MACRO RESEARCH: FABRICATION OF
NANOPARTICLES FOR DRUG DELIVERY
Cristina Sabliov, Associate Professor
Biological and Agricultural Engineering
November 19, 2009
NANOPARTICLES
A. H. FARAJI, P. WIPF. 2009. NANOPARTICLES IN CELLULAR DRUG DELIVERY. BIOORGANIC AND MEDICINAL CHEMISTRY. 17: 2950-2962.
KUMARI, A., S. K. YADAV, AND S. C. YADAV. 2009. BIODEGRADABLE POLYMERIC NANOPARTICLES BASED DRUG DELIVERY
SYSTEMS- REVIEW. COLLOIDS AND SURFACES B: BIOINTERFACES. DOI: 10.1016/J.COLSURFB.2009.09.001.
POLYMERIC NANOPARTICLES IN DRUG
DELIVERY
Polymeric nanoparticle
< 1000 nm
Free drug
Drug: entrapped in the
polymeric matrix
Surfactant: stabilizes
nanostructure
Polymeric matrix:
transport vehicle
Polymer molecules
Controlled release:
diffusion of antioxidant
molecules and
degradation of
polymeric matrix
Synthesis of
nanoparticle
Surfactants
Targeting: ligands aid in
targeted nanoparticle
delivery to the site of action
Additives
Protection: nanostructure
protects drug against
adverse external conditions
during delivery
Intestinal absorption:
improved due to
nanoparticle size or
increased drug solubility
SABLIOV, C. M. AND C. E. ASTETE. 2008. CHAPTER 17: ENCAPSULATION AND CONTROLLED RELEASE OF ANTIOXIDANTS AND VITAMINS VIA
POLYMERIC NANOPARTICLES. IN DELIVERY AND CONTROLLED RELEASE OF BIOACTIVES IN FOODS AND NUTRACEUTICALS (ED. N. GARTI).
WOODHEAD PUBLISHING. ISBN 9781845691455.
CHEMICAL
METHODS
ACOSTA, E. 2009. BIOAVAILABILITY OF
NANOPARTICLES IN NUTRIENT AND
NUTRACEUTICAL DELIVERY. CURRENT
OPINION IN COLLOID & INTERFACE
SCIENCE. 14: 3-15.
NANOPARTICLE SYNTHESIS

Emulsion evaporation
Mixing
Organic solvent,
polymer, and
bioactive
component
Homogenization
Oil in
water (o/w)
Water and
surfactant
Evaporation
Nanoparticles
Sonication
MATERIAL SELECTION- POLYMERS
Alginic acid
o Polysaccharide with
mannuronic and
guluronic acid
o Negative charges from
carboxylic groups
o Thickening agent
PLGA



Poly(lactic-co-glycolic)
acid
Biocompatible and
biodegradable
Mostly used for
biomedical
applications
Chitosan
N-deacetylated
derivative of chitin
Degrades to non-toxic
compounds
Positively charged



HOH 2C
O
HOH 2 C
HO
O
CH 2OH
O
O
HO
NH 2
OH
O
HO
NH2
HO
n
8
NH 2
NANOPARTICLE PROPERTIES
Size and size distribution
 Zeta potential (+ or -)
 Drug entrapment efficiency
(hydrophobic or hydrophylic)
 Drug release properties
(drug-polymer interactions
and polymer degradation)
 Degradation properties (bulk
eroding or surface eroding)
 Nanoparticle-cell interactions
(charge, size)
 Biotoxicity (adhesion, uptake,
translocation)

hydrophilic
hydrophobic
constant release
% of drug
released
Burst effect
0
or
Time (hours or days)
a
b
PROJECT I: IMPROVED DELIVERY OF
ANTIOXIDANT LIPOPHILIC VITAMIN
PROJECT II: ANTIMICROBIAL
POLYMERIC NANOPARTICLES
PROJECT III: IMPROVED
FUNCTIONALITY OF HYDROPHOBIC
NATURAL COLORANT
PROJECT I
IMPROVED DELIVERY OF
ANTIOXIDANT LIPOPHILIC VITAMIN
Imola Zigoneanu, Carlos Astete, and Abitha
Murugeshu
ORAL DRUG DELIVERY
ACOSTA, E. 2009. BIOAVAILABILITY OF NANOPARTICLES IN NUTRIENT AND
NUTRACEUTICAL DELIVERY. CURRENT OPINION IN COLLOID & INTERFACE
SCIENCE. 14: 3-15.
A. H. FARAKI, P. WIPF. 2009. NANOPARTICLES IN CELLULAR
DRUG DELIVERY. BIOORGANIC AND MEDICINAL CHEMISTRY.
17: 2950-2962.
MODEL LIPOPHILIC BIOACTIVE
(ALPHA-TOCOPHEROL)

