Summary of techniques used by Group (4) Paper

"Engineering theranostic nanovehicles capable of targeting cerebrovascular

amyloid deposits"
Carbodiimide crossliner Chemistry
Crosslinking is the process of chemically joining two or more molecules by a covalent
bond. The technique, often called bioconjugation when referring to its use with proteins
and other biomolecules, is an essential component of many proteomics methods,
including creation of detectable probes for Western blotting and E!"# and strategies for
investigating protein structure and interactions.
Carbo$yl%reactive chemical groups in biomolecular probes for labeling and crosslinking
carbo$ylic acids to primary amines include the carbodiimide compounds E&C and &CC.
'ery few types of chemical groups are known to provide specific and practical conjugation
to carbo$ylic acids ()C**+,, such as occur in proteins and many other biomolecules.
Carbonyldiimida-ole (C&!, can be used in non%aqueous conditions to activate carbo$ylic
acids for direct conjugation to primary amines ().+/, via amide bonds.
The most readily available and commonly used carbodiimides are the water%soluble E&C
for aqueous crosslinking and the water%insoluble &CC for non%aqueous organic synthesis
methods.
Chemical structures of carbodiimides E!C and !CC"
Carbodiimides (E!C and !CC)
E&C and other carbodiimides are -ero%length crosslinkers0 they cause direct conjugation
of carbo$ylates ()C**+, to primary amines ().+/, without becoming part of the final
crosslink (amide bond, between target molecules.
E&C crosslinking reactions must be performed in conditions devoid of e$traneous
carbo$yls and amines. #cidic (p+ 1.2 to 2.2, 3E" buffer (1%morpholino%ethane%sulfonic
acid, is most effective, but phosphate buffers at p+ 4 5./ are also compatible with the
reaction chemistry. .%hydro$ysuccinimide (.+", or its water%soluble analog ("ulfo%.+",
is often included in E&C coupling protocols to improve efficiency or to create a more
stable, amine%reactive intermediate (see ne$t section,.
6ecause peptides and proteins contain multiple carbo$yls and amines, direct
E&Cmediated crosslinking usually causes random polymeri-ation of polypeptides.
.evertheless, this reaction chemistry is used widely in immobili-ation procedures (e.g.,
attaching proteins to a carbo$ylated surface, and in immunogen preparation (e.g.,
attaching a small peptide to a large carrier protein,.
!!C
"ame manner as E&C, +owever, because &CC is not aqueous%soluble, it is primarily
used in manufacturing and organic synthesis applications rather than in the typical protein
research biology lab. 7or e$ample, most commercially available, ready%to%use .+"%ester
crosslinkers and labeling reagents are manufactured using &CC. 6ecause water is
e$cluded, the resulting .+" ester can be prepared and stabili-ed as a dried powder
without appreciable hydrolysis. &CC is also commonly used in commercial peptide
synthesis operations.
Source#
$hermo%scientific
-http://www.piercenet.com/method/chemistry-crosslinking
%Crosslinking Technical Handbook
&onic gelation or coacervation of hydrophilic polymers
The method involves a mi$ture of two aqueous phases, of which one is the polymer
chitosan, a di%block co%polymer ethylene o$ide or propylene o$ide (8E*%88*, and the
other is a poly anion sodium tripolyphosphate. !n this method, positively charged amino
group of chitosan interacts with negative charged tripolyphosphate to form coacervates
with a si-e in the range of nanometer. Coacervates are formed as a result of electrostatic
interaction between two aqueous phases, whereas, ionic gelation involves the material
undergoing transition from liquid to gel due to ionic interaction conditions at room
temperature.
