BRAIN DELIVERY OF PROTEINS BY BRAIN DELIVERY OF PROTEINS BY THE INTRANASAL ROUTE OF THE INTRANASAL ROUTE OF

ADMINISTRATION USING CATIONIC ADMINISTRATION USING CATIONIC LIPOSOMESLIPOSOMES

Presented by Mattia M. MigliorePresented by Mattia M. MiglioreFebruary 23, 2007February 23, 2007

Graduate Materials Links SymposiumGraduate Materials Links SymposiumNortheastern University, Boston MA 02115Northeastern University, Boston MA 02115

Introduction:: Neurodegenerative diseases cause the progressive Neurodegenerative diseases cause the progressive

destruction of either peripheral or central nervous destruction of either peripheral or central nervous system neurons and result in significant cognitive system neurons and result in significant cognitive and/or motor dysfunction. and/or motor dysfunction.

Include Alzheimer’s disease, Parkinson’s disease, Include Alzheimer’s disease, Parkinson’s disease, ALS, brain cancer, Huntington’s disease, and ALS, brain cancer, Huntington’s disease, and multiple sclerosis.multiple sclerosis.

Neurotrophic factors are growth factors that Neurotrophic factors are growth factors that stimulate neuronal regeneration and/or prevent stimulate neuronal regeneration and/or prevent neuronal cell death.neuronal cell death.

Introduction (cont.):

The clinical use of neurotrophic factors and other The clinical use of neurotrophic factors and other therapeutic proteins has been limited due to their therapeutic proteins has been limited due to their inability to cross the blood-brain barrier. inability to cross the blood-brain barrier.

Currently, administration is limited to invasive Currently, administration is limited to invasive intracerebral infusions.intracerebral infusions.

The purpose of the present study was to develop a The purpose of the present study was to develop a cationic liposomal system for nasal delivery of cationic liposomal system for nasal delivery of proteins to the brain.proteins to the brain.

The intranasal route of administration was The intranasal route of administration was selected because it can bypass the blood-brain selected because it can bypass the blood-brain barrier, avoids systemic absorption, and limits barrier, avoids systemic absorption, and limits potential peripheral side effects. potential peripheral side effects.

Two different preparations of cationic Two different preparations of cationic liposomes were generated containing a liposomes were generated containing a fluorescently tagged model protein, Alexa-488 fluorescently tagged model protein, Alexa-488 ovalbumin.ovalbumin.

Ovalbumin (OVAL) was selected because its Ovalbumin (OVAL) was selected because its molecular weight (45KDa) is similar to the molecular weight (45KDa) is similar to the molecular weight of several neurotrophic molecular weight of several neurotrophic factors. factors.

Rationale:

Aim # 1Aim # 1: To characterize and optimize a : To characterize and optimize a nanoparticle formulation for intranasal nanoparticle formulation for intranasal delivery of OVAL.delivery of OVAL.

Aim # 2Aim # 2: To determine brain delivery, both : To determine brain delivery, both qualitatively and quantitatively.qualitatively and quantitatively.

Aim # 3Aim # 3: To determine brain distribution of : To determine brain distribution of OVAL, and protein integrity.OVAL, and protein integrity.

Aim # 4Aim # 4: To determine co-localization of : To determine co-localization of OVAL with a dopamine neuronal marker, OVAL with a dopamine neuronal marker, tyrosine hydroxylase.tyrosine hydroxylase.

Specific Aims:

Specific Aim # 1:

Liposome Liposome Characteristics Characteristics

Prep # 1Prep # 1

(1 (1 g/g/l)l)

Prep # 2 Prep # 2

(1 (1 g/g/l)l)

Prep # 2 Prep # 2

(2 (2 g/g/l)l)

Particle size (nm)Particle size (nm) 1215 ± 40.71215 ± 40.7 121 121 ± 0.9± 0.9 126 126 ± 9.8± 9.8

Zeta Potential (mV)Zeta Potential (mV) 36 36 ± 2.17± 2.17 34 34 ± 1.67± 1.67 56 56 ± 1.24± 1.24

Loading Efficiency Loading Efficiency (%)(%)

94 %94 % 99 %99 %

Data presented as mean ± SEM

Intranasal Alexa 488-OVAL (no nanoparticles)

24 hr time point, striatum(20x)

Intranasal Alexa 488-OVAL (Preparation # 1)

24 hr time point, striatum (20x)

Intranasal Alexa 488-OVAL(Preparation # 2)

24 hr time point, SN (20x)

Specific Aims # 2: Qualitative Determination of Protein Brain Delivery:

Specific Aim # 2: Quantitative Analysis

of Protein Brain Delivery:

Time course of brain uptake of 111In-OVAL (1 µg/ µl) for liposomal preparations or control (PBS)

Control Prep #1 Prep #2 0.0000.0010.0020.0030.0040.0050.0060.0070.0080.0090.0100.011