Antioxidant lipophilic
vitamin


Prevents damage from
chemical reactions related
to cancer, diabetes,
cardiovascular disease,
inflammatory responses,
degenerative diseases,
aging, liver injury,
cataract, etc.
Tocopherols +tocotrienols
= Vitamin E

α- tocopherol is the most
biologically active form
R1
Tocopherol
HO
R2
O
R1
Tocotrienol
HO
R2
O
Tocopherol
&
Tocotrienol
R1
R2
α
CH3
CH3
β
CH3
H
γ
H
CH3
δ
H
H
Vitamin E Transport
Alpha-tocopherol
Low aqueous
solubility
Premature
elimination
Entrapment in a
delivery vehicle
• ensures solubility
and transport in
aqueous media
• mucoadhesion
increases
gastrointestinal
retention
• controlled release of
lipophilic substance
from designed
matrix
Ingested
Intestinal Fluid
Basal lamina
Lymph
+NH
−
−
−
− −−
−
−
− −−
+
+NH
+NH
3
3
+
3
+NH
−
−
−
−
−
+NH
+NH
+NH
+NH
+NH
3
+NH
3
3
3
+
3
+NH
+NH
3
+NH
3
+
+NH
3
3
+NH
3
3
+NH
3
+
+NH
+NH
3
+
+NH
+NH
3
+NH
+NH
+NH
+NH
3
3
+NH
3
+NH
3
+NH
3
+
+NH
3
3
+NH
3
+
3
3
+NH
3
3
3
RATIONALE

This project aimed to prolong the gastrointestinal
residence time of lipophilic vitamins by entrapping
the vitamins in mucoadhesive cationic
Chitosan/PLGA nanoparticles.


Mucoadhesive particle systems
were desired because they
decrease translocation into the
cells, and toxicity
Lecithin (FDA approved),
Chitosan & PLGA (FDA approved
for medical purposes)
CHITOSAN EFFECT ON NPS
Chitosan Concentration (w/v %)
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
20000
80
60
40
20
10000
0
5000
-20
-40
0
Average Diameter -60
Average z
-5000
-80
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
Chitosan Concentration (w/v %)
3 regions observed
 0.6 w/v% chitosan concentration selected for positively
charges, stable particles

Zeta Potential z (mV)
Average Diameter (nm)
15000
60
300
200
100
0
0.30
30
0.25
PLGA
Chi/PLGA
0
0.20
-30
-60
0
24
16
8
tocopherol initial loading (%)
αT theoretical
loading (%
w/w relative
to PLGA)
0
8
16
24
PDI (a.u.)
400
 potential (mV)
Average Diameter (nm)
ΑLPHA-TOCOPHEROL INITIAL LOADING EFFECT
0.15
0
24
16
8
tocopherol initial loading (%)
PLGA/Lecithin/a-TOC
Chi/ PLGA/Lecithin/a-TOC
Entrapment
Efficiency (% of αT
theoretical loading)
Size (nm)
Zeta (mV)
Size (nm)
Zeta (mV)
0
52 ± 0.83
58 ± 2.97
59 ± 1.66
83.2 ± 2.35
83.3 ± 2.35
82.8 ± 2.35
85.8 ± 2.35
-50.6 ± 2.62
-53.4 ± 2.62
-56.3 ± 2.62
-58.3 ± 2.62
340.9 ± 10.72
303.2 ± 10.72
343.9 ± 10.72
343.7± 10.72
60.9 ± 2.62
56.5 ± 2.62
59.7 ± 2.62
56.0 ± 2.62
GI PH CHANGE OVER A FEEDING CYCLE
7
6
5
Gastric
pH
4
3
After a meal
2
1
Stomach
0
0

