concentrations in the CSF, or the use of positron emission

tomography (PET) scanning.22,50–52

Despite the above listed limitations, the potential benefits of this

pain-free and direct approach are clear and, hence, N-to-B transport of peptides is becoming a

promising alternative to the other established methods for CNS delivery.

3. Direct transport of P/P drugs from nose-to-brain

Owing to the aforementioned challenges, there has been much discussion during the last decades

about whether peptides can efficiently employ the nose-to-brain pathway.53

Nowadays, after

numerous studies published and patents filed, we can say that peptides and proteins can be

transported to the CNS directly through the nose. Fig. 4 depicts the growing interest in this scientific

area, as found in a PubMed database search that selected exclusively articles reporting in vivo studies

(172 articles).54

Interestingly, almost one third of the registered studies were published in the last 3

years.

The first study describing the direct delivery of a protein molecule to the brain through the olfactory

nerve pathway was published by Frey et al. in 1995.8,55

This affirmation was based on the observed

accumulation of the radiolabeled nerve growth factor (NGF) in the olfactory bulb shortly after its

intranasal administration in rats.8 Years later, a breakthrough study was published, in which the

authors showed that insulin and other peptides like melanocortin (4–10) could efficiently reach the

CSF, with undetectable serum levels, after their intranasal administration in 36 healthy human

volunteers.56

Their study was considered to be the first proof of the existence of the olfactory track in

humans, even if Merkus and van den Berg pointed out the necessity of comparing the nose-to-brain

and the intravenous routes in order to have a clear understanding of the distribution of drugs into the

brain.50

Figure 4. Number of publications on nose-to-brain peptide delivery, reporting in vivo studies

[PubMed database].

0

10

20

30

40

50

60

70

1995-1999 2000-2004 2005-2009 2010-2014 2015-present

Nu

mb

er o

f P

ub

lica

tio

ns

Year

in vivo studies

Table 1 illustrates the studies that reported in vivo data showing the nose-to-brain transport of

protein or peptide molecules, administered either as simple aqueous solution, with permeation

enhancers, or in the form of drug delivery systems. Overall, the most explored protein delivered to

the brain via this direct route is insulin, followed by oxytocin and hypocretin-1. These well

characterized peptides have crucial regulating functions in the CNS, owing to the wide distribution

of their receptors in the CNS, and numerous clinical trials have shown their potential in the treatment

against several disorders, such as cognitive and behavior disorders, narcolepsy, and other important

neurological disorders, i.e. Alzheimer’s disease and Parkinson’s disease.57–67

With regard to the

preclinical data, the limited pharmacokinetic data (37 publications) make very difficult the drawing

of clear conclusions regarding the bioavailability of the different peptides in the brain after their

intranasal administration. Still, while most of the studies have reported enhanced brain delivery of

the protein molecules after their intranasal administration in comparison to their intravenous

administration, the bioavailability of these molecules remained low, generally lower than 1%.55,68

Intranasal co-administration or conjugation with permeation enhancers, such as cell permeating

peptides (i.e. penetratin, low molecular weight protamine, Pz-peptide, Tat), polyethylenimine,

chitosan, lauroyl carnitine, cyclodextrins, Pluronic P85 or peppermint oil, has been a frequent

strategy to enhance the bioavailability of the intranasally (i.n.) administered protein molecules.39,40,42–

48,69–80 For example, some authors reported that the co-administration of chitosan (0.25%) with NGF

led to an increase of up to 13 times in the brain bioavailability of the intranasally administered NGF,

relative to the i.n. free peptide solution.39,42

Similarly, other authors found a 60% enhanced olfactory

bulb uptake of exendin after its co-administration with cyclodextrin, compared to the intravenously

administered free peptide.70

On the other hand, there have been attempts to obtain direct visual evidence of the route that the

proteins follow after their intranasal administration, employing mainly radioactivity based imaging

techniques.8,29,43,44,46,48,69,70,75,77,81–95

These studies provided undeniable evidence of the peptides’

ability to reach the CNS following the olfactory and/or trigeminal pathway.

Table 1. Peptide/protein drugs transported directly from the nose to the brain

P/P drug MW

(Da)

Disease Animal Model Ref.

Insulin 5,800 AD Mice, rats, rabbits,

sheep

46,47,75,94,96–

104

Oxytocin 1,000 CBD, ASD,

PTSD, SD, SCZD

Rats, pigs, vampire

bats, monkeys,

macaques

105–109

Erythropoietin 30,400 AD, CI, Epilepsy Mice, rats 85,88,93,110–

114

Human nerve growth factor 26,500 AD Mice, rats 8–10,39,73,115–

120

Basic fibroblast growth factor 18,000 PD Rats 74,121–124

NAP neuropeptide

(NAPVSIPQ)

825 AD, MCI, SCZD,

FTD

Mice, rats 82,125–130

Vasoactive intestinal peptide 2,800 AD Mice, rats 69,81,131,132

Insulin-like growth factor I 7,650 AD, HD, CI Mice, rats 28,85,133–136

Glucagon- like peptide I 4,100 OB Mice 70,71,93,137

Exendin (9-39) 3,400 CHI Mice, rats 43,70,71,137

Leptin 16,000 OB Rats 48,84,138

Interferon – β1Β 18,500 MS Rats, monkeys 29,71,83

Brain derived neurotrophic

factor

26,900 AD, HD, ASD Mice, rats 42,88,139

Neurotoxin I 6,900 Pain management Mice, rats 140–142

S14G-HN (humanin

derivative)

