Targeted drug delivery

TARGETED DRUG DELIVERY
Presented by:- CHRISTY P GEORGE
M pharm,
BHARATI VIDYAPEETH UNIVERSITY
POONA COLLEGE OF PHARMACY,PUNE
1

CONTENTS
I. OVERVIEW OF TARGETED DRUG DELIVERY
II.
III.
IV.
V.
VI.
VII.
VIII.
I. OVERVIEW OF TARGETED DRUG DELIVERY
II. ACTIVE TARGETING
III. PASSIVE TARGETING
IV. MAGNETIC TARGETING
V. ULTRASOUND BASED TARGETING
VI. SITE SPECIFIC TARGETING
I. BRAIN TARGETING
II. COLON TARGETING
VII.DISEASE BASED TARGETING
I. TUMOR TARGETING
VIII.CASE STUDY
2

DRUG TARGETING- OVERVIEWDRUG TARGETING- OVERVIEW
 “Targeting the drugs (and DDS) involves the
improvement of the specificity of the system towards
the pharmacologically relevant target in the body”
 Targeted drug delivery systems (TDDS) involve
administration of the DDS to the patient, delivery of
the DDS at the target (pathological) site , release of
the active ingredients in/around the target, and
avoiding nonspecific toxicity in normal cells.
 The concept of targeted drugs is not new, but dates
back to 1906 when Ehrlich first postulated the ‘magic
bullet’.
3

TARGETING
AIMS
AVOID TOXICITY OR ADVERSE REACTIONS
OF THE DRUG/GENE ON NONSPECIFIC
NORMAL CELLS AND FACILITATING
ADMINISTRATION OF LOWER DOSES TO
ACHIEVE THERAPEUTIC/DIAGNOSTIC
BENEFITS
FACILITATE THE THERAPEUTIC SUBSTANCE
TO REACH THE SITE OF ACTION FROM THE
SITE OF ADMINISTRATION WHERE THE
TARGET SITE CAN BE ORGAN, TISSUE,
CELL, OR ORGANELLES
RELEASE THE THERAPEUTIC PAYLOAD IN ITS
ACTIVE FORM IN AND AROUND SITE OF ACTION AT
REQUIRED CONCENTRATION
PROTECT THE DRUG OR GENE
FROM THE DETRIMENTAL
ENVIORNMENTAL EFFECTS LIKE PH
OR ENZYMES
4

WHY TO TARGET ?
•
membrane bounding, biological instability,
• Pharmaceutical drug instability in conventional dosage form solubility
,biopharmaceutical low absorption, high-membrane bounding, biological instability,
pharmacokinetic / pharmacodynamic short half life, large volume of distribution,
low specificity, clinical, low therapeutic index.
5

IDEAL CHARACTERISTICSIDEAL CHARACTERISTICS :

biocompatible, biodegradable, and
physicochemical stable
invitro

cells or tissues or organs and should
have uniform capillary distribution.

drug release.
 Drug release does not effect the drug
action.

 Minimal drug leakage during transit.
 biodegradable
or readily eliminated from the body
without any problem and no carrier
induced modulation of diseased state.

system should be easy or reasonably
simple, reproductive and cost effective.
IDEAL CHARACTERISTICSIDEAL CHARACTERISTICS :
 It should be nontoxic,
biocompatible, biodegradable, and
physicochemical stable invivo and
invitro.
 Restrict drug distribution to target
cells or tissues or organs and should
have uniform capillary distribution.
 Controllable and predicate rate of
drug release.
 Drug release does not effect the drug
action.
 Therapeutic amount of drug release.
 Minimal drug leakage during transit.
 Carriers used must be biodegradable
or readily eliminated from the body
without any problem and no carrier
induced modulation of diseased state.
 The preparation of the delivery
system should be easy or reasonably
simple, reproductive and cost effective.
ADVANTAGESADVANTAGES :
•
may be simplified.
• Toxicity is reduced by delivering
a drug to its target site, there by
reducing harmful systemic effects.
•
smaller dose to produce the desire
effect.
•
metabolism.
• Enhancement of the absorption of
target molecules such as peptides
and particulates.
•
conventional drug delivery system.
•
concentration(controlled release).
•
cells that compare to normal cells
ADVANTAGESADVANTAGES :
• Drug administration protocols
may be simplified.
• Toxicity is reduced by delivering
a drug to its target site, there by
reducing harmful systemic effects.
• Drug can be administered in a
smaller dose to produce the desire
effect.
• Avoidance of hepatic first pass
metabolism.
• Enhancement of the absorption of
target molecules such as peptides
and particulates.
• Dose is less compared to
conventional drug delivery system.
• No peak and valley plasma
concentration(controlled release).
• Selective targeting to infections
cells that compare to normal cells.
6

