Transcript General Method - iSpatula Pharmacy

PART 1
Selected Topics In Physical
Pharmacy
A. Physico-Chemical Characterization of
Some Surfactant Containing Systems
1. Polymeric Micelles
• PM are under investigation mainly as
carriers for anti-tumor drugs, contrast
agents for diagnostic imaging, enzymes, in
gene delivery, …
• Polymeric micelles, PM, belong to the
class of “nanomedicine”,
• In drug delivery, PM are classified under
the “nanocarriers”,
• Table: Summary of different
nanotherapeutic technologies proposed for
cancer therapy
• General Structure of the PM
Schematic illustration of the core-shell
architecture of a polymer micelle and its
dimensions
General Structure of the PM
Ref.: Pharmacology & Therapeutics 112 (2006) 630–648
Advantages of PM as nanodrug carriers
• Drug protection
Drugs incorporated in the core of the PM are
protected from harsh biological environments
(e.g. low pH and hydrolytic enzymes),
• Drug Solubilization
Hdrophobic drugs can be imbibed into the
hydrophobic inner core of the PM, significantly
improving their water solubility.
• Stability (low CMC & long circulation period)
PM have low CMC or CAC
“The lower is the CMC value of a given amphiphilic
polymer, the more stable are micelles even at low
concentration of an amphiphile in the medium. This is
especially important from the pharmacological point of
view, since upon the dilution with the large volume of the
blood only micelles with low CMC value still exist, while
micelles with high CMC value dissociate into unimers
and their content precipitates in the blood.”
V.P. Torchilin / Advanced Drug Delivery Reviews 54 (2002) 235 –252
• The critical association concentration,
CAC defines a threshold concentration for
PM assembly.
• Polymeric micelles may not necessarily
dissociate immediately after extreme
dilution following intravenous injection into
the body because they have a remarkably
low CAC, (10–6 – 10–7 M), which is 1000folds lower than that of surfactant micelles.
Stability- cont.
PM dissociation is kinetically slow. This property allows the micelles to
circulate in the bloodstream until accumulation at target tissues.
For example: some PEG-b-PDLLA PM micelles showed a remarkably
prolonged blood circulation (t1/2 about 18 hr) after intravenous
administration, and maintained 25% of the injected dose in the
circulation at 24-hr post-injection.
Also, the presence of hydrophilic polymers on the surfaces of PM is
known to hinder protein adsorption and opsonization of the particles
by the reticuloendothelial system (RES) which is responsible for
engulfing and clearing old cells, miscellaneous cellular debris,
foreign substances, and pathogens from the bloodstream.
• Safety of the PM
The constituent block copolymers might be
finally excreted into the urine due to their
molecular weight being lower than the
threshold of glomerular filtration,
suggesting the safety of polymeric
micelles with a low risk of chronic
accumulation in the body.
•
Targetability
In cancer therapy and diagnostic imaging,
PM have special importance, why ?
PM physico-chemical properties.
PM (and other nanocarriers) size allows for
passive tumor targeting (EPR).
“Tumor blood vessels are generally characterized
by abnormalities accompanied by decreases
lymphatic drainage and renders the vessels
permeable to macromolecules. Because of the
decreased lymphatic drainage, the permeant
macromolecules are not removed efficiently, and
are thus retained in the tumor. This passive
targeting phenomenon, has been called the
‘‘enhanced permeation and retention (EPR)
effect’’. EPR effect results in passive
accumulation of macromolecules and nanosized
particulates (e.g. polymer conjugates, polymeric
micelles, dendrimers, and liposomes) in solid
tumor tissues, increasing the therapeutic index
while decreasing side effects.”
J.H. Park et al. / Prog. Polym. Sci. 33 (2008) 113–137
• The PM structure allows for active
targeting.
Specific ligands such as antibodies can be
attached to the hydrophilic block polymer.
• Figure: Multicomponent targeting strategies.
Nanoparticles extravasate into the tumour stroma
through the fenestrations of the angiogenic vasculature,
demonstrating targeting by enhanced permeation and
retention.