- AD Mice, rats 90,143

Hypocretin-I (orexin A) 3,500 Narcolepsy Rats, monkeys 86,95,144

Glial cell-derived neurotrophic

factor

15,000 PD Mice, rats 145–147

Bovine serum albumin 66,500 - Mice 76,92

Transforming growth factor β1 25,000 PD, CI Mice, rats 121,148

Vascular endothelial growth

factor

38,200 AD Rats 87,149

Pituitary adenylate cyclase-

activating peptide

4,500 PD, CI Mice 44,91

Thyrotropin-releasing hormone 362 Epilepsy Rats 150,151

Substance P 1,347 PD Rats 152,153

NEMO-binding domain

peptide

2,841 HIE Rats 45,154

Osteopontin 35,423 CI Rats 155,156

Arginine-vasopressin 1,080 CBD, pro-social

effects

Rats, monkeys 157,158

Glucagon-like peptide II 3,766 Depression Mice 79

Ovalbumin 45,000 - Rats 159

Leucine-enkephalin 555 Pain management Mice 160

Neurotrophin-4 22,400 MS Rats 88

interleukin-1 receptor

antagonist

17,000 CI Rats 161

Ciliary neurotrophic factor 22,700 AD, HD Rats 88

Transforming growth factor –a 5,000-

35,000

CI Rats 162

Growth differentiation factor 5 27,400 PD Rats 163

Galamin-like peptide 6,500 OB Mice 164

Calcitonin gene-related peptide 3,800 Migraine Rats 165

Exendin-4 4,186 CI Mice 166

Urocortin 4,700 PD Rats 167

Hexarelin 887 GHD Rabbits 89

Neuropeptide Y 4,253 PTSD,

Depression

Rats 168

Neuropeptide S 2,206 Anxiety disorder Mice 169

Cystatin C-peptide 13,000 AD Mice 170

H102 Peptide - AD Rats 171

D1–D2 interfering peptide - MDD Rats 172

Apelin-13 1,550 CI Mice 173

Human b-Amyloid Peptide 4,514 AD Rats 174

Cyclosporine-A 1,200 AD, PD Rats 175

TNF-alpha inhibitory single-

chain antibody fragment

ESBA105

26,300 AD, PD, MS Mice 72

Horseradish peroxidase 40,000 - Rats 55

V24P(10–40) - AD Mice 77

R8-Ab(25–35) - AD Mice 80

Elastin-like polypeptide - - Mice 78

AD: Alzheimer’s Disease, ASD: Autism Spectrum Disorder, CBD: Cognitive and behavior disorders, CHI:

Congenital Hyperinsulinism, CI: Cerebral ischemia, FTD: Fronto-temporal dementia, GHD: Growth

hormone deficiency, HD: Huntington’s disease, HIE: Hypoxic-ischemic encephalopathy, MCI: Mild

cognitive impairment, MDD: Major depressive disorder, MS: Multiple sclerosis, OB: Obesity, PD:

Parkinson’s disease PTSD: Post-traumatic Stress Disorder, SCZD: Schizophrenia, SD: Sexual dysfunction

Given the still limited number of studies in animal models, it is not surprising that the clinical

development of the peptide nose-to-brain formulations is still at an early stage. Nevertheless, as

shown in Table 2, it is encouraging to realize that several peptides are making their way along the

clinical development path, with some of them, i.e. insulin and oxytocin, having reached Phase IV

clinical studies.59,67,176–178

In the case of insulin, the indication selected in the phase IV clinical

studies focused on its role in energy metabolism.178

So far the disclosed studies have shown that a

single dose of intranasal insulin can increase the peripheral insulin sensitivity and reduce the hepatic

fat in healthy humans.179,180

Apart from this, intranasal insulin is currently being evaluated for its

memory-ameliorating effect in Alzheimer’s suffering patients, as well as its action against different

CNS disorders in humans, like Parkinson's disease, multiple system atrophy and psychiatric

conditions such as schizophrenia and major depressive disorder.64

With regard to oxytocin, it is

known that it works as a brain neurotransmitter, thereby influencing the social behavior and

emotional function in different species. Oxytocin has been tested against several disorders, such as

stress disorders, social dysfunction, cognitive and behavior disorders, Autism Spectrum Disorder

(ASD) and sexual dysfunction in clinical studies.65,181

The most advanced indication (Phase IV

clinical studies) is the control of satiety in people with schizophrenia, as well as its effect on pain

threshold.182,183

The first results of these on-going studies have shown that a single dose of intranasal

oxytocin can significantly increase the satiety feeling and, subsequently, reduce food consumption in

schizophrenia patients. No results have been posted yet on its effect on pain sensitivity.

Table 2. P/P drugs under clinical studies for N-to-B delivery

P/P drug Disease Stage of

development

Ref.

Insulin AD, OB, PD,

MS, SCZD,

MDD

Phase I 56,59,184–192

Phase II 52,184,186,188,193–199

Phase III 191,198,200

Phase IV 178

Oxytocin CBD, ASD,

PTSD, SD,

SCZD, Pain

management

Phase I 37,201–211

Phase II 212–217

Phase III 218–220

Phase IV 182,183

Arginine-vasopressin CBD, pro-

social effects

Phase I 56,97,203,206,221,222

Melanocortin (4–10) OB Phase I 56,184,223,224

Phase II 184

Cholecystokinin OB, AD, CBD Phase I 221,225,226

Phase II 227

NAP neuropeptide AD, SCZD Phase I 228

Phase II 229,230

Hypocretin-I (orexin A) OB, AD, PD,

narcolepsy

Phase I 231–233

Hexarelin GHD Phase I 234

Neuropeptide Y OB Phase I 184

Insulin-like growth

factor-I

DB, OB Phase I 195

AD: Alzheimer’s Disease, ASD: Autism Spectrum Disorder, CBD: Cognitive and behavior

disorders, DB: Diabetes, GHD: Growth hormone deficiency, MDD: Major depressive

disorder, OB: Obesity, PD: Parkinson’s disease PTSD: Post-traumatic Stress Disorder,

SCZD: Schizophrenia, SD: Sexual dysfunction

Another interesting point to note is that most of the peptide drugs presented in Table 2, have already

been tested in numerous clinical trials, using systemic administration routes and, thus, their safety

and pharmacological/toxicological effects have been assessed. Still, a new evaluation of the toxicity

and safety of these drugs would be required in the case of intranasal delivery.235

These studies may,

additionally, include histological assessments of local tissues and potentially affected brain areas.

Apart from this, the majority of the P/P drugs in Table 2 have been administered intranasally to

humans with the help of liquid delivery devices, such as spray pumps or nebulizers/atomizers.

Detailed description of the principles and the types of these devices can be found elsewhere.14

This

fact introduces additional regulatory requirements for the development of a intranasal product, such

as droplet size distribution, dose uniformity, plume geometry and spray pattern studies.236,237

4. The potential of nanotechnology for nose-to-brain peptide delivery

The contribution of nanotechnology to this field is crucial since not only it allows the protection of

the delicate therapeutic cargo from degradation, but most importantly it improves the uptake by the

olfactory mucosa and the access to the CNS, based either on passive or active targeting. As a result,

the use of drug delivery nanocarriers has led to enhanced drug concentrations and extended half-life

times with the subsequent improved therapeutic effect of the delivered molecules. According to our

records, Gao et al. was the first group to prove in 2007 that poly(ethylene glycol)-poly(lactic acid)

nanoparticles (PEG-PLA NPs) functionalized with wheat germ agglutinin (WGA) could deliver a

neuropeptide against Alzheimer’s disease directly from the nose to the brain.131

Since then, a variety

of nano-based drug delivery systems have been developed with the objective of improving the nose-

to-brain delivery of a variety of peptide drugs, most of them summarized in Tables 3 and 4. These

delivery carriers made of either polymers (Table 3), mainly polyesters and chitosan, or lipids (Table

4), were engineered to promote the transport across the neural pathway of different drug molecules.

Nevertheless, the number of research articles in this field is limited, a fact that explains the lack of

nose-to-brain potential nanomedicines in clinical development.