APROACHES IN TARGETED DRUG DELIVERY
ACTIVE
TARGETING
PASSIVE
TARGETING
DISEASE BASED
STRATEGIES
LOCATION BASED
STRATEGIES
LIGAND BASED

LIGAND BASED
EXTERNAL STIMULI
EXTERNAL STIMULI
LIKE:-
•MAGNETIC
FIELD
•LIGHT
•ULTRASOUND
•TEMPERATURE
CANCER

DISEASES
CANCER
INFECTIOUS
DISEASES
BBB





BBB
COLON
RETINA
SKIN TARGETED
PULMONARY
INTRACELLULAR
7

PASSIVE TARGETING (physiology based targeting)
• Passive targeting is present naturally in the human body. Hormones,
•
•
• Passive targeting is present naturally in the human body. Hormones,
neurotransmitters, growth factors, etc. have a natural tendency to go and target
the receptors at their sites of action, e.g., insulin and insulin receptors. This
concept can be applied to the drugs too.
• The accrual of drugs/drug-carrier systems at the intended site of action by the
action of physicochemical and physiological factors is passive targeting.
• Certain tissues under diseased conditions present opportunities in terms of
modified physiologies which can be exploited by passively targeting nano carriers.
8

• E .g. of passive targeting:- The nano carriers
phagocytes. This fact can be used to passively
affect the RES (e.g., leishmaniasis and malaria)
• E .g. of passive targeting:- The nano carriers
are largely affected to clearance by the
reticulo-endothelial system (RES) comprising
of macrophages and mononuclear
phagocytes. This fact can be used to passively
target the macrophages and even lymph
nodes and spleen to treat infections that
affect the RES (e.g., leishmaniasis and malaria)
9

ACTIVE TARGETING
• Modifications and functionalization on the drugs or drug carriers
afford them affinity towards specific receptors/markers on cells,
tissues or organs.
• Factors considered in selecting moiety to be attached
• Disease
• Intended target organ
• larger presence of targetable components on the target organ/cell
than in normal cells e.g., transferrin receptors in tumor
• Modifications on the drugs or drug carriers can involve the use of
ligands such as peptides, antibodies, sugars, lectins, etc.
• On administration to the body, the targeting moieties will enable
the drug/drug-carriers to efficiently reach only the intended sites of
action and avoid nonspecific accumulations and related side effects.
10

There are different approaches to actively target drugs to the site of action
like ligands, immune reaction, external stimuli etc
There are different approaches to actively target drugs to the site of action
like ligands, immune reaction, external stimuli etc
11

LIGANDS IN DRUG TARGETING
 MCROMOLECULES
o ANTIBODY
o IMUNOTOXINS
o OLIGONUCLEOTIDES
o CD4
o INTERLUKINS AND INTERFERONS
o TRANSFERRIN
o FOLIC ACID AND FOLATE
o INSULIN
 ENZYMES
 GLYCOPROTEINS AND GLYCOSYLATED PROTEINS
 PEG, POLY-LYSINE AND RELATED POLYMERS
 VESCICLES ESPECIALLY LIPOSOMES
12

13
CONJUGATION
Covalent Non- covalent
Homo or hetro bifunctional
cross linkers are used
Mostly used in conjugation
with liposome's
Most commonly used for
targeting purpose
Less chances for changing
of the properties
Hydrophobic, electronic,
immunospecific, interactions
are utilized

Characterization of ligand drug conjugates
• Changes in shape-SEM,
transmission electron, atomic
force microscopy
• Size- gel exclusion chromatography
• Toxicity
• Antigen binding capacity
• Catalytic activity
• Stability
• In vivo or ex vivo studies- cell line
studies in tumor cells 15
Depends on
type of
ligands used

STIMULI RESPONSIVE
• External stimulus, such as magnetic fields and ultrasound,
employed to perform imaging, to
•
• External stimulus, such as magnetic fields and ultrasound,
acting on nano carriers, are employed to perform imaging, to
target and release drugs from the nano carriers at the
intended site of action.
• It has various advantages like
 Real-time targeting,
 Targeting deep-seated tissues,
 Simultaneous imaging and therapy.
16

MAGNETICALLY MODULATED DRUG DELIVERY
They are of different types
Magnetic nano particles
Magnetic microspheres
Magnetic emulsions
Magnetic resealed erythrocytes
Magnetic implants
17