• The particles carry multiple antibodies, which further
target them to epitopes on cancer cells, and direct
antitumour action.
• Nanoparticles are activated and release their cytotoxic
action when irradiated by external energy.
The red blood cells are not shown to scale; the volume
occupied by a red blood cell would suffice to host 1–10
million nanoparticles of 10 nm diameter.
NATURE REVIEWS | CANCER VOLUME 5, 2005, 161-171
PART 2
Polymeric Micelles
• PM are formed from the association of block
copolymers.
• Block copolymers micelles have the capacity to
increase the solubility of hydrophobic molecules
due to their unique structural composition, which
is characterized by a hydrophobic core sterically
stabilized by a hydrophilic shell.
The hydrophobic core serves as a reservoir in
which the drug molecules can be incorporated
by means of chemical, physical or electrostatic
interactions, depending on their physicochemical
properties.
Block vs. Random
Diblock vs Multiblock
natural (dextran) and synthetic (polystyrene) polymeric
micelle.
Daniel Taton and co-workers at the University of Bordeaux, France, 2007
According to the type of intermolecular
forces driving the micelle formation, PM
are classified as:
• amphiphilic micelles; formed by non polar
hydrophobic interactions,
• polyion complex micelles, PICM; resulting
from electrostatic interactions,
• micelles formed by metal complexation.
Non polar hydrophobic interactions
Micellization is driven by a gain in entropy of the solvent
molecules as the hydrophobic components withdraw from
the aqueous media.
Examples:
Polymers contain
• polyester as Poly(lactic acid) (PLA),…
• poly(amino acid) PAA derivative as Poly(aspartic acid)
(PAsp),… (neutral or conjugated to hydrophobic group).
,…....
• Polyethers as the poloxamer family { (poly(ethylene
glycol)-b-poly(propylene oxide)-bpoly( ethylene glycol))
(PEG-b-PPO-b-PEG)
All are biocompatible and biodegradable approved by the
FDA for biomedical applications in humans.
Electrostatic interactions
The self-assembly of PICM, proceeds
through the electrostatic interaction between
polycations and polyanions leading to
neutralization of oppositely charged
polyions.
The presence of the polymer hydrophilic
segment prevents precipitation.
Examples:
• Polymers having protonated amines at physiological pH
like poly(ethyleneimine) (PEI),…..
Micelle Preparation
Among the methods used in PM preparation are
1. Direct dissolution method
Suitable for moderately hydrophobic copolymers, such as
poloxamers, and for PICM.
dissolving the block copolymer along with the drug in an
aqueous solvent.
The copolymer and drug are dissolved separately in an
injectable aqueous vehicle.
Micelle formation is induced by combining the two solutions
to appropriate drug–polymer /charge ratios.
2. Indirect method using organic solvent
Applies to amphiphilic copolymers which are not
readily soluble in water.
Organic solvent common to both the copolymer
and the drug
is used. (such as dimethylsulfoxide, acetone,….
The mechanism by which micelle formation is
induced depends on the solvent-removal
procedure.
a. Dialysis method: the copolymer mixture can be
dialyzed against water. Slow removal of the
organic phase triggers micellization.
b. Solution-casting method: evaporation of the
organic phase to yield a polymeric film where
polymer–drug interactions are favored.
Rehydration of the film with a heated aqueous
solvent produces drug-loaded micelles.
c. Emulsion (O/W) method using non-watermiscible organic solvent like dichloromethane.
d. Freeze drying method:
one-step procedure based on the dissolution
of both the polymer and the drug in a
water/tert-butanol (TBA) mixture with
subsequent lyophilization of the solvents.
Drug-loaded micelles are formed
spontaneously upon reconstitution of the
freeze-dried polymer–drug cake in an
injectable vehicle.
Common drug-loading procedures: (A) simple
equilibrium, (B) dialysis, (C) O/W emulsion, (D) solution
casting, and (E) freeze-drying.