We have analyzed the tendency in the nose-to-brain nanomedicine field, and the results are

summarized in Figure 5. It should be noted that 87 % of the 172 publications analyzed, refer to the

administration of drugs in the form of a simple solution or after co-administration with permeation

enhancers to facilitate the drugs transport across the olfactory epithelium. Nevertheless, the trend is

changing now and the number of nano-based drug delivery systems explored to nose-to-brain

peptide/protein delivery increases year by year. The majority of these delivery carriers are either

polymeric nanoparticles, mainly made of PLA and derivatives, and lipid-based nanocarriers, with a

predominance of liposomes and lipid nanoparticles. A tendency is also observed towards the

functionalization of the nanocarriers.

Figure 5. Representation of the publication trends in N-to-B peptide delivery. (NPs: nanoparticles,

NLCs: nanostructured lipid carries, GNLs: gelatin core lipid carriers)

NPs

50.0%

GNLs

18.3%

NCLs

13.6%

Liposomes

9.0%

Emulsions

4.55%

Cubosomes

4.55%

Peptide

Solution

87.0%

Nanocarriers

13.0%

Type

Penetration Enhancement

172 publications in total

Subtype

Polymer Type

without

permeation

enhancers

87.0%

with

permeation

enhancers

13.0%

PEG-PCL

9.1%

PEG-

PLGA

18.2%

PEG-

PLA

36.3%

PLA

18.2%

Gelatin

9.1%

Chitosan

9.1%

4.1 Importance of the chemical and physicochemical characteristics of the nanocarriers

Although it has been generally accepted that the particle size is a key factor in the capacity of

nanocarriers to overcome mucus barriers and well-organized epithelia, the recent work by Ahmad et

al. provided a good illustration of this effect. The authors studied the biodistribution of

nanoemulsions of different sizes after intranasal administration in rats, by fluorescence imaging, and

concluded that nanocarriers with a particle size around 100 nm were able to be transported along the

olfactory or trigeminal route, whereas nanoemulsions with a larger droplet size were not able to

follow the olfactory pathway.238

Apart from this, a number of studies have also reported the

efficiency of nanocarriers with a size up to 200 nm in terms of facilitating the transport of molecules

in animal models.16,129,239–242

These results are aligned with the reported morphological studies of the olfactory epithelium of

different species, which have revealed that the average diameter of olfactory axons is around 200

nm, with many axons to have diameters even less than 100 nm, while in humans this range goes

from 100 nm up to 700 nm.16,243,244

This fact alone sets a size constraint for the effective transcellular

transport of the nanocarriers via the olfactory axons to the brain. In agreement with this

morphological constraint, the size of the majority of the nanocarriers developed for the peptide

delivery through the intranasal route is in the range of 70-150 nm (Table 3&4).

On the other hand, the effect of the zeta potential on the performance of the nanocarriers has not

been clearly elucidated yet. Apart from the already known fact that the use of positively charged

nanocarriers will likely adhere to the mucus layer, due to the presence of negatively charged mucus

proteins in the region, the potential benefit of this adhesive/retention behavior remains to be

clarified.41,245

In a recent study, the authors sought to describe this effect by tracking the localization

of fluorescently labeled poly(lactic-co-glycolic acid) nanoparticles (negative) and chitosan-coated

PLGA NPs (positive) in different brain areas, following their intranasal administration in rats.246

The

conclusion of this study was that while both type of carriers were found in the brain, the surface

charge and composition of the nanocarriers had a significant effect on their transport pathway, with

negative NPs showing a preference for the olfactory pathway, while the positive nanoparticles for

the trigeminal pathway. While these conclusions remain to be confirmed, it should be highlighted

that the comparison of these nanocarriers was not done in a rigorous manner as they had a different

particle size (118 nm for PLGA nanoparticles and 213 nm, for chitosan-coated PLGA nanoparticles).

Irrespective of the differences in the surface charge, a critical parameter that determines the fate of

the nanocarriers after their intranasal administration is their composition.16

For example, in a study

gelatin-nanostructured lipid carriers (GNLs) stabilized with Poloxamer 188 (size of ~170 nm and

zeta potential of –27 mV) were compared with gelatin nanoparticles (GNPs), (size of ~120 nm and

zeta potential of –17 mV), with regard to their capacity for the nose-to-brain transport of bFGF in

hemiparkinsonian rats.123

The results showed the superiority of the Poloxamer 188-containing

formulation, which was attributed to the known permeation and mucodiffusive properties of the

surfactant. Other studies have evaluated the effect of various coatings, i.e. chitosan and polysorbate

80 around the nanoparticles on their olfactory transport.16,136,141,142,247,248

The results showed that the

presence of polysorbate-80 or chitosan could potentially enhance the nanocarriers’ interaction with

the olfactory mucosa. As in the case of poloxamer, this positive behavior of polysorbate 80 was

attributed to its permeation and mucodiffusive properties, whereas chitosan apart from increasing the

hydrophilicity on the surface of the carrier, can significantly prolong also its retention time in the

olfactory area.239

However, these mechanistic behaviors remain to be elucidated.

4.2 Polymer-based nanocarriers

Polymeric nanocarriers, made either of natural or synthetic polymers, have recently attracted

significant research attention for N-to-B peptide delivery, since they offer a plethora of advantages,

like enhanced stability, the ability to protect and control the release of their therapeutic payload and

multiple possibilities for surface modification. However, so far only few examples have been

reported in the literature, including polylactic/glycolic acid (PLGA) nanoparticles, PEG-PLGA

nanoparticles, PLA NPs modified with chitosan or polysorbate 80, PEG-poly (ε-caprolactone) (PEG-

PCL) nanoparticles, chitosan nanoparticles, and gelatin nanoparticles. Table 3 summarizes the

physicochemical characteristics and the PK/PD behavior of the different polymer-based carriers

employed so far in the literature. The performance of these nanocarriers, after their i.n.

administration, was further enhanced in some cases via their surface modification with targeting

moieties, which will be discussed below.

The advantages of using polymer-based nanocarriers for the N-to-B delivery of small hydrophobic

drugs have been explored by different authors since 2000.249–251

This know-how was then transferred

to N-to-B peptide delivery, with Gao et al. being the first to prove the capacity of PEG-PLA NPs

functionalized with WGA, to facilitate the transport of a peptide drug, the vasoactive intestinal

peptide, to the brain in 2007. In their study, an almost 4-fold increase in the concentration of the

peptide in the brain was reported, when delivered associated to PEG-PLA NPs, reaching a 7-fold

increase with the help of the active targeting functionalization, in comparison to the peptide solution

after its intranasal administration in mice.131

Following this pioneering study, several authors have

investigated the effect of the surface composition of PLA/PLGA particles in their capacity to

overcome the N-to-B barriers. Interestingly, the potential of PLA or PLGA nanoparticles for

peptide/protein delivery, as well as the effect of the particle size and the PEGylation on the transport

of nanoparticles across the nasal mucosa was first reported by our group almost 2 decades ago.252,253

Years later, in 2009, the effect of the particle size on the performance of PLA particles for N-to-B

peptide delivery for the N-to-B transport of Thyrotropin-releasing hormone (TRH) was also

reported.151

The evaluation of the uptake of the particles by fluorescent microscopy led to the

conclusion that 100 nm PLA nanoparticles could gain access to different brain regions through the

olfactory epithelium, whereas the larger ones (~560 nm) could not. This explained the enhanced

therapeutic effect of the neuropeptide in terms of suppressing stage IV seizures in epileptic rats.150,151

Moreover, different authors employed PEG-PLGA of a 120 nm size in order to protect Urocortin

(UCN) and basic fibroblast growth factor (bFGF) respectively, from degradation and facilitate their

intranasal delivery to the CNS.122,167

Additionally, Zhang et al. presented PK results, showing an

almost 1.5-fold increase of the AUCs of bFGF in different brain areas in the case of the i.n.

administration of the bFGF-loaded NPs, relative to i.n. administered bFGF solution in rats.122

This

increase reached up to 3 times more in the case of the i.n. administration of lectin modified bFGF-

NPs, and will be further discussed below.