18
Magnetic fields are believed to be harmless to biological systems. So targeted drug
delivery can be attained by using magnetic carriers which can be guided with help of
magnetic field in the biological system
They provide advantages like
Magnetic fields are believed to be harmless to biological systems. So targeted drug
delivery can be attained by using magnetic carriers which can be guided with help of
magnetic field in the biological system
They provide advantages like
Decreased dose
Controlled drug release
Adaptable to any part of the body
Targeting
Disadvantages
Expensive
Specialized magnets are
required
Magnetite and other materials
used gets deposited in tissues

19
MAGNETIC NANO PARTICLES
 Magnetic nano particles (MNP) as imaging agents for
magnetic resonance imaging, magnetic drug
targeting and hyperthermia treatment is well
explored in the field of targeted drug delivery.
 The MNP can be either metallic or bimetallic or
super paramagnetic iron oxide nano particles
(SPION)
 SPION are widely studied for biomedical applications
because of nontoxic nature, ability to be
functionalized with different targeting coatings and
can encapsulate drugs in reasonable quantity.

Optimizing the MNP as well as the external magnet is of prime importance
because on application of magnetic field , they must be able to generate enough
magnetic moments and magnetic gradient that the MNP can overcome the force of
the blood-flow (rating from 0.05–50 cm/s) depending on the target area.
The MNP have found several uses in thrombolytic therapy intravascular imaging
and cardiovascular diseases, tumor imaging and treatment, as well as delivery across
the blood–brain barrier
20

ULTRASOUND MEDIATED DRUG DELIVERY
Ultrasound has been used previously for contrast imaging, and it is explored at
length for use in drug delivery. Ultrasound mediated targeting can lead to disruption
of the drug-loaded carriers (micro bubbles, micelles, etc.) causing drug release
21

Inverse Targeting :
• In this type of targeting attempts are made
to avoid passive uptake of colloidal carrier
by RES (Reticulo Endothelial Systems) and
hence the process is referred to as inverse
targeting.
• To achieve inverse targeting, RES normal
function is suppressed by pre injecting large
amount of blank colloidal carriers or
macromolecules like dextran sulphate
• This approach leads to saturation of RES
and suppression of defence mechanism. This
type of targeting is a effective approach to
target drug(s) to non-RES organs.
Dual Targeting :
• In this targeting approach
carrier molecule itself
have their own
therapeutic activity and
thus increase the
therapeutic effect of drug.
• For example, a carrier
molecule having its own
antiviral activity can be
loaded with antiviral drug
and the net synergistic
effect of drug conjugate
was observed.
Double Targeting :
• Temporal and spatial methodologies are combined to target a carrier
system, then targeting may be called double targeting.
• Spatial placement relates to targeting drugs to specific organs, tissues,
cells or even subcellular compartment. whereas temporal delivery refers to
controlling the rate of drug delivery to target site
22

BRAIN TARGETING IS UNIQUE DUE TOBRAIN TARGETING IS UNIQUE DUE TO
• Presence of blood brain barrier constituting tight junctions, pericytes,
astrocytes and continuous basal membrane
• Anatomically, the endothelial cells of the BBB are distinguished from
those in the periphery by increased mitochondrial content, a lack of
fenestrations, minimal pinocytotic activity, and the presence of tight
junctions
• endothelium is characterized by exhibiting a high transepithelial
electrical resistance (1500–2000 Ωcm2)
• The BBB makes the brain practically inaccessible for polar molecules and
small ions. (transport proteins for the transport glucose and amino acids)
• BBB is also an enzymatic barrier making drug delivery even more
challenging as drugs are exposed to cytosolic and membrane-associated
enzymes, such as γ-glutamyl transpeptidase, alkaline phosphatase,
aromatic acid decarboxylase, dipeptidyl(amino)peptidase IV, and
aminopeptidase A and N directed at metabolizing neuroactive agents
24

CHEMICAL DELIVERY
PRODRUGS
•
crossing the bbb
• excellent example of a drug that exploits an
endogenous carrier is levodopa, a lipid
insoluble precursor of dopamine, used for the
treatment of Parkinson’s disease.
PRODRUGS
•Produgs gets converted to active drug after
crossing the bbb
•An excellent example of a drug that exploits an
endogenous carrier is levodopa, a lipid-
insoluble precursor of dopamine, used for the
treatment of Parkinson’s disease.
DRUG CONJUGATES
Therapeutic compounds
are able to cross the BBB
after association or
conjugation to specific
ligands
Recepter directed
Insulin receptor
Transferrin receptor
Cationic derivatives
BBB endothelium possesses anionic sites that
attract cationic substances to the membrane
surface
Lipoidal derivatives
27