Journal of Controlled Release 109 (2005) 169–188
Ref.: © 2004 IUPAC, Pure and Applied Chemistry 76, 1321–1335
PART 3
PM Physico-Chemical
Characterization
Determination of critical micelle
concentration (CMC)
Pyrene fluorescence for the determination of
CMC and studying the interior of the PM
One of the mostly used methods in PM characterization.
For a pyrene molecule P;
P > (excitation)> P*
P* + P > P (excimer)
Pe/Pm: measure of the ease of excimer (e)
formation from the monomer (m)
Excimer formation is function of microviscosity of
the micelle core,
Excimer formation is sensitive to pyrene
concentration because it involves an interaction
between two pyrene species.
The emission spectrum of the pyrene monomer in
the 350- to 420-nm region consists of five
primary vibronic bands, usually designated as
I1–I5, from shorter to longer wavelengths.
band 1: shows significant intensity enhancements
in polar environments;
band 3: shows minimal variation in intensity with
polarity changes.
General Method
Dissolve Pyrene in organic solvent,
Add to concentration series of the polymer
solution*,
Evaporate the organic solvent,
Measure fluorescent spectra using fluorescence
spectrophotometer.
* the final concentration of pyrene is in the 10−7 M range.
CMC is determined using the ratio of peak
intensities at 338 and 333 nm (I338/I333) from
pyrene’s excitation spectra.
A number of I338/I333 values is been obtained by
varying the polymer concentration.
When the polymer concentration is low, the I338/I333
value is the same as that of pyrene in water.
When the polymer concentration increases, the
red shift from 333 to 338 nm in the pyrene
excitation spectra indicates the movement of
pyrene into a more hydrophobic environment.
Figure: Plot of the intensity ratio I338/I333 and I1/I3, which were
obtained from the excitation and emission spectra, respectively, as a
function of log C. The cmc was taken from the intersection of the
horizontal line at low polymer concentrations with the tangent of the
curve at high polymer concentrations.
Measurement of lower critical solution
temperature (LCST) or the cloud point
Turbidity method using UV–vis
Spectrophotometer by monitoring the
transmittance at 500 nm at preset heating
rate.
LCST, why?
Hydrophilic polymer + water
 hydrogen bond (exothermic),
Hydrophobic polymer,
 Surrounded by water clusters (low entropy),
At higher temperatures
 Release of water molecules (increase in S),
 Hyrophobic interaction (increase in S)
 polymer precipitation.
Figure - Schematic representation of hydrophobic interaction
Ref.: Physical Pharmacy Book
J.X. Zhang et al. / Colloids and Surfaces B: Biointerfaces 43 (2005)
123–130
Particle size distribution of PM in aqueous solution of 0.5%
at: (a) 25 ◦C; and (b) 45 ◦C. LCST=32.6 C.
Self-assembly and thermally-induced change of a copolymer in
aqueous solution.
Note: the students get confused from the above figure, we should
distinguish between increase in concentration and increase in
temperature.
Diameter changes of a PM as a function of temperature
Micelle size: can be determined using light
scattering methods,
Micelle morphology: can be observed using
transmission electron microscopy (TEM),
……..
Micellar drug solubilization
Surfactants and amphiphilic block copolymers can
greatly affect the aqueous solubility of compounds
by providing a hydrophobic reservoir where they
can partition.
Water solubility of some hydrophobic drugs was
enhanced by a factor of 300 when incorporated
into the core of some PM.
The partitioning of some chemotherapeutic agents
into the hydrophobic PM phase was highly favored
with partition coefficients as high as 5.0x104.
G. Gaucher et al. / Journal of Controlled Release 109 (2005) 169–188
Measurement of drug solubilized in the PM:
By dissolving the lyophilized PM in
organic solvent like DMSO.
Drug concentration is then measured using
suitable analytical method.
Micelle stability
Involves:
Storage stability;
Dilution stability;
In vivo stability: Adsorption of Protein at PM
surface >>PM clearance from the blood.
OR
PM-protein binding>>disrupt micelle
cohesion>> premature drug release.
Storage stability
lyophilized drug-loaded PM are stored at specific
temperature and humidity conditions and the
samples are monitored for time-dependent changes in
particle size and
drug content
during the storage period.
