Inhalable Nano Composite

Particles for Treatment of

Lung Cancer
Keishiro Tomoda, Takehisa Nakajima, Keiji Hirota,
Hiroshi Terada and Kimiko Makino
Center for Drug Delivery Research
and
Faculty of Pharmaceutical Sciences
Tokyo University of Science
8th CRS Symposium
Feb. 26th-27th, 2008, B.V.Patel Perd center,
Armedabad, India

Lung cancer
Lung cancer is a common malignancy and
remains the leading cause of cancerrelated deaths in both men and women.
Approximately 15 % of all cancers arise
in the lungs, and 52,000 deaths/ year
result from this disease in Japan.
38,000 deaths/year in men and 14,000
deaths/year in women.

The main causes of lung cancer include carcinogens

(such as those in tabacco smoke, particularly
cigarettes, asbestos), ionizing radiation,
and viral infection.
This exposure causes cumulative changes to the DNA
in the tissue lining the bronchi of the lungs.
As most tissue becomes
damaged, eventually a cancer
development.

Frequency of histological types

of lung cancer
Histological type Frequency (%)
Non-small cell lung carcinoma 80.4
Small cell lung carcinoma
16.8
Carcinoid
0.8
Sarcoma
0.1
Unspecified lung cancer
1.9

Cross section of a human lung.

The white area in the upper lobe is cancer;
the black areas indicate the patient was a smoker.
http://en.wikipedia.org/wiki/Lung_cancer

Non-small cell lung carcinoma (NSCLC) 80%

Sub-types of non-small cell lung cancer[16]
Histological sub-type

Frequency of
all lung cancers (%)

Squamous cell lung carcinoma

31.1

Adenocarcino Adenocarcinoma (not

ma
otherwise specified)

23.2

Bronchioloalveolar carcinoma

3.0

Adenosquamous carcinoma

1.2

Papillary adenocarcinoma

0.7

Mucoepidermoid carcinoma
Adenoid cystic carcinoma

[19]

[20]

Other specified adenocarcinoma

Large cell carcinoma

0.1
0.04
1.1
10.7

Giant cell and spindle cell carcinoma

0.4

Other/unspecified non-small cell lung carcinoma

8.9

Non-small cell lung carcinoma is staged from

IA ("one A", best prognosis) to IV ("four", worst
prognosis).
The American Joint Committeee on Cancer (AJCC)
recommends TNM staging followed by further
grouping.
TX: The primary tumour cannot be assessed, or there are malignant cells in the sputum or
bronchoalveolar lavage but not seen on imaging or bronchoscopy.
Tis: Carcinoma in situ.
T0: No evidence of primary tumour.
T1: Tumour less than 3 cm in its greatest dimension, surrounded by lung or visceral pleura
and without bronchoscopic invasion into the main bronchus.
T2: A tumour with any of:More than 3 cm in greatest dimension. Extending into the main
bronchus, but more than 2 cm distal to the carina. Obstructive pneumonitis, but not involving
the entire lung.
T3: A tumour with any of: - Invasion of the chest wall, diaphragm, mediastinal pleura, or
parietal pericardium. Extending into the main bronchus, within 2 cm of the carina, but not
involving the carina.
Obstructive pneumonitis of the entire lung.
T4: A tumour with any of: - Invasion of the mediastinum, heart, great vessels, trachea,
oesophagus, vertebra, or carina. Separate tumour nodules in the same lobe. Malignant pleural
effusion.

The mainstay of treatment for these patients is

surgical resection.
The patients who present with non-small cell lung
carcinoma, 75 % have advanced, unresectable
disease. Of the 25 % who are able to undergo
surgery (stage I or II), only half are alive 5 years
after surgery.
Patients with stage IIIA disease have been
treated with either radiation or, in selected
cases, surgical resection.
Median survival with radioation alone is only 9-13
months, and the 5-year survival rate is only
5-10 %. The radiation therapy has poor curative
potential.