Another tendency observed is the surface modification of PLA NPs with either PEG-containing

surfactants, like polysorbate 80 (P80), or chitosan. This trend, in the case of P80, originated from the

reported improved performance of the P80-coated nanocarriers in the CNS drug delivery, which was

later adopted in N-to-B peptide delivery.254–256

Accordingly, P80-coated PLA NPs with a particle

size of 60 nm were employed for the nose to brain administration of Neurotoxin-I in rats, resulting in

an 1.5/1.8-fold increase in the peptide bioavailability relative to i.v. administration of the same

nanoparticles and peptide solution, respectively.140

In a similar study, the N-to-B transport of the

same peptide was evaluated upon its incorporation into P80-coated PLA nanoparticles (size was not

mentioned in the report) and the brain uptake showed a 2.2/3 times higher drug transport in

comparison to the i.v. administration of the same particles and the i.n. administration of the free

peptide in mice, respectively. This enhanced delivery could potentially be attributed to the PEG-

containing surface coating of the NPs that facilitates their diffusion through the olfactory mucosa

since it raises the hydrophilicity of the carrier, or to its general surfactant-like properties and its

interaction with the endothelial cells.141

Chitosan, on the other hand, is considered to be an attractive polymer due to its biocompatibility and

bioadhesion and penetration enhancing properties.257

It is worth mentioning that our group pioneered

in the development of CS nanoparticles and nanocapsules for nasal drug delivery in the mid-

90s.258,259

Regarding its use in N-to-B peptide drug delivery, chitosan-coated PLA nanoparticles,

having a particle size of 140 nm and a positive surface charge of +34 mV, were shown to

significantly enhance the brain uptake of the neurotoxin-I peptide (~1.8-fold relative to the uncoated

NPs), an effect that was attributed to the mucoadhesive and cell permeating properties of chitosan.142

In another study, Kumar et al. investigated the performance of trimethyl chitosan nanoparticles

(TMC NPs) with a mean size of 440 nm and a positive zeta potential of +15 mV for the intranasal

delivery of Leucine-enkephalin (Leu-Enk). The authors reported a high peptide uptake in different

brain regions, however this conclusion was simply based on qualitatively microscopic

observation.160

Both of the studies mentioned did not provide a systemic comparison though, and

thereby proof that the delivery of the respective peptides is only due to direct passage of the

therapeutic molecules via the olfactory or trigeminal nerves to the brain and not through the nasal

epithelium also.

Another biodegradable and FDA approved biomaterial that has received minor attention is gelatin.

Joachim et al. used gelatin nanoparticles (GNPs) to study their ability to transfer Osteopontin (OPN)

to the brain after their intranasal application. The selection of gelatin was based on the authors claim

that this protein can passively target several brain areas in case of ischemic stroke.155,260

According to

the expectations, the results of this work showed an enhanced response for the peptide associated to

the nanoparticles as compared to the free peptide administered intranasally.

Moreover, nanoparticles made of PEG-poly (ε-caprolactone) and surface-modified with lactoferrin,

with mean diameter less than 90 nm, have been used to study the delivery of NAP, a model

octapeptide, from the nose directly to the brain of Altzheimer’s disease animal models. Throughout

this study, a rapid accumulation of the NPs in various brain areas was showed based on fluorescent

microscopy, whereas the enhanced memory amelioration effect of the Lf-NPs was further proven by

a Morris water maze experiment.129

Depending on the polymer chosen and the composition of the nanocarriers, different properties can

be attributed to the systems. A general characteristic of the polymer-based carriers employed so far

for the N-to-B peptide delivery is that their diameter is within the range of 70-200 nm. Overall, the

surface functionalization of the carriers either with PEG, pegylated surfactants, chitosan and/or

targeting moieties makes a significant contribution to the enhancement of the peptide bioavailability

in the brain. However, a strict comparison among these nanocarriers is difficult to make due to the

different nature of the studies performed to evaluate them.

Table 3. Overview of polymer-based nanocarrier systems for N-to-B peptide delivery.

Nanocarrier Drug Size

(nm)

Z-pot

(mv)

Targeting

molecule

Animal Pharmacokinetics

outcome/

Therapeutic effect

Ref.

D,L- PLA NPs Thyrotropin-

releasing

hormone

~100 - - Rats Significant suppressed

seizures in epileptic rats

Enhancement of

neuroprotective effect

150,151

PLA NPs

coated with

polysorbate 80

NT-I 65 -29 - Rats 1.8-fold increased

bioavailability compared

to i.v. peptide solution

140

PLA NPs

coated with

polysorbate 80

NT-I - - - Mice 3-fold increased brain

concentration of NT-I

relative to the i.n. solution

141

PLA NPs

modified with

chitosan

NT-I 140 +34 - Rats 9.5-fold increased brain

concentration relative to

the i.n. solution and 1.8-

fold to unmodified PLA-

NPs

142

PEG-PLA NPs Vasoactive

intestinal

peptide

100 -

120

- WGA Mice ~7-fold increased

bioavailability relative to

i.n. peptide solution

131

PEG–PLGA

NPs

Urocortin 120 -20 OL Rats ~6-fold enhancement of

the therapeutic effect

relative to i.n. peptide

solution and 3 fold relative

to the unmodified NPs

167

PEG-PLGA

NPs

bFGF 120 -32 STL Rats 1.6- to 3.2-fold higher

AUCs in olfactory bulb,

cerebrum and cerebellum

of rats relative to i.n.

bFGF solution

122

N-trimethyl

chitosan NPs

Leucine –

encephalin

443 +15 - Mice Significant brain uptake

relative to i.n. solution

Significant enhancement

of the therapeutic effect

160

Gelatin NPs Osteopontin 183 - - Rats Enhancement of

therapeutic effect

155

PEG-co-PCL

NPs

NAP peptide 70 -

90

-24 Lf Mice

/Rats

~2.5- fold increased AUC

of coumarin in brain areas

relative to the i.n.