50
Duodenum
100
150
Time (mins)
Gastric


Basal = 1.3- 1.7
Fed = can rise to ~ 5.0

Average of 1.5 and 4.0

200
250
Intestinal


Basal = 5.9 – 7.4
Fed = drop to ~ 5.5

Average of 5.5 and 6.5
Russell, T., R. Berardi, et al. (1993). "Upper gastrointestinal pH in seventy-nine healthy, elderly, North American men and women."
Pharmaceutical Research 10(2): 187-196.
300
STABILITY OF PARTICLES
aT Emulsion
PLGA(aT)
NPs
Chi/PLGA(aT)
NPs
Average Diameter (nm)
3500
3000
2500
40
 potential (mV)
PH
2000
1500
1000
a)

0
-20
-40
500
-60
0
-80
b)
2
4
6
8
pH

20
10
12
2
4
6
8
10
pH
PLGA particles were negatively charged and approached
zero below pH 2
Chi/PLGA particles approached zero between pHs 5 -8
12
STABILITY OF PARTICLES OVER TIME
2000
20
pH 1.5
pH 4.0
pH 5.5
pH 4.0
pH 5.5
pH 6.5
0
-20
}
}
Average Diameter (nm)
 potential (mV)
40
Chi/PLGA
PLGA
-40
pH 1.5
pH 4.0
pH 5.5
pH 4.0
pH 5.5
pH 6.5
1500
1000
}
}
Chi/PLGA
*
PLGA
500
-60
0
0
5
10
15
Time (hrs)




20
25
0
3
6
Time (hrs)
25
PLGA particles were stable for 24 hrs under pHs 5.5 and 6.5
Chi/PLGA particles remained stable for 24 hrs under pHs 1.5 and 4.0
Chi/PLGA particles aggregated after 24 hrs in pH 5.5, but remained
stable for 6 hrs; Chi/PLGA particles precipitated at pH 6.5
PLGA particles precipitated at pH 1.5,
PLGA PARTICLE TEM MORPHOLOGY UNDER
GASTRIC PHS
PLGA
33K
pH 1.5
0 hrs
PLGA
33K
pH 4.0
1 hrs
2 hrs
PLGA PARTICLE TEM MORPHOLOGY UNDER
INTESTINAL PHS
PLGA
33K
pH 5.5
0 hrs
PLGA
33K
pH 6.5
6 hrs
24 hrs
CHI/PLGA PARTICLE TEM MORPHOLOGY AT
UNDER GASTRIC PHS
Chi/PLGA
100K
pH 1.5
0 hrs
Chi/PLGA
100K
pH 4.0
1 hrs
2 hrs
CHI/PLGA PARTICLE TEM MORPHOLOGY UNDER
INTESTINAL PHS
Chi/PLGA
33K
pH 5.5
0 hrs
Chi/PLGA
20 K & 33K
pH 6.5
6 hrs
24 hrs
ALPHA-TOCOPHEROL GASTRIC RELEASE
FROM 16 % INITIAL LOADING PARTICLESEFFECT OF SYSTEM
Cumulative released (%)
70
Chi/PLGA
PLGA
60
50
40
30
20
pH 1.5
pH 4.0
10
a)
0
0
1
2
Time (hrs)
3
ALPHA-TOCOPHEROL INTESTINAL RELEASE
FROM 16 % INITIAL LOADING PARTICLESEFFECT OF SYSTEM
NSD in release rate
between pHs and
systems
 Slower initial
release from
Chi/PLGA
19% (P) vs. 9% (CP)
pH 5.5
 29% (P) vs. 7 % (CP)
pH 6.5
 54% (P) vs. 58% CP
after 5 days

Cumulative Released (%)

80
60
40
20
PLGA
Chi/PLGA
0
0
20
40
60
80
100
120
Time (hrs)
PROJECT I
GASTROINTESTINAL RELEASE OF ALPHATOCOPHEROL FROM 16% INITIAL LOADING
PARTICLES
Alpha-tocopherol released
100
80
60
40
PLGA
Chi/PLGA
Basal
Fed
20
0
0
20
40
60
Time (hrs)
80
100
120
140
PROJECT I
CONCLUSIONS