Chemotherapy is also used for patients with advanced

disease and in neoadjuvant and adjuvant therapy,
but its effect in prolonging survival remains unclear.
New approaches to the treatment of this disease are
clearly necessary and the discovery of active new
cytotoxic agents could have a major impact on overall
survival rates from cancer.
Unfortunately, non-small cell lung carcinoma is among
the most chemoresistant of all neoplasms.

Chemotherapeutic agents with single-agent activity

in NSCLC are
cyclophosphamide, vincristine, cisplatin, mitomycin C,
vinblastine, vindesine, carboplatin, etoposide
and ifosfamide.
The most widely used combinations in NSCLC have
been cisplatin-etoposide,
mitomycin C-vinblastine-cisplatin,
vinblastine-cisplatin,
cyclophosphamide-cisplatin- vinblastine,
and ifosfamide-carboplatin-etoposide.

The outlook for patients with primary or

metastatic pulmonary involvement is generally
poor and has not been improved substantially
even with the availability of new
chemotherapeutic agents.
The reasons for treatment failure are diverse.
One factor is the inability to achieve therapeutic
concentrations of drugs at the tumor site,
since the anticancer drugs have been administered by
a intravenous infusion.

One potential approach to improving therapeutic

efficacy is targeted drug dose intensification using
regional therapy.
The regional drug delivery also has a potential for
minimizing the systemic side effects of cytotoxic
agents delivered in this way.

Pulmonary delivery of anti cancer drugs

to the tumor site in the lungs.

Nanocomposite
particles
Lung cancer
Macrophage

should reach
deep in the
lungs
should escape
from alveolar
macrophages
should be
internalized by
lung cancer
cells

Aerosol Droplets

Type I cell

Alveolar M

Epithelial lining fluid

Tubular myelin

Interstitium

Type II cell

Endothelial cell
Capillary Lumen

Route of solute
diffusion

How, can we deliver anti-cancer

drugs deep in the lungs?
Medication using DPI can achieve high local levels
of drug in epithelial lining fluid of the airways and
low respiratory tract. Drugs administered locally to
the lungs, via DPI are attractive in that they
achieve higher levels in the lungs with fewer
systemic side effects.
The powder composed of therapeutic drugs should
escape from the recognition by alveolar
macrophages.
Biocompatible and biodegradable carrier is needed
to reach deep in lung.

PLGA
O
H

H
C

O
O

C
n

C H3

H
C
H

OH
m

+H 2 O
O

O
HO

H
C

OH

HO

H
C
H

CH3
lactic acid

glycolic acid

OH

p=0.01

140

Phagocytosis

120

p<0.0001
p=0.006 p<0.0001

100
80

p=0.05

60
40
20
0
8.9
6.2
3.5
1.5
Particle diameter (m)

The Anti-cancer drug-loaded NS

When TAS103-loaded PLGA nano particles

are used as an inhaled formulation,

they can overcome the mucociliary clearance.
they can be escaping from the recognition of alveolar
macrophages and can be taken up by other cells like
cancer cells.
From these unique properties inhaled nanoparticle
system has potential as novel therapeutic
treatments.
Their mass median aerodynamic diameter (MMAD) is
not suitable for inhalation since their size is too small.
particles having 2-3 m of diameter are the most
effective properties for deposition to the deep lung
site .

Maximum size
for transition

Depositioned
particle size
trachea
5m

pharynx
larynx

25m

60m

trachea

Primary bronchiole
Primary bronchiole

bronchus Secondary bronchiole

2m

bronchiolus Terminal bronchiole

Respiratory
bronchiole
alveolus
Alveolar duct

20m
6m
2m

To utilize nanoparticles for inhalation treatment,

the nanocomposite particles having 2-3 m of diameter
were prepared as a complex of nanoparticles and additives,
trehalose.
After reaching the site, they are decomposed into
drug-loaded nanoparticles,
since the sugar moiety is soluble in alveolar lining fluid.