unmodified NPs

Enhanced memory

amelioration effect

129

PEG: Poly(ethylene glycol), PLA: polylactic acid, NPs: Nanoparticles, PLGA: poly(lactic-co-glycolic acid),

PEG-co-PCL: PEG-poly (ε-caprolactone), WGA: Wheat germ agglutinin, OL: Odorranalectin, Lf: Lactoferrin,

STL: Solanum tuberosum lectin, NT-I: Neurotoxin-I, bFGF: basic fibroblast growth factor

4.3 Lipid-based nanocarriers

Lipid-based nanocarriers describe a broad category of drug delivery systems that includes liposomes,

nanoemulsions and solid lipid nanoparticles. From our knowledge, Migliore et al. were the first, in

2009, to explore the use of lipid-based nanocarriers for the N-to-B delivery of protein drugs.159

Following this study, nanoemulsions, liposomes, cubosomes, as well as mixed systems like lipid

nanoparticles with solid matrix or nanostructured lipid carriers (NLC) have been successfully

employed for the N-to-B peptide delivery and analyzed below (Table 4).

In the pioneering work by Migliore et al., the authors investigated the potential of cationic liposomes

with a size of 300 nm, consisting of dioleoylphosphatidylcholine (DOPC), cholesterol and

stearylamine for the N-to-B transport of ovalbumin − a model protein – in rats.159

The i.n.

administered liposomal formulation exhibited increased residence time at the olfactory mucosa and

this was translated into a 10-fold protein concentration in the brain, as compared to the protein

solution administered by the same route. The authors attributed these positive results to the

electrostatic interactions of the positively charged carrier with the negatively charged mucus

proteins. Other authors reported the fact that pegylated liposomes with a size of 110 nm and a neutral

charge facilitated the direct transport of the H102 Peptide to the brain after intranasal administration

to a Alzheimer’s disease rat model.171

In this study, the performance of the liposomal formulation

was compared with that of the free peptide administered intranasally in the presence of chitosan. The

results showed that the i.n. liposomal formulation was significantly more effective than the chitosan

solution in terms of enhancing the access of the H102 peptide to the olfactory bulb, cerebellum,

cerebrum and hippocampus.

Cubosomes are a nanoparticulate system, consisting of amphiphilic lipids and surfactants that are

organized in a cubic nanostructure. These systems contain liquid-crystal phases that may facilitate

the dissolution of hydrosoluble peptides. Wu et al. explored the efficacy of pegylated cubosomes,

consisting of 1-Monoolein (Glycerol Monooleate), Poloxamer 407 and maleimide–PEG–oleate,

functionalized with a lectin, for the N-to-B delivery of the S14G-HN peptide, a novel peptide against

Alzheimer’s disease and Cerebral ischemia.143

The results showed a concentration dependent

enhancement of the neuroprotective effect of the S14G-HN peptide, following their intranasal

administration in rats.

Although a number of reports have described the possibility to deliver small hydrophobic molecules

from the nose to the brain using nanoemulsions, the only work describing a peptide-based

nanoemulsion formulation was applied to Cyclosporine A (CsA).175,261,262

The developed

nanoemulsions in this study consisted of flax-seed oil as the oil phase, known for its neuronal

regulating properties, egg phosphatidylcholine, polysorbate 80 and stearylamine. Their size was 270

nm and their zeta potential was +57 mV. The i.n. administration of CsA-loaded nanoemulsion

resulted in a significantly higher (6 to 18 times) drug concentration in the olfactory bulb and also in

the brain compared to the levels obtained following i.n. and i.v. administration of the free peptide, as

well as i.v. of the peptide loaded nanoemulsion.

Another lipid based system used for the N-to-B peptide delivery is the so called nanostructured lipid

carriers (NLCs), which consist of a combination of solid and liquid lipids. Gartziandia et al.

developed NLCs coated with chitosan, with a size of 114 nm and a positive zeta potential of +28 mV

for the delivery of human insulin-like growth factor-I (hIGF-I).136

The authors reported an enhanced

residence time of the nanocarriers in the nasal epithelium owing to the mucoadhesive properties of

chitosan and a high brain accumulation based on the results obtained using fluorescence imaging

techniques. In a later study, this system was also used by the same group for the N-to-B delivery of

the glial cell-derived neurotrophic factor (GDNF), achieving a significant improvement in terms of

behavior (movement recovery) and neuroprotection, in comparison to the GDNF solution, in a

hemiparkinsonian rat model.146

A different type of lipid nanoparticles, is the one containing a gelatin core (GNLs) surrounded by a

shell of phospholipids, cholesterol and Poloxamer 188.123,124,152,153

These nanoparticles, with a

diameter of 170 nm and a surface charge of -30 mV, were tested for their capacity to enhance the N-

to-B transport of the Substance P peptide, intended to treat hemiparkinsonian rats.152,153

The results

showed an enhanced response of the peptide when delivered through the nose. The same delivery

vehicle was also tested for the N-to-B delivery of bFGF in order to treat cerebral ischemia.124

The

results indicated that the intranasal administration of this formulation led to a significant increase

(~1.5-fold) in the concentrations of the peptide in different brain regions (pallium, hippocampus,

striatum, olfactory bulb), whereas no increase was observed after the i.v. administration of the bFGF

solution or the bFGF loaded GNLs. Furthermore the therapeutic effect of the i.n. administered bFGF

GNLs was verified by the reported improvement in the neurological deficit score and locomotor

activity of rats.

Overall, from these studies it could be concluded that the efficient N-to-B peptide delivery does not

depend solely on the nanocarrier size, but also on its composition. Compared to the other N-to-B

lipid-based peptide delivery technologies, the highest rise of peptide concentration in the brain,

relative to the i.n administration of the peptide solution, was reported for cationic liposomes (10

times higher) followed by cationic nanoemulsions (6 times higher) with diameters bigger than 200

nm (Table 4). This result could be attributed to the increased residence time achieved by these

positively charged formulations in the olfactory region and more importantly to the presence of

surfactants like polysorbate 80, known for its cell permeation enhancing effects. In conclusion,

proper selection of lipids and surfactants can positively affect the peptide delivery through the

olfactory route. Still, the limited number of studies performed so far does not allow us to draw

further conclusions. Focused mechanistic studies are needed to shed light on the complex

interactions of the different nano-based systems with the olfactory epithelium, the importance of the

surface characteristics of the carriers and their effect on the in vivo efficacy of the systems.

Table 4. Overview of lipid-based nanocarrier systems for N-to-B peptide delivery.

Nanocarrier Drug Size

(nm)

Z-pot

(mv)

Targeting

molecule

Animal Pharmacokinetics

outcome/

Therapeutic effect

Ref.

Cationic

liposomes

OVA 299 +19 - Rats 10-fold brain levels &

>4-fold AUCbrain/AUCblood

relative to i.n. peptide

solution

159

Liposomes H102

Peptide

110 -3 - Rats 1.6-3-fold higher AUC

values in specific brain

areas than the i.n. solution

(no drug in the brain after

i.v. admin.)