COLLOIDAL DELIVERY
LIPOSOMES
Liposomes
composed of a
phospholipid
that may act as a
carrier for both
hydrophilic and
hydrophobic drugs.
The biophysical
properties of
liposomes, such as size
and surface chemistry,
may be varied in order
to control distribution,
including distribution
to the brain
LIPOSOMES
Liposomes are
composed of a
phospholipid bilayer
that may act as a
carrier for both
hydrophilic and
hydrophobic drugs.
The biophysical
properties of
liposomes, such as size
and surface chemistry,
may be varied in order
to control distribution,
including distribution
to the brain
POLYMER NANOPARTICLES
Manufactured from a wide range of
materials such as poly(D,L
co lactic acid),
poly(methyl) (PMMA),
poly(alkyl) , as well as
from natural polymers such as
chitosan
and gelatin. These
are able to prolong drug blood
residence time and protect
from enzymatic degradation
POLYMER NANOPARTICLES
Manufactured from a wide range of
materials such as poly(D,L-lactide-
co-glycolic acid), poly(L-lactic acid),
poly(methyl)methacrylate (PMMA),
poly(alkyl)cyanoacrylate, as well as
from natural polymers such as
chitosan, dextran, starch, albumin,
and gelatin. These nanoparticles
are able to prolong drug blood-
residence time and protect drug
from enzymatic degradation
MONOCYTES
They have unusual
ability to cross bbb.
infected brain cells
have augmented
population density of
monocytes
also can be used
MONOCYTES
They have unusual
ability to cross bbb.
infected brain cells
have augmented
population density of
monocytes which
also can be used
29

BIOLOGICBIOLOGIC
PSEUDO NUTRIENTS
They are polar small
molecular structure
mimicking nutrients that
normally undergo carrier
mediated transport
through bbb
e.g. hexoses,
monocarboxylic acid
PSEUDO NUTRIENTS
They are polar small
molecular structure
mimicking nutrients that
normally undergo carrier
mediated transport
through bbb
e.g. hexoses,
monocarboxylic acid
CHIMERIC PEPTIDES
In this aproach drug
delivery to brain is by
anchoring drug to a
peptide or protien
vector
Some of the vectors used
are
Cationized albumin
CHIMERIC PEPTIDES
In this aproach drug
delivery to brain is by
anchoring drug to a
peptide or protien
vector
Some of the vectors used
are
Cationized albumin
ANTIBODY DIRECTED
ENZYME PRODRUG
THERAPY
(1) an activating enzyme
is specifically
delivered to intended
site of action with a
targeting antibody
(2) a subsequent
administration of
substrate prodrug
ANTIBODY DIRECTED
ENZYME PRODRUG
THERAPY
(1) an activating enzyme
is specifically
delivered to intended
site of action with a
targeting antibody
(2) a subsequent
administration of
substrate prodrug
30

GIT:
•Stomach
•Small intestine
•Large intestine
Colon
 Rectum
 Anal canal
Colon consists:
• Cecum
• Ascending
colon
• Transverse
colon
• Descending
colon
• Sigmoid colon
ANATOMY OF COLON
32

Major metabolic processes occurring in the colon are hydrolysis and reduction
Enzymes in Colon
Reducing enzymes Hydrolytic enzymes
 Nitroreductase Esterases
 Azoreductase Amidases
 N-oxide reductase Glycosidases
 Sulphoxide reductase Glucuronidase
 Hydrogenase Sulfatase
 Azoreductases, which reduces azo-bonds selectively and
 Polysaccharidases which degrades the polysaccharides
MICROBIAL FLORA IN COLON
Bacterial count in the colon is much higher around 1011-1012 CFU/ml.
 400 species of microorganisms are present
 Fundamentally anaerobic in nature.
 Predominant species: Bacteroides, Bifidobacterium and Eubacterium.
33

WHY COLON TARGRETING?
• A number of diseases like inflammatory
bowel diseases (IBD) like Crohn’s disease
and ulcerative colitis, colon cancer,
irritable bowel syndrome (IBS),
amoebiasis, etc. and desired transport of
proteins and peptide drugs require the
use of colon targeted drug delivery
systems (CDDS).
• The general routes of reaching the colon
are via the oral delivery or the rectal
delivery. Using the rectal mode of
administration is usually uncomfortable
for the patient and can often result in
irregular dose distribution.
• Using regular oral modes of delivery can
degrade the drugs by acid actions in the
stomach and alkaline and enzyme activity
in the small intestine.
34