23 m
nanocomposite
particle

Nanoparticles have highly scattering, highly affected by

static electricity, and are difficult to be used.
Water adsorption, Decomposition
Binder

Nanoparticles

Degradation

Nanocomposite particles
Carrier+nanoparticles
Assembly of nanoparticles

Volume [%]

16
14
12
10
8
6
4
2
0

107

138
179
232
Particle diameter [nm]

300

Size distribution of primary nanoparticle, 200 nm

40
35

Volume [%]

30
25
20
15
10
5
0

296

367
455
Particle diameter [nm]

565

702

Size distribution of primary nanoparticle, 400 nm

Mini Spray Dryer B-290

It is an outer mixing twofluid nozzle, using gas of 5

to 8 bar.
The nozzle has a diameter
of 0.7 mm. The integrated
ruby stone leads to a
precise and reproducible
spray cone.

nanocomposite
particle

Double Distilled
Water

A: SEM image of nanocomposite particles prepared at 70 C

using the primary nanoparticles with the size of 200 nm.
The weight ratio of the primary nanoparticles was 0.5.
B: SEM image of nanoparticles after decomposition of nanocomposite particles prepared at 70 C using the primary
nanoparticles with the size of 200 nm.
The weight ratio of the primary nanoparticles was 0.5.

A: SEM image of nanocomposite particles prepared at 80 C

using the primary nanoparticles with the size of 400 nm.
The weight ratio of the primary nanoparticles was 0.5.
B:SEM image of nanoparticles after decomposition of nanocomposite particles prepared at 80 C using the primary
nanoparticles with the size of 400 nm.
The weight ratio of the primary nanoparticles was 0.5.

Particle diameter [nm]

10000
8000
6000

Particle diameter [nm]

1000
800
600
400
200
0
0.1

4000

0.3
0.5
0.7
0.9
The weight ratio of primary particles

2000
0

0.2
0.4
0.6
0.8
1
The weight ratio of primary nanoparticles

The effects of inlet temperature and the weight ratio of primary nanoparticles
on the decomposition efficiency of nanocomposite particles.
The size of primary nanoparticles was 400 nm and the inlet temperatures were
80 () and 90 () C.
The magnified graph for the particle size range between 0 and 1000 nm
is shown in the inset.

4000
3000

500
Particle diameter [nm]

Particle diameter [nm]

5000

400
300
200
100
0
0.1

0.3
0.5
0.7
0.9
The weight ratio of primary particles

2000
1000
0

0.2
0.4
0.6
0.8
1
The weight ratio of primary nanoparticles

The effects of inlet temperature and the weight ratio of primary nanoparticles
on the decomposition efficiency of nanocomposite particles.
The size of primary nanoparticles was 200 nm and the inlet temperatures were
70 () and 80 () C.
The magnified graph for the particle size range between 0 and 500 nm is
shown in the inset.

Aerodynamic diameter
Deq : Equivalent volume diameter, obtained by
the measurement with laser scattering
and diffraction particle sizing method
Daer : Aerodynamic diameter
Daer=Deq(p / 0)1/2
p : particle density
0 : unit density sphere
: particle shape function

Table 1, Mass median diameter of nanocomposite particles, dmass.

Size of
primary
nanopartic
les [nm]

Inlet
temperat
ure
[C]

0.1

0.2

0.3

0.4

0.45

0.5

0.55

0.6

0.7

0.8

0.9

1.0

200

70

1.77

1.70

1.77

1.99

1.63

2.09

1.99

2.21

2.03

1.77

1.91

1.85

80

13.70

3.24

3.55

2.47

3.89

2.84

2.40

2.58

2.31

2.17

1.74

80

1.61

1.78

1.51

1.88

1.78

1.77

1.87

1.80

1.85

1.85

2.04

2.19

90

2.12

2.47

2.07

1.77

1.77

1.86

1.58

1.99

2.52

1.91

1.98

1.80

400

Weight ratio of primary nanoparticles in nanocomposite particles

Table 2, density of nanocomposite particles, .