!n the current paper, the formulation components were optimi-ed to yield the smallest
si-e nanoparticles. 7or this purpose, 9: acetic acid aqueous solutions containing various
chitosan concentrations; <.9:, <.92:, <./:, <./2: and <.=: (w>v, were prepared. The
p+ of each chitosan solution was adjusted to 2.2 using 9. .a*+. # <.<9: solution of
T88 was also prepared in purified water. .anoparticles were formed spontaneously upon
addition of = ml of T88 solution to ? ml of chitosan solution under constant stirring (2<<
rpm, at room temperature.
"ource;
%Calvo 8, @emunan%ope- C, 'ila%Aato A, #lonso 3A. Chitosan and chitosan>ethylene
o$ide)propylene o$ide block copolymer nanoparticles as novel carriers for proteins and
vaccines. 8harm @es. 9BB50 91;91=9)91=?.9<.9</=>#;9<9/9/CB<5//2 D8ub3ed;
B=2C225E
%E.F. #gyare, G.. Curran, 3. @amakrishnan, C.C. Hu, A.7. 8oduslo, F.F. Fandimalla,
&evelopment of a smart nano%vehicle to target cerebrovascular amyloid deposits and
brain parenchymal plaques observed in #l-heimerIs disease and cerebral amyloid
angiopathy, 8harm. @es. /2 (/<<C, /?51)/?C1.
E'&S( technique
% Principle#
9. #ntibody coating
"pecific capture antibody is immobili-ed on high protein%binding plates by overnight
incubation. 8lates are blocked with irrelevant protein e.g. albumin.
/. 8rotein capture
"amples and standard dilutions are added to the wells and will be captured by the
bound antibodies.
=. &etection antibody
"pecific biotinylated detection antibody is added to the wells to enable detection of the
captured protein.
1. "treptavidin%en-yme conjugate
"treptavidin conjugated with alkaline phosphatase or horseradish pero$idase is added
to the wells and will bind to the biotinylated antibody.
2. #ddition of substrate
Colorimetric substrate is added to the wells and will form a colored solution when
cataly-ed by the en-yme.
?. #nalysis
#bsorbance is measured in an E!"# reader and the amount of protein in the samples
is determined.
quart) crystal microbalance%dissipation#
principle;
The active component of a JC3 is a thin quart- crystal disk sandwiched between a
pair of electrodes.
The application of an #C voltage over the electrodes causes the crystal to oscillate at
its acoustic resonance frequency.
When the #C voltage is turned off, the oscillation decays e$ponentially (Krings downK,.
This decay is recorded and the resonance frequency (f, and the energy dissipation
factor (&, are e$tracted.
& is defined as the loss of energy per oscillation period divided by the total energy
stored in the system.
& is equal to the resonance bandwidth divided by the resonance frequency.
Changes in the resonance frequency (Lf, are primarily related to mass uptake or
release at the sensor surface.
When employed as a mass sensor, the instrument has a sensitivity of about <.2
ng>cm/ according to the manufacturer. Changes in the dissipation factor (L&, are
primarily related to the viscoelasticity (softness,.
The softness, in turn, often is related to structural changes of the film adhering at the
sensor surface.
methodology;
9. The gold>quart- crystal surface was equilibrated with distilled water
/. #M1< fibril suspension (<.2 mg>ml, was passed over the crystal at a flow rate of
9<< Nl>min.
=. 6inding of #M1< fibrils to the gold surface was monitored by recording the
frequency changes in the sensors .
1. the flow of #M1< fibril suspension was continued until the frequency plateaued.
2. &istilled water was again passed over the crystal for 9< min toremove the
loosely bound #M1< fibrils.
?. T.' or CC. suspension (9< mg>ml, was passed over the #M1< fibril bed
adsorbed to the crystal surface.
5. The mass of #O1< fibrils adsorbed on was calculated as follows;
*lo+ Cytometry
,%*luidics system
*ne of the fundamentals of flow cytometry is the ability to measure the properties of
individual particles. When a sample in solution is injected into a flow cytometer, the
particles are randomly distributed in three%dimensional space. The sample must
therefore be ordered into a stream of single particles that can be interrogated by the
machinePs detection system. This process is managed by the fluidics system.