FACTORS GOVERNING COLONIC DELIVERY
Gastrointestinal transit
Small intestinal transit
Colonic transit
Gastric emptying
Stomach and intestinal pH
Colonic micro flora and enzymes
35

The most commonly used targeting mechanisms



•
The most commonly used targeting mechanisms
are:-
pH – dependent delivery
Time - dependent delivery(Pulsincap device)
Pressure- dependent delivery
• Bacteria—dependent delivery(Sulfasalazine-In
the colon, it degrades into 5-ASA
APROACHES IN COLON TARGETING
36

Evaluation
In vitro models
In vitro test for intactness of coating and
carriers in simulated conditions of
stomach and intestine
step1
Drug release study in 0.1N HCL for 2
hours (mean gastric emptying)
step 2
Drug release study in phosphate buffer
for 3 hours (mean small intestine transit
time)
In vitro enzymatic degradation test
Method 1:
Drug release in buffer medium containing
enzymes(e.g.pectinase, dextranase) or
rat or guinea pig or rabbit fecal
contents
Amount of drug release in particular time
directly proportional to the rate of
degradation of polymer carrier.
Method 2:
Incubating carrier drug system in
fermenter
Suitable medium containing colonic
bacteria (streptococcus faecium or
B.ovatus)
Amount of drug released at different time
intervels determined
Clinical evaluation is also performed
38

TUMOR CELL MICROENVIRONMENT
 Tumor is uncontrolled growth of abnormal cells characterized by mutations
proteins

 Tumor is uncontrolled growth of abnormal cells characterized by mutations
which help the cells to proliferate, avoid apoptosis and develop survival proteins
 compared with normal tissue tumor tissues has got significant differences
 vascular abnormalities and angiogenesis
 oxygenation,
 perfusion,
 pH and metabolic states
40

ANGOGENISIS
Angiogenesis is defined as the formation
of new blood vessels from existing ones.
In the angiogenesis process, five phases
can be distinguished:
1. endothelial cell
activation, 2. basement membrane
degradation, 3. endothelial cell
migration, 4. vessel formation, and 5.
angiogenic remodeling.
VASCULAR ABNORMALITIES
The abnormal vascular architecture plays
a major role in drug targeting at tissue
(1) Extensive angiogenesis and hyper
vasculature
(2) Lack of smooth-muscle layer,
pericytes
(3) Defective vascular architecture:
fenestrations
(4) No constant blood flow and direction
(5) Inefficient lymphatic drainage that
leads to enhanced retention in the
interstitium of tumors
(6) Slow venous return that leads to
accumulation from the interstitium of
tumor
pH
Tumors exhibit a lower extracellular pH
than normal tissues. The low extracellular
tumor pH mostly arises from the high
glycolysis rate in hypoxic cancer cells
The relatively basic intracellular
compartment may in turn favor the
ionization of the molecule, thereby
promoting the cytosolic accumulation of
the drug
TARGRTING
41

Many different targeted therapies have been approved for use in cancer treatment
These are:
(a) structural
modifications for improving the physicochemical properties in accordance with
structure–activity relationships (SAR)
(b) conjugating homing ligands for predetermined bio-distribution patterns and
(c) involvement of carrier based approaches
PASSIVE METHODS
Enhanced permeation and
retention effects
 Phagocytosis of particulate
carrier by mononuclear
phagocytosis systems (MPS)
Localization in the organs of
reticuloendothelial System (RES)
Low extracellular ph
Relative micro-acidosis
Mild hyperthermia
ACTIVE METHODS






antibodies
ACTIVE METHODS
Vitamin based
Peptide based
Lectin based
Transferrin based
Albumin based
Monoclonal
antibodies
43

Monoclonal antibodies
• A great amount of work has been done with antibodies to target
•
•
•
•
• A great amount of work has been done with antibodies to target
drugs to specific cells, especially for cancer therapy. Theoretically,
targeting with antibodies is ideal because antibody-antigen
interactions are very specific
• Monoclonal antibodies are monospecific antibodies that are made
by identical immune cells that are all clones of a unique parent cell
• Monoclonal antibodies have been produced which react with a
wide range of human cancers, including carcinomas of the colon,
rectum, breast, ovary, lung, pancreas, and bladder, malignant
melanomas, bone and soft tissue sarcomas, and leukemia's.
• Saccharides of the cell surface are important tumor associated and
differentiation antigens
• Monoclonals are useful in the detection of glycosylation changes
and to study the biochemistry of these changes
44