171

Pegylated

Cubosomes

S14G-HN

(humanin

derivative)

100 -

120

-14 OL Rats 1.7-3.5-fold increase of the

coumarin distribution in

brain regions relative to the

unmodified cubosomes

Significant enhancement of

therapeutic effect

143

Oil-in-Water

Nanoemulsion

CsA 270 +57 - Rats 6-fold increased brain

concentration relative to i.n

CsA solution

175

Gelatin

NCL

bFGF 170 -27 - Rats 1.5-fold enhancement of

peptide brain concentration

and its therapeutic effect

relative to i.n. peptide

solution (no transport)

123

Gelatin

NCL

SP 172 -30 - Rats Significant enhancement of

therapeutic effect

152,153

Gelatin

NCL

bFGF 128 -15 - Rats Increased peptide

concentration in brain

relative to i.n. peptide

solution

Enhanced response

124

Chitosan

coated NCL

hIGF-I 114 +28 - Mice Increased residence time in

the nasal epithelium and

brain-accumulation

136

Chitosan

coated NCL

GDNF 137 +30 - Rat Significant enhancement of

neuroprotective effect

146

OVA: Ovalbumin, bFGF: basic fibroblast growth factor, SP: Substance P, CsA: Cyclosporine-A, hIGF-I:

Human insulin-like growthfactor-I, GDNF: glial cell-derived neurotrophic factor, NCL: nanostructured lipid

carriers

5. Strategies to enhance N-to-B drug transport

5.1 Olfactory targeting strategies

As mentioned above, several studies have explored the use of covalently linked biorecognitive

ligands in order to enhance the N-to-B transport of the nanosized drug delivery carriers. Table 5

summarizes the targeting moieties that have been described in the literature for targeting the

olfactory region. The most commonly used targeting ligands have been proteins that have receptors

in the olfactory region, i.e. lactoferrin, or glycoproteins, i.e. lectins. Lactoferrin (Lf) has received a

significant attention due to the high expression of its receptor (LfR) in the brain endothelial cells and

neurons.49,129,263

For example, Liu et al., studied the N-to-B transport of lactoferrin conjugated PEG-

PCL NPs (< 90 nm), loaded with a neuroprotective octapeptide (NAP), in an Alzheimer’s disease

animal model, and observed a significant neuroprotective and memory ameliorating effect.129

According to the in vivo biodistribution study performed with fluorescent nanoparticles in rats, Lf-

functionalized NPs exhibited a rapid accumulation in various brain areas, in comparison to the

unmodified NPs. Other authors showed the capacity of the same type of nanoparticles (120 nm) for

the N-to-B delivery of rotigotine, a dopamine agonist used to treat Parkinson’s disease, in a mice

model. The results showed a two times higher drug concentration in different brain areas, relative to

the ones achieved with the unmodified NPs.264

Following the study by Ferrari et al. showing the affinity of several lectins for the N-acetyl-D-

glucosamine and sialic acid residues in the olfactory mucosa, a number of works has been oriented

to show that the functionalization of peptide-loaded nanoparticles with specific lectins, such as

wheat germ agglutinin (WGA), solanum tuberosum lectin (STL), ulex europeus agglutinin I (UEA-

1), is a useful approach to N-to-B delivery.41,131,265,266

For example, the functionalization of PEG-

PLA nanoparticles with WGA was reported to double the N-to-B transport of the Vasoactive

intestinal peptide associated to them, in comparison to the transport observed for the unmodified

nanoparticles.131

In a different study, the authors studied the brain distribution of the labeled (with

125I and coumarin) WGA-functionalized PLGA nanoparticles after nasal administration to rats.

267

The conclusion was that the WGA functionalized PEG-PLA NPs could move through the olfactory

epithelium and reach the olfactory bulb in 5 minutes, suggesting their transport through the

extraneuronal pathway via the olfactory epithelium. In addition, a strong radioactivity signal was

found in deeper brain areas (e.g., striatum, hippocampus, medulla, cerebellum, pons) at 30 min post-

administration.

PEG-PLGA nanoparticles have also been functionalized with STL.122,266,268

The resulting

nanoparticles showed an almost 2.5 times higher brain targeting efficiency (AUCBrain/AUCBlood) for

the STL functionalized PEG-PLGA NPs relative to the values attained for the unmodified NPs after

their i.n. administration in rats.266

Some authors have argued about the potential immunotoxicity of lectins and suggested the use of

smaller peptides with lectin-like function, like Odorranalectin (OL), a small peptide (1.7 KDa) from

frog skin secretions.269

This lectin recognizes and specifically binds to L-fucose, which is highly

expressed in the olfactory mucosa. In vivo fluorescence imaging studies showed that the interaction

of fluorescent PEG-PLGA nanoparticles (95 nm), as well as that of pegylated cubosomes (100-200

nm) functionalized with OL, with the olfactory epithelium was significantly improved (up to a 3.5-

fold increase) as compared to the one observed for the unmodified NPs.143,167

Table 5. Examples of targeting ligands and nanocarriers used for Nose-to-Brain drug delivery

Nanosystem Targeting ligands Animal

model

Ref.

PEG-PCL NPs Lactoferrin Rats/Mice 129

PEG-PLGA NPs Lactoferrin Mice 264

PEG-PLGA NPs STL Rats 122,266,268

PEG-PLA NPs WGA Rats/Mice 131,251,267,270

PEG-PLA NPs UEA I Rats 271

PEG-PLGA NPs OL Rats/Mice 167

Pegylated Cubosomes OL Rats 143

PEG: Poly(ethylene glycol), PEG-PCL: PEG-poly (ε-caprolactone), PLA: polylactic acid, NPs: Nanoparticles, PLGA: poly(lactic-glycolic acid), STL:

Solanum tuberosum lectin, WGA: Wheat germ agglutinin, UEA I: Ulex

europeus agglutinin I, OL: Odorranalectin

5.2 Cell-penetrating peptides (CPPs)

CPPs are traditionally positively charged oligopeptides, containing arginine and lysine residues, that

have been long recognized for their ability to interact with biological membranes and enable the

efficient cellular uptake of biomacromolecules, via different endocytosis pathways or direct

cytoplasmic translocation.272

Recent research on N-to-B drug delivery has proven the ability of these

peptides to significantly increase the permeation of several nanosystems through the olfactory

epithelium (Table 6). Among them, the human immunodeficiency virus Tat peptide

(GRKKRRQRRRPQ) is the most commonly used so far.273–277

Tat peptide’s cell penetrating

properties have been directly associated to the presence of the guanidinium groups of arginine,

which can induce both electrostatic and hydrogen bonding with the cell-surface.278