Essentially, the fluidics system consists of a central channel>core through which the
sample is injected, enclosed by an outer sheath that contains faster flowing fluid. #s
the sheath fluid moves, it creates a massive drag effect on the narrowing central
chamber. This alters the velocity of the central fluid whose flow front becomes
parabolic with greatest velocity at its center and -ero velocity at the wall (see 7igure
The effect creates a single file of particles and is called hydrodynamic focusing. Qnder
optimal conditions (laminar flow, the fluid in the central chamber will not mi$ with the
sheath fluid. The flow characteristic of the central fluid can be estimated using @eynolds
.umber (@e, Without hydrodynamic focusing the no--le of the instrument (typically
5< N3, would become blocked, and it would not be possible to analy-e one cell at a time.
Where & R tube diameter, ' R mean velocity of fluid, p R density of fluid, and R viscosity of fluid. When @e
S /=<<, flow is always laminar. When @e T /=<<, flow can be turbulent, which accelerates diffusion.
-% .ptics and detection
#fter hydrodynamic focusing, each particle passes through one or more beams of light.
ight scattering or fluorescence emission (if the particle is labeled with a fluorochrome,
provides information about the particlePs properties. The laser and the arc lamp are the
most commonly used light sources in modern flow cytometry.
/% Signal processing
When light hits a photodetector a small current (a few microamperes, is generated. !ts
associated voltage has an amplitude proportional to the total number of light photons
received by the detector. This voltage is then amplified by a series of linear or logarithmic
amplifiers, and by analog to digital convertors (#&Cs,, into electrical signals large enough
(2)9< volts, to be plotted graphically.
og amplification is normally used for fluorescence studies because it e$pands weak
signals and compresses strong signals, resulting in a distribution that is easy to display on
a histogram. inear scaling is preferable where there is not such a broad range of signals
e.g. in &.# analysis.
The measurement from each detector is referred to as a UparameterP e.g. forward scatter,
side scatter or fluorescence.
4% Electrostatic cell sorting
# major application of flow cytometry is to separate cells according to subtype or epitope
e$pression for further biological studies. This process is called cell sorting or 7#C"V
analysis.
#fter the sample is hydrodynamically focused, each particle is probed with a beam of
light. The scatter and fluorescence signal is compared to the sort criteria set on the
instrument. !f the particle matches the selection criteria, the fluid stream is charged as it
e$its the no--le of the fluidics system.
Electrostatic charging actually occurs at a precise moment called the Ubreak%off pointP,
which describes the instant the droplet containing the particle of interest separates from
the stream.
!n our 8aper;
The e$tents of intracellular accumulation of #7?15%!gG1.9 and #7?15%T.'s were
determined by 7#C"Calibur (6ecton &ickinson, with e$citation and emission
wavelengths set at ?2/ and ??C nm, respectively as fluorescent dyes were added
such as fluorescein isothiocyanate and #le$a 7luor ?15.
"ource; !ntroduction to 7low Cytometry by 3isha @ahman 8h.&.
Confocal 'aser Scanning 0icroscopy
!n a confocal laser scanning microscope, a laser beam passes through a light source
aperture and then is focused by an objective lens into a small (ideally diffraction
limited, focal volume within or on the surface of a specimen. !n biological applications
especially, the specimen may be fluorescent. "cattered and reflected laser light as
well as any fluorescent light from the illuminated spot is then re%collected by the
objective lens. # beam splitter separates off some portion of the light into the detection
apparatus, which in fluorescence confocal microscopy will also have a filter that
selectively passes the fluorescent wavelengths while blocking the original e$citation
wavelength. #fter passing a pinhole, the light intensity is detected by a photodetection
device (usually a photomultiplier tube (83T, or avalanche photodiode,, transforming
the light signal into an electrical one that is recorded by a computer.