Dilution stability
The effect of dilution on the micelles can be studied by
incubating the micelles in buffer solution at say 10-fold
dilution at the required temperature for certain period.
After filtration, the incubation solution is then analyzed
for the presence of the drug.
In vitro Drug release from the PM
In vitro release profiles of the loaded drug from
the PM is examined at specific conditions
(vehicle, temperature, pH, ) using dialysis
membrane of suitable MWCO.
At predetermined time intervals, the amount of
released drug is determined using suitable
analytical method.
PART 4
Evaluation of Polymeric Micelles from Brush
Polymer with Poly(ε-caprolactone)-bPoly(ethylene glycol) Side Chains as Drug
Carrier
Biomacromolecules 2009, 10, 2169–2174
Introduction
Polymeric micelles self-assembled from amphiphilic copolymers have
attracted significant attention in the fields of biomedical applications.
They have been developed as delivery systems of drug as well as
contrast agents in diagnostic imaging applications.
In an aqueous environment, the hydrophobic component of the copolymer
is expected to segregate into the core of the micelles, while the
hydrophilic component forms the corona or outer shell. The
hydrophobic micelle core serves as a microenvironment for
incorporation of various therapeutic compounds, while the corona can
act as a stabilizing interface between the hydrophobic core and the
external medium.
Although various micelles have been developed for in vitro and in vivo
studies and applications, the majority of works have been focused on
micelles formed by intermolecular aggregation of amphiphilic linear
block copolymers. Micelles from polymers with other architectures as
drug delivery vectors are less studied, especially brush-like
macromolecules.
However, it has been reported that the architecture of polymer may
determine the static and dynamic stability, morphology, size and size
distribution of the micelles, and further affect the performance of
micelles including drug loading and release rate, even in vivo
circulation and distribution.
Cylindrical brush polymers have recently attracted
considerable attention from polymeric chemists.
In this work, we synthesized cylindrical brush polymers
PHEMA-g-(PCL-b-PEG) with poly(2-hydroxyethyl
methacrylate) (PHEMA) as the backbone and poly(εcaprolactone)-bpoly( ethylene glycol) (PCL-b-PEG)
amphiphilic block copolymers as the side chains.
The micelle formation of these polymers was studied, and
doxorubicin (DOX) was used to evaluate the drug loading
and release behavior from the micelles.
The internalization and cytotoxicity of drug-loaded micelles
against A549 human lung carcinoma cells were also
investigated.
Experiments and Results
Experimental
Materials
Different polymers and reagents.
Methods
Preparation of Micelles
Micelles were prepared by a dialysis method.
Briefly, PHEMA-g-(PCL-b-PEG) or PCL-b-PEG (10 mg) was
dissolved in 2 mL of dimethyl sulfoxide (DMSO) and stirred for 2
h at room temperature.
Then, the polymer solution was added dropwise into 5 mL of
ultrapurified water under vigorous stirring. Two hours later, the
solution was transferred into dialysis membrane tubing and
dialyzed for 24 h against water to remove the organic solvent.
Transmission Electron Microscopy (TEM) TEM
was performed on a transmission electron microscope for the
measurement of micelle morphology and dimensions.
Result
The formation of micellar nanoparticles was
confirmed by TEM observations. Formed
micelles are well-dispersed and display
spherical morphology. They are relatively
uniform in size and the average diameters
are approximately 45 nm.
Fluorescence Measurements.
The fluorescent probe method was employed to
determine the critical micellization concentration
(CMC) of the micelles as followed.
A predetermined amount of pyrene solution in acetone
was added into a series of volumetric flasks, and the
acetone was then evaporated completely.
A series of copolymer solutions at different
concentrations ranging from 1.0 × 10-5 to 1.0 mg mL1 were added to the flasks, while the concentration of
pyrene in each flask was fixed at a constant value
(6.0 × 10-7 mol L-1).
The excitation spectra were recorded at 25 °C at λem
of 390 nm.