PLGA particles were stable under fed gastric, and all intestinal
conditions for 24 hrs (but unstable under basal gastric conditions)
Chi/PLGA particles were stable under all gastric for 24 hrs, and fed
intestinal conditions for 6 hrs (but unstable under basal intestinal
conditions)
Chi/PLGA particles released most of the alpha-tocopherol in the
intestine and only half of the amount entrapped was released in 5
days
Chi/PLGA are better for increasing the particle retention time in the
GI tract, AND subsequently expected to increase drug bioavailability
ANTIMICROBIAL POLYMERIC
NANOPARTICLES
Nipur Patel
ITRACONAZOLE (ITZ)
ITZ acts by impairing the synthesis of ergosterol,
essential component of the fungal cell membrane
 Antifungal effect of ITZ is limited due to low
bioavailability (60%)
 It is insoluble in aqueous media (S∼1 ng/mL at
neutral pH and S= 4g/mL at pH 1)
 Hypothesis: entrapping Itraconazole into PLGA
nanoparticles improves its solubility, insures a
controlled release of the drug over time, and hence
improve its antifungal efficacy

PLGA NPS WITH ENTTRAPPED ITRACONAZOLE: PROPERTIES
NP Type
Unloaded PLGA NPs
Theoretical
Loading
(% w/w)
Size
(nm)
Pdi
(au)
Zeta
(mV)
0.010±0.031
0.213±0.035
-33.14±8.40
-31.89±5.30
0.114±0.042
-24.28±2.49
Empty
0
PLGA1
12.5
201.32±5.33
232.11±1.76
PLGA2
25
199.75±4.66
90
80
70
% Released
Sporanox/PLGA NPs
60
50
40
30
20
10
0
0
20
40
60
Time (Hrs)
80
100
120
PLATE QUALITATIVE ANTIMICROBIAL ACTIVITY
Itraconazole/water
Itraconazole/Triton X sol.
Itraconazole/NP conc. x
Itraconazole/NP conc. 10x
GFP QUANTITATIVE INHIBITION ACTIVITY
Fluorescence (low conc)
Fluorescence (higher conc)
1000
1200
900
1000
800
800
Fluorescence Units
Fluorescence Units
700
600
500
400
300
400
200
0
200
0
2
4
6
8
10
-200
100
0
-100
600
0
2
4
6
8
10
Time (Days)
-400
Time (Days)
GFP A.flavus
Water-ITZ (0.3)
PLGA-ITZ NPs (0.3)
GFP A.flavus
Water-ITZ (0.003)
Tx-ITZ (0.3)
Tx-ITZ (0.003)
PLGA-ITZ NPs (0.003)
Blank NPs
Tx-ITZ Emulsion
INHIBITION AS FUNCTION OF TIME AND
CONCENTRATION
500
0
100
200
300
400
500
Water-ITZ
300
200
100
10
8
5
4
10
2
15
Conc
.
20
(mg/m
l)
25
30
Ti
m
e
6
PLGA-ITZ NPs
ay
s)
Water-ITZ
0
0
800
400
600
200
10
5
4
10
2
15
Conc
. (mg
20
/ml)
25
30
0
10
0
8
6
0.05
4
0.10
0.15
Conc
2
0.20
. (mg
/ml)
0.25
0.30
0
Ti
m
e
6
100
(D
a
8
200
ys
)
0
0
100
200
300
400
(D
ay
s)
400
Units
Fluorescence
300
0
200
400
600
800
Ti
m
e
Units
Fluorescence
PLGA-ITZ
(D
Units
Fluorescence
400
CONCLUSIONS-IMPROVED ANTIMICROBIAL
ACTIVITY



Antifungal particles measuring ~200 nm were
synthesized from PLGA with entrapped Itraconazole
Release of the antifungal was sustained over 100 hrs
Antifungal properties of the PLGA NPs were superior
to those of free Itraconazole or emulsified antifungal

Studies needed to identify mechanism of action
IMPROVED FUNCTIONALITY OF
HYDROPHOBIC COLORANT
Carlos Astete
C. E. Astete, C. M. Sabliov, F. Watanabe, and A. Biris. 2009. Ca2+ crosslinked alginic acid nanoparticles for
solubilization of lipophilic natural colorants. Journal of Agricultural and Food Chemistry. DOI:10.1021/jf900563a.
The food and pharmaceutical industries
mainly use synthetic colorants
FDA approved synthetic colorants:
Name
Color
Comments
FD&C Blue #1
Bright blue
Allergic reactions
FD&C Blue #2
Royal blue
Poor water solubility
FD&C Green #3
Sea green
Allergic reactions, and it
is not used in EU
FD&C Red #3
Cherry red
It may be carcinogenic
FD&C Red #40
Orange-red
Allergic reactions
FD&C Yellow #5
Lemon
yellow
Allergic and intolerance
reactions
FD&C Yellow #6
Orange
Allergic reaction
Disadvantages:
Advantages:
• Water soluble
• Uniform color
distribution
• Not expensive
• Color
combinations
• Stable over time
• Ease of
manipulation
• Health issues
G Mazza (2000), Health aspects of natural colorants, In: Natural food colorants: science and technology. Ed by Gabriel Lauro, Marcel and Dekker Inc., New York, 289-314
Natural colorants can overcome the health
issues of synthetic colorants