45
Transferrin receptor- Transferrin, a serum
glycoprotein, transports iron through the
blood and into cells by binding to the
transferring receptor and subsequently
being internalized via receptor-mediated
endocytosis. The transferrin receptor is a
vital protein involved in iron homeostasis
and the regulation of cell growth. The high
levels of expression of transferrin receptor
in cancer cells, which may be up to 100-fold
higher than the average expression of
normal cells
Folate receptor-binds to the vitamin folic
acid and folate–drug conjugates or folate
grafted nanocarriers with a high affinity
and carries these bound molecules into
the cells via receptor-mediated
endocytosis. Folic acid is required in one
carbon metabolic reactions and
consequently, is essential for the synthesis
of nucleotide bases. The alpha isoform,
folate receptor-α is overexpressed on 40%
of human cancers
Lectins- proteins of non-
immunological origin
which are able to
recognize and bind to
carbohydrate moieties
attached to glycoprotein's
expressed on cell surface.
Cancer cells often express
different glycoproteins
compared to normal cells.
Peptides employed for tumor targeting could
be either monomeric, homodimeric,
heterodimeric oligomeric or tetrameric in
nature. The tumor specific peptides could be
broadly categorized into two categories, one
targeting tumor cell surface while other
targeting tumor vasculature

Liprosomes loading paclitaxel for brain-targeting delivery by intravenous
administration: In vitro characterization and in vivo evaluation
Bo Tang, Guihua Fang, Ying Gao, Yi Liu, Gang Cheng , School of Pharmacy,
Shenyang Pharmaceutical University, China
Objective:- In this study, a lipid–protein nanocomplex (liprosome) was evaluated for its
potential use for brain-targeting drug delivery. Liprosome was fabricated with the
desolvation–ultrasonication method and characterized in terms of particle size, size
distribution, zeta potential, morphology, crystal state of the drug, and in vitro release. The
in vivo distribution of paclitaxel loading lipid–protein nanocomplex (PTX-liprosome) and
Taxol were compared after i.v. administration in mice
Preparation of the PTX-liprosome
Liprosomes are nanocomplexes formed by combination of protien(bovine serum albumin)
and lipid(egg yolk lecithin).A desolvation–ultrasonication technique was used to prepare
the PTX-liprosome. Anhydrous ethanol was used as a solvent for PTX and PL 100 M. Briefly,
BSA (0.20 g) was dissolved in 5 mL of deionized water with magnetic stirring. PTX (0.01 g)
and PL 100 M (0.10 g) were dissolved in 0.3 mL of anhydrous ethanol, respectively. Then,
the PTX ethanol solution was added dropwise to the BSA solution with magnetic stirring,
and the PL 100 M ethanol solution was also added dropwise to the PTX-BSA solution with
magnetic stiring. The resultant mixture was further ultrasonicated at 300 W for 3 min using
a probe sonicator in an ice bath with a 3 s pulse-on period and a 3 s pulse-off period. After
sonication, anhydrous ethanol was evaporated under vacuum using a rotator RE-2000
49

Rational behind the study
• Therapeutic efficacy of PTX against brain cancer has been disappointing
• From literature it was found that Polymeric nano particles are an effective
• Compared to synthetic polymeric nano carriers, natural nano carriers have
degradability and biocompatibility. Of these, proteins have also been used
extensively as carriers for drug delivery particularly human serum albumin
•
• In addition, albumin nano particles preferentially accumulate at malignant
•
• Therapeutic efficacy of PTX against brain cancer has been disappointing
because of drug-resistance and poor penetration across the BBB
• From literature it was found that Polymeric nano particles are an effective
carrier system for drug transport across the BBB
• Compared to synthetic polymeric nano carriers, natural nano carriers have
attracted much interest because of their decreased cytotoxicity, higher
degradability and biocompatibility. Of these, proteins have also been used
extensively as carriers for drug delivery particularly human serum albumin
(HSA)
• HSA contains regions for binding many organic and inorganic molecules,
and these domains are characterized by deep hydrophobic pockets with
a negative charge.
• In addition, albumin nano particles preferentially accumulate at malignant
and inflamed tissues because of passive targeting due to the enhanced
permeability and retention effect and the active targeting mediated by
gp60 receptor-mediated transcytosis
• On the basis of these observations PTX was prepared as liprosomes
50