Size of
primary
nanopartic
les [nm]

Inlet
temperat
ure
[C]

0.1

0.2

0.3

0.4

0.45

0.5

0.55

0.6

0.7

0.8

0.9

1.0

200

70

1.41

1.40

1.45

1.43

1.44

1.47

1.44

1.41

1.23

1.29

1.25

1.16

80

1.37

1.29

1.12

1.07

1.10

1.27

1.10

0.98

0.91

0.93

0.99

0.70

80

1.18

1.07

0.99

1.10

1.10

1.11

1.05

1.10

1.12

1.14

1.09

0.93

90

1.49

1.44

1.44

1.44

1.33

1.31

1.45

1.39

1.38

1.35

1.31

1.34

400

Weight ratio of primary nanoparticles in nanocomposite particles

Particle aerodynamic diameter [m]

5
4
3
2
1
0

0.2
0.4
0.6
0.8
1
The weight ratio of primary nanoparticles

The effects of inlet temperature and the weight ratio of primary

nanoparticles on the aerodynamic diameter of nanocomposite
particles.
The size of primary nanoparticles was 200 nm and the inlet
temperatures were 70 () and 80 () C.

Particle aerodynamic diameter [m]

5
4
3
2
1
0

0.2
0.4
0.6
0.8
1
The weight ratio of primary nanoparticles

The effects of inlet temperature and the weight ratio of primary

nanoparticles on the aerodynamic diameter of nanocomposite
particles.
The size of primary nanoparticles was 400 nm
and the inlet temperatures were 80 () and 90 () C.

DPI (Dry powder inhaler)

device
DPI device Jethaler]

Cascade impactorArtificial lungs

8 stages

Stage
Daero(m)

0
>11

1
7.011.0

Stage
Daero(m)
2.1

3
3.34.7

Stage
BF
Daero(m)

6
0.651.1

28.3 (L/min) 5 sec.

Andersen nonviable impactor

4
2.13.3

2
4.77.0
5
1.1

7
0.130.65

<0.13

30

FPF [%]

25
20
15
10
5
0

0.2
0.4
0.6
0.8
1
The weight ratio of primary nanoparticles

FPF values of nanocomposite particles prepared with the primary nanoparticles

with the size of 200 nm at 70 C (), and prepared with the primary nanoparticles
with the size of 400 nm at 80 C ().

Evaluation of anti-canceractivity of
TAS-103/PLGA Nanocomposite particles
free drug solution
or
nanocomposite suspension
incubation
for 72 hr at 37
washing

A549 cell culture

in F-12K

crystal violet
in PBS

spectrophotometric measurement
Scheme of cell study

5% TAS-103/PLGA NP
10% TAS-103/PLGA NP

TAS-103/PLGANanocomposite particles
CH3
H3C

NH

2HCl

HO
Structure of TAS-103

6-{ [ 2 - (dimethylamino) ethyl ] amino } -3- hydroxyl -7H indeno

[2,1-c] quinolin -7- one dihydrochloride (TAS-103, Taiho
Pharmaceutical Co., Ltd.) .
The drug is a potent dual-inhibitor of topoisomerases and
and is highly effective against solid tumors

Evaluation of anti-canceractivity of
TAS-103/PLGA Nanocomposite particles
free drug solution
or
nanocomposite suspension
incubation
for 72 hr at 37
washing

A549 cell culture

in F-12K

5% TAS-103/PLGA NP
10% TAS-103/PLGA NP

crystal violet
in PBS

spectrophotometric measurement

[%]

120
110
100
90
80
70
60
50
40
30
0

control
free TAS-103
5% nanocomposite
10% nanocomposite

3 10 20 30 100
Concn. [nM]

Cell cytotoxicity of TAS-103/PLGA nanocomposite particles

against A549 cell line after 72 hrs (meanS. D.)