!n our 8aper;
The in vitro C## model was prepared by pre%incubating the hC3EC> &= monolayer
with /2 Ng>ml fluorescein isothiocyanate (7!TC,% &utch #M1< protein for =< min. The
7!TC%&utch #M1< was aspirated, =< Ng>ml #le$a 7luor ?15 (#7?15,%T.'s were added
and incubated for ?< min at 2: C*/ and =5 WC with minimal shaking. The T.'s were
/92 removed, the monolayer was washed with +ankIs balanced salt solution (+6"",,
and fi$ed using 1: paraformaldehyde. The Transwells X were stained with &#8!,
mounted, and imaged with an #$iovert 9<<3micro% scope equipped with Yeiss "3
29< laser confocal microscope (&#8!, E$>Em ; =2<>15< nm0 7!TC, E$>Em; 1CC>2=2
nm0 #7?15, E$>Em; ?2/> ??C,
"ource;
%Wikipedia
-www.olympusconfocal.com/theory/LSCMIntro
!ynamic single photon emission computed tomography (SPEC$) #
!t is a technique that uses tracers to obtain images that reflect fundamental
biophysiologic functions of perfusion and etabolism in body organs.
This is done by quantifying the temporal changes of radionuclide concentration in
tomographic sections through angular sampling of projections.
This technique, in conjunction with mathematical model%based analysis of regional
kinetics of the radiotracer, allows for the quantitation of functional parameters, which
are estimated by utili-ing a time series of dynamic reconstructed images.
8hysiologic parameter estimation using kinetic analysis is well established in positron
emission tomography (8ET,.
The development and use of kinetic analysis in dynamic "8ECT
applications have been limited by low sensitivity, poor spatial resolution and poor
temporal resolution, which are consequences of the need to mechanically rotate the
detector head for most studies.
Magnetic resonance imaging
3agnetic resonance imaging (3@!,, nuclear magnetic resonance imaging (.3@!,, or
magnetic resonance tomography (3@T, is a medical imaging technique used in
radiology to investigate the anatomy and function of the body in both health and
disease. 3@! scanners use strong magnetic fields and radiowaves to form images of
the body. The technique is widely used in hospitals for medical diagnosis, staging of
disease and for follow%up without e$posure to ioni-ing radiation.
How MRI works
To perform a study the patient is positioned within an 3@! scanner which forms a
strong magnetic field around the area to be imaged. 3ost medical applications rely on
detecting a radio frequency signal emitted by e$cited hydrogen atoms in the body
(present in any tissue containing water molecules, using energy from an oscillating
magnetic field applied at the appropriate resonant frequency. The orientation of the
image is controlled by varying the main magnetic field using gradient coils. #s these
coils are rapidly switched on and off they create the characteristic repetitive noises of
an 3@! scan. The contrast between different tissues is determined by the rate at
which e$cited atoms return to the equilibrium state. E$ogenous contrast agents may
be given intravenously, orally or intra%articularly.
3@! requires a magnetic field that is both strong and uniform. The field strength of the
magnet is measured in tesla ) and while the majority of systems operate at 9.2T
commercial systems are available between <./T)5T. 3ost clinical magnets are
superconducting which requires liquid helium. The lower field strengths can be
achieved with permanent magnets, which are often used in KopenK 3@! scanners for
claustrophobic patients.
Phantom
#n artificial object of known si-e and composition that is imaged to test, adjust or
monitor an 3@! systems homogeneity, imaging performance and orientation aspects.
# phantom is usually a fluid%filled container or bottle often filled with a plastic structure
of various si-es and shapes.
Contrast agents
The most commonly used intravenous contrast agents are based on chelates of
gadolinium. !n general, these agents have proved safer than the iodinated contrast
agents used in Z%ray radiography or CT.
Role of agarose
8hantoms for evaluation of nuclear magnetic resonance (.3@, imaging systems were
made from water%based agarose gels.