In a recent study,

the authors described a nanostructured lipid carrier (particle size of 206 nm and surface of +22 mV)

coated with Tat-conjugated chitosan, for the intranasal delivery of glial cell-derived neurotrophic

factor (GDNF).147

According to the results, the surface modification of the system with the Tat

peptide led to a significant enhancement of the therapeutic effect of the administered protein, in

terms of behavior, histological evaluation and microgliosis reduction in a mouse model of

Parkinson’s disease. On the other hand, insulin-loaded PLGA NPs, surface modified with the

cationic Tat peptide (~200 nm, +11 mV), were able to accumulate nearly 6.5 times more in the

olfactory bulb in a matter of a few hours, in contrast to the non-modified NPs, that exhibited poor

transport from the nose to the brain.276

Other studies have been oriented to analyze the potential of the Tat peptide to improve the N-to-B

transport of surface-conjugated MPEG-PCL nanomicelles with a size <100 nm and a slightly

positive surface charge.273

The same group employed this carrier for the nose-to-brain delivery of an

anti-tumor drug (camptothecin) and a small interfering ribonucleic acid.274,275,277

In a follow up

study, a similar system (50-80 nm, 10-15 mV) was employed, where MPEG-PCL micelles,

conjugated with a different arginine-based CPP, were tested for their ability to improve the N-to-B

delivery of macromolecules, using dextran as a model drug.279

A comparison was also made with a

system containing a hydrophobic derivative of the CPP (stearyl modification, STR) (100 nm, 20

mV), proving the possibility of targeting different brain areas, depending on the hydrophilicity of the

CPP. Using in vivo fluorescence imaging, it was found that both systems reached the olfactory bulb

in the short period of 15 minutes, in contrast to the labeled dextran control for which almost no

fluorescence was observed in the brain. More interestingly, the more hydrophobic complex

accumulated mostly in the forebrain, whereas the regular CPP complex could be found throughout

the brain. Still, the mechanisms involved throughout this brain translocation remain unknown.

Another CPP type that has attracted attention for nose-to-brain delivery are short peptide fragments

of the well-known CPP protamine, produced by enzymatic degradation, called low molecular weight

protamine (LMWP, CVSRRRRRRGGRRRR). The advantage of these chain-shortened peptides is

their significant lower toxicity and immunogenicity than their parent protein. For example, it has

been reported that the attachment of LMWP onto the surface of PEG-PLGA nanoparticles led to a

significant enhancement of the N-to-B delivery of coumarin.280

Taking all these results into account,

we can conclude that the CPPs hold a potential in terms of facilitating the nanoparticle-mediated

transport of macromolecules from the nose directly to the brain.

Table 6. Examples of nanocarriers surface-modified with CPPs for Nose-to-Brain drug delivery

Nanosystem Surface modification Animal

model

Ref.

Chitosan coated NCL Tat Mice 147

PLGA NPs Tat Mice 276

Nanomicelles MPEG- PCL Tat Rats 273–275,277

Nanomicelles CH2R4H2C Rats 279

PEG-PLA NPs LMWP Rats 280

NCL: nanostructured lipid carriers, PLGA: poly(lactic-glycolic acid), PEG-PCL: PEG-

poly (ε-caprolactone), PLA: polylactic acid, LMWP: low molecular weight protamine

6. Comparative analysis of in vivo studies

In a review by Merkus and van den Berg, the authors defined the principles for a reliable animal

study aiming to prove the direct N-to-B transport. These principles are: (i) the selection of realistic

administration volumes and concentrations (ii) the comparison of the data with those obtained upon

intravenous administration of the same formulation, (iii) the pharmacokinetic analysis in plasma and

CNS and (iv) studying the brain biodistribution of the administered formulations.50

Other authors

defined specific parameters to express the efficacy of different N-to-B delivery approaches, i.e. the

drug targeting efficiency % (DTE %), which represents the efficiency of the drug to reach the brain

compared to the blood, following intranasal versus parenteral administration, and the direct transport

percentage % (DTP %), which represents the percentage of the drug that reaches the brain due to

direct N-to-B transport, with respect to the total amount of drug found in the brain%), as described

by the following equations:13,242,281,282

Drug targeting efficiency % (DTE%)

𝐷𝑇𝐸% =(𝐴𝑈𝐶𝑏𝑟𝑎𝑖𝑛 𝐴𝑈𝐶𝑏𝑙𝑜𝑜𝑑⁄ )𝑖𝑛𝑡𝑟𝑎𝑛𝑎𝑠𝑎𝑙

(𝐴𝑈𝐶𝑏𝑟𝑎𝑖𝑛 𝐴𝑈𝐶𝑏𝑙𝑜𝑜𝑑⁄ )𝑝𝑎𝑟𝑒𝑛𝑡𝑒𝑟𝑎𝑙∙ 100%,

where AUCbrain and AUCblood are the area under the curve of the drug in the brain or blood

respectively, estimated each time for the corresponded route and

Direct transport percentage % (DTP%)

𝐷𝑇𝑃% =(𝐵𝑖𝑛−𝐵𝑥)

𝐵𝑖𝑛∙ 100%,

where Bin is the brain AUC after the intranasal administration, Bx is the brain AUC fraction from the

blood after intranasal administration and equals to Bx = (Bin/Ppar)·Pin, where Ppar is the blood AUC

following parenteral administration, and Pin is the blood AUC following intranasal administration.

The variety of pharmacokinetics/pharmacodynamics parameters analyzed in the different literature

reports (Table 3&4) renders the comparison of the in vivo performance of nanocarriers very difficult.

After a thorough screening of the selected publications, it is apparent that not all of them provide

adequate information to substantiate the direct N-to-B peptide transport, according to the criteria set

by Merkus and van den Berg.50

In fact, most of the reports do not disclose accurate

pK/biodistribution data, but rather indirect efficacy or semiquantitative biodistribution results (Fig.

6-7).124,129,136,143,150–153,155,160,167

Figure 6. (A) Mean concentration of cyclosporine-A (CsA) in the brain and (B) Brain-to-blood

concentration ratio of cyclosporine-A (CsA) after intranasal (IN) or intravenous (IV) administration

of CsA loaded nanoemulsion (CsA-NE) or CsA-solution (CsA-S) to rats. (*p < 0.05 or *p < 0.01 vs

control groups). Adapted with permission from [175].

[B]

[A]

In addition, some studies lack of proper controls, such as a systemic comparison or biodistribution

data, a fact that makes it inappropriate to conclude about the direct N-to-B transport

mechanism.152,155,160

It is, however, important to highlight that a significant number of studies have

reported direct evidence of the ability of nanocarriers to travel through the olfactory pathway and

deliver their payload to the different brain areas, employing fluorescence and radioactivity-based

imaging techniques (Fig. 7).122,129,131,136,140,142,143,151,159,160,167,276

Nevertheless, quantitative information

on the N-to-B transport of the nanocarriers is still missing in the literature.