Sl
no
Characterization
of liprosomes
Method of analysis
1 Size distribution
and zeta
potential
The size distribution and polydispersity index (P.I.) of liprosome
were measured by dynamic light scattering using a NicompTM
380 submicron particle sizer (Particle Sizing System, Santa
Barbara, CA, USA). The zeta potential of the liprosomes was
measured by electrophoretic light scattering using the same
device.
2 Particle
morphology
The morphology of liprosome was assessed using TEM (JEOL,
Tokyo, Japan) at 90 kV.
3 X-ray
photoelectron
spectroscopy
(XPS)
To identify the surface properties of the PTX-liprosome, X-ray
photoelectron spectroscopy (XPS) was conducted on an ESCALAB
250
4 Differential
scanning
calorimetry (DSC)
The thermal properties of the PTX-liprosome were determined
with a differential scanning calorimeter (Shimadzu, Kyoto, Japan)
51

5 X-ray powder diffraction (XRPD) The physical state of PTX in the PTX-
liprosome was further determined
with an X-ray diffractometer
(Geigerflex, Rigaku Co., Japan).
6 Fourier transform infrared spectroscopy
(FTIR)
The intermolecular interactions of
PTX, BSA, and PL 100 M were
investigated with a FTIR system
(Bruker IFS-55, Bruker, Switzerland)
using the KBr disk method.
Sl
no
Charaterisation METHOD
1 Determination
of PTX
concentration
The PTX concentrations were determined using a modified HPLC
method on a HPLC system (LC-10Avp SHIMAZU, Japan), which
was equipped with a UV–vis detector (SPD-10Avp SHIMAZU,
Japan) and a Diamonsil C18 reversed phase column
2 Entrapment
efficiency (EE)
and loading
capacity (LC)
To release the PTX from PTX-liprosome, acetonitrile was added
to precipitate BSA by bath sonication for 5 min. After
centrifugation at 12,000 rpm for 10 min, 20 mL of the
supernatant was injected into an HPLC system to determine the
drug content in the liprosome
52

3 In vitro drug release The release behavior of PTX from the PTX-liprosome
suspen-sions was studied in vitro using a dialysis method
with phosphate buffered saline (PBS, 0.01 M, pH 7.4)
containing 0.5% w/v Tween 80
4 In vitro hemolysis
assay
To evaluate the hemolytic potential of the PTX-liprosome,
an in vitro hemolysis test was performed. All of the
experiments were performed using the venous blood from
the ear vein of a healthy rabbit
IN VIVO ANALYSIS
EXPERIMENTAL PROCESS:- Sixty-four mice were divided randomly into two groups
receiving Taxol and PTX-liprosome suspensions via lateral tail vein injections at 20
mg PTX/kg body weight. At 0.083, 0.25, 0.5, 1, 2, 4, 6, and 8 h after administration,
four mice from each group were anesthetized with diethyl ether, 0.5 mL of blood
was collected into a heparinized centrifuge tube from the retrobulbar venous plexus
of the mice. The animals were then sacrificed, and the heart, liver, spleen, lung,
kidney, and brain were removed. The blood was centrifuged (4000 rpm, 10 min) to
obtain the plasma samples. The tissue samples were washed with physiological
saline and dried on filter paper. The plasma and tissue samples were stored at 20 C
until analysis by hplc technique
53

RESULTS AND DISCUSSION
Preparation and
physical properties
The amount of BSA strongly affected the EE of the PTX-
liprosome. Significant increase in the EE at the BSA of 0.20 g
was observed whereas a concomitant decrease in the EE at the
BSA of 0.30 g was noted.
increased particle size, whereas proportionate decrease in the
EE was observed with increase in the drug amount
The optimal PTX-liprosome has a high entrapment efficiency
(>90%), small particle size (approximately 113 nm), and narrow
distribution
Transmission
electron microscopy
(TEM)
liprosome displays a typical multilayer structure with a
spherical shape.
X-ray photoelectron
spectroscopy (XPS)
These results suggest that the PL 100 M formed the majority of
the surface layers of the PTX-liprosome and that the PTX and
BSA conjugate was inside the PTX-liprosome.
54

FTIR
analy
sis
FTIR was used to evaluate the intermolecular interactions between PTX, BSA, PL
100 M. The spectra of PTX, BSA, PL 100 M, blank liprosome, and PTX-liprosome.
Hydrogen bonds are formed between amide groups in BSA and hydroxyl groups in
PTX.
 hydrogen bonds are also formed between carbonyl groups in the PTX and
hydroxyl groups of the amino acids in BSA
existence of electrostatic interactions between the CN+(CH3)3 group of PL 100 M
and surface COO group of BSA
DSC DSC analysis was conducted for PTX, BSA, PL 100 M, blank liprosome, and PTX-
liprosome.
DSC thermogram of PTX endothermic peak at 223.8 C and one exothermic peak at
244.3 C, which were attributed to the melting and decomposition of PTX,
respectively. Compared to the PTX, the endothermic peak of PTX in the PTX-
liprosome disappeared, indicating a significant conversion from the crystalline state
to an amorphous or molecular state in the PTX-liprosome during preparation.
 The DSC thermogram of BSA showed a denaturation peak at 97.5 C and a melting
temperature at approximately 220 C (Kang et al., 2009). However, the denaturation
peak is significantly shifted to a lower temperature in the blank liprosome (85.8 C)
and PTX-liprosome (87.8 C), indicating interaction forces among PTX, BSA, and PL
100 M.
56