111Indium Ovalbumin Brain Levels (1ug/ul): 6 hr time point

% a

dmin

iste

red

dose

rec

over

ed/

g of

tis

sue

Control Prep #1 Prep #2 0.000

0.001

0.002

0.003 P < 0.01

111Indium Ovalbumin Brain Levels (1 ug/ul): 24 hr time point

% a

dmin

iste

red

dose

rec

over

ed/

g of

tis

sue

Control Prep #2 0.000

0.025

0.050

0.075

0.100

111Indium Ovalbumin Brain Levels (1 ug/ul): 1 hr time point

% a

dmin

iste

red

dose

rec

over

ed/

g of

tis

sue

Control Prep #2 0.000

0.005

0.010

0.015

0.020

0.025

0.030

0.035

111Indium Ovalbumin Brain Levels (1 ug/ul): 4 hr time point

P= 0.0468

% a

dm

inis

tere

d d

ose

rec

ove

red

/g

of

tiss

ue

111In OVAL in Preparation #2 (1 µg/µl) 111In OVAL in Preparation #2 (2 µg/µl)

Biodistribution Study:

Control Liposomal0.0

0.1

0.2

0.3

111Indium Ovalbumin Brain Levels: 1 hour time point

P= 0.0227

% a

dm

inis

tere

d d

ose r

eco

vere

d/

g o

f ti

ssu

e

Control Liposomal0.0000

0.0025

0.0050

0.0075

0.0100

111Indium Ovalbumin Brain Levels: 6 hr time point

P= 0.0390

% a

dm

inis

tere

d d

ose r

eco

vere

d/

g o

f ti

ssu

e

Control Liposomal0.00000

0.00025

0.00050

0.00075

0.00100

0.00125

0.00150

0.00175

111Indium Ovalbumin Brain Levels: 24 hr time point

P= 0.0387

% a

dm

inis

tere

d d

ose r

eco

vere

d/

g o

f ti

ssu

e

Control Liposomal0.0000.0010.0020.0030.0040.0050.0060.0070.0080.0090.0100.0110.0120.0130.014

111Indium Ovalbumin Brain Levels: 4 hr time point

P= 0.0005

% a

dm

inis

tere

d d

ose r

eco

vere

d/

g o

f ti

ssu

e

Time course of brain uptake of 111In-OVAL (2 µg/µl) for liposomal Preparation # 2 or control (PBS)

Intracellular uptake of Alexa 488-OVAL in the substantia nigra afterintranasal administration. Alexa 488-OVAL (Preparation # 2), 24 hr time point (40x).

Localization of Alexa 488-OVAL in the corpus striatum after intranasal administration. Alexa 488-OVAL (Preparation # 1), 24 hr time point (20x).

Specific Aim # 3: Protein Brain Distribution

Alexa-488 ovalbuminbrain distribution

Olfactory bulb

Frontal

Striatum

Midbrain

Posterior

Specific Aim # 3: Protein Integrity

Intranasal OVAL(Preparation # 2, 2 μg/μl)

1 hr time point, striatum (20x).

Intranasal OVAL(Preparation # 2, 2 μg/μl)

24 hr time point, SN (40x).

Specific Aim # 4: Co-localization of Alexa-488 OVAL with Tyrosine Hydroxylase

Intranasal Alexa 488-OVAL(Preparation # 2)

24 hr time point, SN (40x)

TH positive dopamine neuronsSN (40x)

Merged image

Conclusions: Liposomal preparations of OVAL effectively deliver the

protein to brain after intranasal administration to rats. The highest brain levels were detected at the shortest time

point, i.e. 1 hr after administration. Liposomal preparations increase brain residence time of the

protein at the 24 hr time point when compared to control. Liposomal OVAL delivered intranasally yields discrete

protein deposits in both striatum and SN, with apparent cellular uptake in the SN by 24 hrs.

Intranasal administration of the 2 µg/µl form of liposomal Preparation #2 provides higher brain

levels and reduced distribution to the GI tract relative to the more dilute form.

Acknowledgements: Work was supported by the Work was supported by the

NCI-NSF IGERT NCI-NSF IGERT Nanomedicine S&T award, and Nanomedicine S&T award, and a 2005 AFPE (American a 2005 AFPE (American Foundation for Pharmaceutical Foundation for Pharmaceutical Education) fellowship.Education) fellowship.

Thesis committeeThesis committee::Dr. Barbara WaszczakDr. Barbara WaszczakDr. Mansoor AmijiDr. Mansoor AmijiDr. Robert CampbellDr. Robert CampbellDr. Rebecca carrierDr. Rebecca carrierDr. Ralph LoringDr. Ralph LoringDr. Robert SchatzDr. Robert Schatz

Dr. Tushar VyasDr. Tushar Vyas

Dr. Amiji’s lab:Dr. Amiji’s lab:

SandipSandip

LillianLillian

MayankMayank Dr. Campbell’s lab:Dr. Campbell’s lab:

Suman and Ashish.Suman and Ashish. Dr. William Hartner.Dr. William Hartner. Dr. Torchilin’s lab.Dr. Torchilin’s lab. Eunice.Eunice. Fran.Fran.