Result
With increasing the polymer
concentration, several changes in
the fluorescence spectra of pyrene
were observed. The intensity ratio
of bands at 338 and 335 nm (I338/
I335) was calculated and plotted
against the polymer concentrations,
giving a sigmoid curve as shown in
Figure 3. I338/I335 remained
nearly unchanged at low polymer
concentrations and increased
dramatically when the polymer
concentration reached a certain
value, exhibiting the characteristic
of pyrene entirely in a hydrophobic
environment, which is actually a
reflection of micelle formation.
Preparation of DOX-Loaded Micelles
DOX-loaded micelles were prepared similarly to
the blank micelles. Briefly, 10 mg of each
polymer was dissolved in 2 mL of DMSO,
followed by adding a predetermined amount
of DOX· HCl and two molar equivalents of
triethylamine (TEA) and stirred at room
temperature for 2 h. Then, the mixed solution
was added dropwise to 5 mL of water. After
being stirred for an additional 2 h, the solution
was dialyzed against water for 24 h. The
solution was further centrifuged at 3000 g for
3 min to remove free DOX.
Determination of DOX Loading Content (DLC) and Loading
Efficiency (DLE)
To measure the amount of DOX trapped in the micelles, an aliquot of
the DOX-loaded micelle solution was lyophilized and dissolved in
DMSO. The concentration of DOX was measured by HPLC
analyses as described below. The percentages of DLC and DLE
were calculated according to the following equations:
Results
Size and size distributions of DOX-loaded
micelles by DLS measurements revealed that
encapsulation of
DOX into micelles increased the diameters of micelles,
as
summarized in Table 3.
In vitro Drug Release
DOX-loaded micelle solution was diluted to 1
mg mL-1 in phosphate buffered saline, and
transferred into a dialysis membrane tubing.
The tubing was immersed in 20 mL of PBS and
shaken at 37 °C. At a predetermined time
interval, the external buffer of the tubing was
collected, and it was replaced by fresh PBS.
The concentration of DOX was determined by
HPLC analyses with a fluorescence detector.
Results
DOX release kinetics was indeed
affected by polymer architectures.
Cell Culture
A549 human lung carcinoma cells (ATCCs) were cultured in
suitable conditions.
Confocal Laser Scanning Microscopy (CLSM)
A549 cells were incubated at 37 °C. Twenty-four hours later, free
DOX (6 µM) and DOX-loaded micelles (final DOX concentration at
6
µM) were added to the cells, and the cells were incubated for 2 h.
The
cells were then observed with a Zeiss LSM510 Laser Confocal
Scanning Microscope imaging system.
Results
In the Figure, cells incubated with free DOX showed strong
fluorescence in cell nuclei, while very weak fluorescence was
observed in cytoplasm, indicating DOX molecules entered the
cells and rapidly accumulated in the nuclei.
On the contrary, after 2 h incubation of DOX-loaded brush
copolymer micelles, intense DOX fluorescence was observed in
the cytoplasm rather than in cell nuclei. It implies that DOX
loaded brush copolymer micelles can be effectively internalized
by A549 cells.
Cytotoxicity Study
Cytotoxicity of DOX-loaded micelles and
free
DOX were measured against A549 cells
where the relative cell viability was
calculated.
Results
As shown in Figure 6, DOX-loaded brush polymer
micelles exhibited relatively lower cytotoxicity to A549
cells when compared with free DOX at the same
dose.
Conclusions
The brush polymers self-assembled into spherical micelles with the
diameter less than 100 nm in aqueous solution.
The brush polymer micelles showed enhanced aqueous stability than
linear PCL-b-PEG diblock copolymer with similar structure to the
side chains as determined by fluorescent probe method. When
used as drug delivery carriers, the brush polymer micelles
exhibited higher DOX loading capacity than the micelles from
linear PCLb- PEG, and the burst drug release in the initial period
was significantly suppressed.
The micelles can be effectively internalized by A549 cells and slowly
released the encapsulated drug molecules.
They showed less potent but effective cell proliferation inhibition
compared to free DOX.
With these advantages, the brush polymer micelles are potential
carriers for efficient drug delivery.