XRPD XRPD was used to evaluate the dispersion of the drug within the
liprosome. XRPD analysis was conducted for PTX, BSA, PL 100 M, blank
liprosome and PTX-liprosome.
 Diffractograms revealed the crystalline nature of the PTX. Sharp
diffraction peaks of the PTX disappeared in the diffractograms of the
PTX-liprosome indicating molecules existed inside the PTX-liprosome in
an amorphous or molecular state.
Partial weak crystalline peaks were displayed between 5 and 10 in
the diffractogram of the PL 100 M which is characteristic of some
crystallinity. These peaks disappeared in the diffractograms of the
blank liprosome and PTX-liprosome, suggesting an interaction
between BSA and PL 100 M, such as electrostatic attraction,
hydrophobic interactions and hydrogen bonds. These interaction
forces are involved in the formation of liprosome and caused the
partial amorphization of the lipid in the liprosome
In vitro
hemolysis
assay
Hemolytic activity of the PTX- liprosome was negligible (<5%), in the
range of 20–240 mg/m
58

IN VITRO DRUG RELEASE
PTX- liprosome exhibits biphasic
release behavior. After the initial
burst release for approximately 6
h, the release rate of the PTX
gradually decreased to nearly
zero-order release. After 36 h, the
cumulative PTX release was
approximately 75%. This special
release pattern is likely due to the
unique multilayer structure. The
burst release of the PTX may be
because of the diffusion of the PTX
encapsulated in the outer layers,
which is likely to be released at a
faster rate, whereas the drug
encapsulated in the inner layer
would requires a longer time for
release. To predict the release
kinetics, several drug release
models, including the zero order,
first order, Higuchi and Weibull
distribution models were adopted 59

Tissue distribution
The tissue targeting efficiency was evaluated using the drug targeting index (DTI)
described and the relative uptake rate (R). The values of R and DTI are defined as follows
60

The
liprosome was determined after
administration. The relative uptake
rate (R) and drug targeting index
(DTI) were used to evaluate the
target efficiency of the PTX
liprosome.
tissues in descending order was brain
> liver > plasma > spleen > kidney >
heart > lung. The DTI of the tissues in
a descending order was brain > liver
> spleen > kidney > heart > lung.
There is a similar trend for the values
of R and DTI. The PTX
clearly targets the brain (
1.329, = 2.190), significantly
increasing drug uptake by the brain
tissue.
The targeting efficiency of the PTX-
liprosome was determined after
administration. The relative uptake
rate (R) and drug targeting index
(DTI) were used to evaluate the
target efficiency of the PTX-
liprosome. The R values for the
tissues in descending order was brain
> liver > plasma > spleen > kidney >
heart > lung. The DTI of the tissues in
a descending order was brain > liver
> spleen > kidney > heart > lung.
There is a similar trend for the values
of R and DTI. The PTX-liprosome
clearly targets the brain (Rbrain =
1.329, DTIbrain = 2.190), significantly
increasing drug uptake by the brain
tissue.
62

CONCLUSION
• Lipid–protein nano complex (liprosome) with a unique
•
•
•
• Lipid–protein nano complex (liprosome) with a unique
spherical multilayer structure was fabricated using a
desolvation–ultra sonication method.
• The liprosome achieved excellent drug entrapment, a
satisfactory particle size and a narrow size distribution. In
vitro hemolysis assays demonstrated that the liprosome
exhibited low cytotoxicity.
• Compared to taxol, the prepared ptx-liprosome is an
effective formulation for increasing drug distribution to
brain tissue while reducing it in other organs.
• Therefore, liprosome is a good drug delivery system for
transporting drugs to the brain and for reducing toxic and
side effects in other organs.
63

Reference
• Targeted Delivery of Small and Macromolecular Drugs by Ajit S. Narang, Ram I.
Mahato
• Targeted Drug Delivery: Concepts and Design by Padma V. Devarajan , Sanyog Jain
• Targeted and controlled drug delivery by S.P Vyas, R.K. Khar
• Research papers
64

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