Free TAS 103, ED50>30 nM

Nanocomposite particles, ED50 10 nM

Cumulative
release amount [%]

Drug release from TAS-103/PLGA Nanocomposite particles

100

80
60
40
20
0

5% NP
10% NP

5% nanocomposite
10% nanocomposite

10
15
Time [day]

20 0

10
15
Time [day]

20

Cumulative release amount of TAS-103 from TAS- Cumulative release amount of TAS-103 from TAS103/PLGA nanocomposite particles in PBS at pH 103/PLGA primary nanoparticles in PBS at pH 7.4
(meanS. D.)
7.4 (meanS. D.)

Slow release was observed from 5% TAS103-loaded PLGA

Nanoparticles, but Initial burst was observed from 10% TAS-loaded
PLGA Nanoparticles.

No difference was observed between nanoparticles and

nanocomposite particles.

In vivo study
compressed
air

sample chamber

Wistar rat
8 weeks old
male

3 mg of
10% TAS-103/PLGA
nanocomposite particles
delivery tube

TAS-103
in physiological saline
Sketch of inhalation and intravenously administration

TAS103 concentrations in Lungs and Plasma were measured.

4000
3000
2000

In lung by I.H.
In plasma by I.H.
In lung by I.V.
In plasma by I.V.

1000
0

1 2 3 4 5 6 7 8
Time [h]

TAS-103 concentration in plasma and lung tissue

after inhalation of nanocomposite particles or
intravenously administration of free drug (meanS.
D.)

D r u g co n cn . [n M ]

Drug concn. [nM]

5000

200
150

In plasma by I.H.
In plasma by I.V.

100
50
0

1 2 3 4 5 6 7 8
Time [h]

TAS-103 concentration in plasma after inhalation

of nanocomposite particles or intravenously
administration of free drug (meanS. D.)

From the comparison between concentrations of TAS-103

in lungs after I.H. and I.V., it is clear that the higher
concentration is obtained by I. H. than by I.V.

Delivery ratio [%]

25
20
15

In lung by I.H.
In plasma by I.H.
In lung by I.V.
In plasma by I.V.

10
5
0

1 2 3 4 5 6 7 8
Time [h]

Delivery ratio of TAS-103 in lung and plasma after

inhalation of nanocomposite particles or
intravenously administration of free drug
(meanS. D.)

When TAS-103 was administered as inhalable nanocomposite

particles, drug delivery ratio to lungs is significantly higher
than that to plasma.
On the other hand, when TAS-103 was intravenously
administered, drug delivery ratio to lungs is lower than that
to plasma.

Conclusion
Inhalable Nano Composite Particles for
Treatment of Lung Cancer
The anti cancer activity was evaluated using the
lung cancer cell line A549.
The value of ED50 3 days after the addition of
the drugs was about 30 nM for free TAS103 and
10 nM for PLGA nanocomposite particles loaded
with 5 % TAS103.
Much higher concentration of TAS103 was
observed in the lungs than that in plasma, when
the nanocomposite particles were pulmonary
administered.

Acknowledgement
Center for Drug Delivery Research
Tokyo University of Science*

Hiroshi Terada
Takehisa NAKAJIMA
Keishiro TOMODA
Keiji HIROTA

Taiho Pharmaceutical
Co. Ltd.
Masahito Komuro
Kenji Kitazato

This work was supported by High-Tech Research Center Project

for PrivateUniversities: matching fund subsidy from Ministry of Education,
Culture, Sports, Science and Technology (MEXT), 2004-2008 and
MEXT (19300177), 2007-2008.

IN VITRO PERMEATION OF GOLD NANOPARTICLES THROUGH RAT SKIN

AND RAT INTESTINE:
EFFECT OF PARTICLE SIZE.
Ganeshchandra S. Sonavane, Keishiro Tomoda and Kimiko Makinoa

Colloids and Surfaces B; Biointerfaces, in press.