[A]

Figure 7. Brain distribution study of PEG-PLGA NPs (Odorranalectin-functionalized (OL-NP) and

non-functionalized (NP)) in mice by in vivo imaging system. (A) Fluorescence images of animal

following i.n. administration at different time points; (B) Fluorescence intensity in the brain versus

time. (*p<0.05) Adapted with permission from [167].

Among the studies analyzed throughout this review, high nose-to-brain delivery efficiency was

reported for both, lipid based and polymer-based systems. Comparatively, the highest performance

of a nanocarrier in terms of enhanced transport of the associated peptide was reported for cationic

liposomes of 300 nm, which resulted in 10 times enhanced bioavailability of OVA in the brain.159

Similarly, PLA nanoparticles coated with chitosan (<150 nm) were found to lead to a 9.5-fold

enhanced concentration of Neurotoxin-I, in the brain.142

Another example, describes a 7-fold peptide

(vasoactive intestinal peptide) transport increase following the i.n. administration of WGA

[B]

functionalized PEG-PLGA nanoparticles as compared to the i.n. administered free peptide

solution.131

Similar transport efficiency was observed for the CsA administered in the form of a

cationic oil-in-water nanoemulsion (~270 nm).175

Among the discussed studies, the one from Zhang et al. is considered to be particularly rigorous as it

complies with all the criteria mentioned above.122

The group employed PEG-PLGA NPs surface

modified with STL, with a particle size of 120 nm and a negative surface charge of -32 mV, for the

N-to-B delivery of bFGF. Radioisotopic tracing method enabled the tracking of the radiolabeled

protein molecule after its i.v. and i.n. administration with or without drug delivery system, proving

the superiority of the i.n. administered nanoformulation. DTE% and DTP% values provided

additional evidence of the ability of the carrier to enhance the nose to brain peptide delivery (DTE%

= 1050% and DTP%=90.44% for the olfactory bulb). The enhanced therapeutic effect was later

verified in rats after the i.n. administration of the peptide loaded nanocarriers.

A general conclusion from these studies is that despite the recognized importance of the particle

size, the composition of the carrier plays a critical role in the fate of the intranasally administered

nanocarriers. Overall, the tendency is to observe that cationic or functionalized nanocarriers perform

better than plain negatively charged nanocarriers. Nevertheless, the variability in the parameters and

criteria, as well as the different animal models employed, do not allow an in-depth comparison

among the different systems. Therefore, more exhaustive comparative studies are required in order

to distinguish among the different nano-based N-to-B peptide delivery systems.

Comparative analysis of the in vivo efficacy of the nano-based DDS vs. permeation enhancers

As mentioned above, various authors have proven the ability of non-covalent permeation enhancers

to facilitate the N-to-B delivery of the co-administered peptide drugs through the olfactory

epithelium. One of the most popular permeation enhancers used for the N-to-B peptide delivery is

chitosan, followed by various cell permeating peptides (i.e. penetratin, low molecular weight

protamine, Pz-peptide). In terms of the peptide bioavailability in the brain, the most promising

results so far have been reported by Morishita’s group who presented up to 20 times increased levels

of insulin in different brain areas after its co-administration with L-penetratin (0.5 mM) as compared

to the i.n. administered free protein solution.46

Following these studies, Vaka and co-workers showed

a 13−14-fold increase in the brain bioavailability of NGF and BDNF respectively, when i.n. co-

administered with chitosan, in the form of a solution.39,42

The same authors showed the ability of

peppermint oil to increase around 8 times the brain concentration of NGF, after their i.n. co-

administration.73

Peppermint oil is also believed to have a tight junctions’ opening effect, promoting

thus the paracellular uptake in the olfactory epithelium.

Unfortunately, we have identified only one report showing the performance of penetration enhancers

vs. nanocarriers using the same peptide drug. In particular, in 2015, Zheng et al. compared the

efficiency of the permeation enhancer chitosan, and a formulation of PEGylated liposomes, with a

particle diameter of 110 nm, for the N-to-B delivery of the H102 peptide. The results showed

significantly higher (1.6–3-fold) values of the drug concentration over the time in different brain

areas for the liposomal formulation, as compared to the AUC values observed for the chitosan

solution. According to the authors, this significant difference in performance was attributed to the

ability of the nanoformulation to provide adequate protection to the peptide from enzymatic

degradation, something that a simple co-administration with a permeation enhancer failed to

provide.171

In another study, the authors explored the use of chitosan as a permeation enhancer for

the i.n. administration of bFGF, achieving up to 1.95 times increased concentrations in different

brain areas as compared to the i.n. administered free peptide in rats.74

Similar results though were

reported by later studies, using gelatin based nanocarriers for the N-to-B transport of the same

peptide.123,124

Overall, it is clear that, more comparative studies based on the same peptide drugs are required in

order to make a clear conclusion on the comparison between nano-based drug delivery systems and

permeation enhancers.

7. Conclusions and Future Perspectives

The increasing number of the in vivo studies published over the last years on the direct nose-to-brain

transport of protein and peptide drugs indicates the growing interest in this route. This modality of

administration is particularly attractive for peptides and proteins as they have the chance to reach the

CNS directly, avoiding the disadvantages and limitations of the systemic route. Several studies have

already proven the ability of peptide and protein drugs to be transported through this route to

different brain areas in animal models, while several of them are already undergoing clinical studies.

However, the bioavailability of these peptides remains quite low. The contribution of

nanotechnology in this field is becoming more and more evident. Nanosized drug delivery carriers

provide a versatile platform with great potential in overcoming the underlying challenges of this

route. In general, the particle size and the composition of the carrier are considered to be important

factors that may influence the fate of the nanocarriers after their intranasal administration.

Specifically, a particle size in the range of 100-200 nm has been explored with the conclusion that

the importance of this size range is also connected with the composition of the nanocarrier.

Regarding the composition, both muco-permeating agents (i.e, PEG and pegylated surfactants) and

cell permeation enhancing agents (i.e, chitosan) were reported to lead to some positive effects.

However, the mechanism underlying this positive outcome has not been elucidated. In addition, a

tendency is observed towards the surface functionalization of the nanocarriers with biorecognitive

ligands. Still, no nanosystem has reached the clinical development phase up to now. The limited

number of in vivo studies in non-primate animals and the differences in nasal anatomy between

human and animals, do not allow us to draw clear conclusions. In conclusion, the field of nano-based

N-to-B peptide delivery is still at an early development stage and its potential is not fully unveiled.

To translate this research from the bench to the clinic, more conclusive studies involving

pharmacokinetic and pharmacodynamic data are required to shed light on the brain targeting

efficiency of the nanosystems and the underlying mechanisms.

Acknowledgements

The work was supported by the European B-Smart Consortium, which received funding from the

European Union’s Horizon 2020 research and innovation programme under grant agreement No

721058. Images were adopted from Servier Medical Art by Servier

(http://www.servier.com/Powerpoint-image-bank) and modified by the authors under the following

terms: CREATIVE COMMONS Attribution 3.0 Unported (CC BY 3.0).

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