Carotenes




β-Carotene
Capsanthin
Lycopene
Xanthophyls



Zaexanthin
Bixin
Lutein

Polyphenols


Curcumin
Chlorophylls


Porphin
Phorbin
Source
Carrots
Paprika
Tomatoes
Corn
Safron
Annatto seeds
Green leaves
Advantages:
• Natural components (low
toxicity)
• Health benefits
(antioxidants, anticancer,
CVD prevention, antiaging)
Limitations:
• Not uniform color distribution
• Poor water solubility
• Expensive
• Poor stability
Hypothesis and Objectives

Hypothesis


Water solubility of a natural oily pigment is improved
by entrapment in a natural water soluble polymer
(alginic acid) crosslinked with Ca2+
Objectives
To synthesize polymeric nanostructures for delivery of
natural pigments (b-carotene)
 To characterize the nanostructures (size, zeta
potential, polydisperse index, morphology) as a
function of Ca2+ concentration, and alginic acid
concentration
 To study the stability of the formed structures

Components used are GRAS
Alginic acid



Polysaccharide with
mannuronic and
guluronic acid
Negative charges from
carboxylic groups
Thickening agent
Lecithin




Phophatidyl choline
Surfactant
Emulsions
Extracted from egg
yolk and soybeans
β-carotene



Natural pigment
Lipophilic
Antioxidant
Approach
Ca2+
CH3
+
CHN
3 CH3
Ca2+
Ca2+
Ca2+
Ca2+
Ca2+
Ca
Ca2
O
2+ O P OO
+
Ca2
COO
+
COO
Ca2+
Ca2+
Ca2+
Ca2+
Ca2+
Ca2+
Ca2+
COO
COO
O
O P OO
Ca2+
CH3
+
CHN
3 CH3
Ca2+
Ca2+
Ca2+
Nanoparticle synthesis
Organic Phase:
Ethyl acetate/
Chloroform
CaCl2
Alginic acid
Stabilization
Aqueous Phase:
Water
Lecithin
Emulsion Sonication
Time: 6 min
Temperature 4-8 °C
in an ice bath
Pigment
nanoparticles
(suspension or dry)
Evaporation under
vacuum
No CaCl2
Alginic acid: 0.35 mg/ml
CaCl2: 0.29 mg/ml
Alginic acid: 0.35 mg/ml
Nanoparticle stability
1000
Solvent: Ethyl acetate
a1
a2
a3
600
400
200
Particle
precipitation
0
0.07
0.35
1.76
3.5
Alginic acid (mg/ml)
300
b1
b2
b3
250
Size (nm)
Size (nm)
800
Solvent: Chloroform
200
150
100
50
0
0
0.07
0.35
1.76
Alginic acid (mg/ml)
3.53
-4
-12
-3
-10
-3
-8
-2
-6
-2
-4
-1
-2
-1
0
Ratio absorbance (1/C0 - 1/C)
ratio absorbance (Ln(C/C0)))
Antioxidant and pH stability
0
0
20
40
60
80
100
120
140
135
-10
-20
130
Alginic acid: 0.07 mg/ml -20
125
-30
120
-40
115
-50
110
-60
105
-70
Size (nm)
-30
130
-40
size
zeta potential
120
-50
-60
110
-70
100
-80
1
2
3
4
pH
5
6
7
Size (nm)
Alginic acid: 0 mg/ml
140
-10
Zeta potential (mV)
150
100
-80
2
3
4
pH
5
6
7
Zeta potential (mV)
Time (hrs)
CONCLUSIONS-IMPROVED FUNCTIONALITY




Water-soluble nanostructures for entrapment of bcarotene were formed with the proposed method
The organic solvent used in the synthesis significantly
affected the size and stability of the structures by
precipitation
The nanostructures were stable
between pH 3 and 7
Ca2+ and polymer concentration
affected drastically the morphology
and functionality of the system,
not as much size and size distribution
ACKNOWLEDGEMENTS