Polymers in Biomedicine • Introduction of Polymers
• Polymeric Biomaterials
• Polymeric Drugs
• Polymer Drug Transporter
• Materials that have one or more properties that can be significantly changed in a
controlled fashion by external stimuli,
• Such as stress
• Temperature
• Moisture
• pH
• electric or magnetic fields
• Akustik sounds
• Example:
• pH-sensitive polymers are materials that change in volume when the pH of the
surrounding medium changes
3. “Smart” Biomaterials
• Hydrogels are crosslinked network polymeric materials that are not soluble but
can absorb large quantities of water.
• These materials are soft and rubbery in nature, resembling living tissues in their
physical properties.
3. Hydrogels
http://www.youtube.com/watch?feature=endscreen&NR=1&v=TpvNEZCvk84 http://www.youtube.com/watch?v=pxIJdjizQes&feature=related
Many hydrogels are smart and respond to external stimuli
https://www.youtube.com/watch?v=iBZAwhxwHX0 https://www.youtube.com/watch?v=by53LP0Yu4c
12/8/2016 4
3. Definition of a Hydrogel
• Water insoluble, three dimensional network of
polymeric chains that are cross-linked by chemical or
physical bonding
• Polymers capable of swelling substantially in aqueous
conditions (eg. hydrophilic)
• Polymeric network in which water is dispersed
throughout the structure
12/8/2016 5
3. Hydrogel Forming Polymers – Hydrophilc Polymers
O
H O O H
H O 2 C
O
O H O
N H
H O
O
O
O
H O O H
N a O 2 C
O
O
O
O
N H O
n
p o l y ( h y a l u r o n i c a c i d ) p o l y ( s o d i u m a l g i n a t e )
n
n
p o l y ( e t h y l e n e g l y c o l )
n
p o l y ( l a c t i c a c i d )
n
p o l y ( N - i s o p r o p y l a c r y l a m i d e )
Natural
Synthetic
3. Characteristics of Hydrogels
• No flow when in the steady-state
• By weight, gels are mostly liquid but behave like solids
• Absorption of large quantities of water
– 1-20% up to 1000 times their dry weight
• Cross linkers within the fluid give a gel its structure
(hardness) and contribute to stickiness (tack).
• Tissue-like bahaviour
3. Hydrogels
Highly swollen hydrogels
• Cellulose derivatives
• Poly(vinyl alcohol)
• Poly(ethylene glycol)
Common structural features
• Many OH (or =O) groups to interact with
• Acidic environments hydrophillic swelling
8
O
n
Poly(ethylene glycol)
3. Biomedical Uses for Hydrogels
• Scaffolds in tissue engineering.
• Sustained-release delivery systems
• Hydrogels that are responsive to specific molecules, such as glucose or
antigens can be used as biosensors as well as in DDS.
• Disposable diapers where they "capture" urine, or in sanitary napkins
• Contact lenses (silicone hydrogels, polyacrylamides)
• Medical electrodes using hydrogels composed of cross linked polymers
(polyethylene oxide, polyAMPS and polyvinylpyrrolidone)
• Lubricating surface coating used with catheters, drainage tubes and gloves
3. Biomedical Uses for Hydrogels
• Breast implants
• Dressings for healing of burn or other hard-to-heal wounds. Wound gels are
excellent for helping to create or maintain a moist environment.
• Reservoirs in topical drug delivery; particularly ionic drugs, delivered by
iontophoresis
• Artificial tendon and cartilage
• Wound healing dressings (Vigilon®, Hydron®, Gelperm®)
• non-antigenic, flexible wound cover
• permeable to water and metabolites
• Artificial kidney membranes
• Artificial skin
• Vocal cord replacement
10
• The polymer chains usually exist in the
shape of randomly coiled molecules.
• In the absence of Na+ ions the negative
charges on the carboxylate ions along
the polymer chains repel each other and
the chains tend to uncoil.
3. Polyacrylate Hydrogel
• Water molecules are attracted to the
negative charges by hydrogen bonding
• The hydrogel can absorb over five
hundred times its own weight of pure
water but less salty water
3. Polyacrylate Hydrogel
• When salt is added to the hydrogel, the chains start to change their shape and water
is lost from the gel
3. Polyacrylate Hydrogel
3. Hydrogel Swelling
• By definition, water must constitute at least 10% of the total weight (or volume)
for a materials to be a hydrogel
• Swelling due to one or more highly electronegative atoms which results in charge
asymmetry favoring hydrogen bonding with water
• Because of their hydrophilic nature, dry materials absorb water
• When the content of water exceeds 95% of the total weight (or volume), the
hydrogel is said to be superabsorbant
12/8/2016 14
• Hydrogels containing interactive functional groups along the main polymeric chains are
usually called ‘‘smart’’ or ‘‘stimuli-responsive’’ hydrogels.
• the polymer conformation in solution is dictated by both the polymer–solvent and
polymer–polymer interactions.
• Good solvent: polymer–solvent interactions dominate and the polymer chains are
relaxed
• Poor solvent, the polymer will aggregate due to a restricted chain movement because of
increased polymer–polymer
3a. Smart Hydrogels
16
3a. Hydrogel Forming “Smart” Polymers
Cross-linked Polyacrylamide
• Thermally and mechanically stabile
• Not degradable
Cross-linked PNiPAM (poly(N-isopropyl acrylamide)
• Finetuning of LCST behavior via copolymerization
• Mechanic stability
• No degradability
Application: 2D Tissue growth
• Synthesized in the 1950s
• Sensitive to both pH and temperature
• T> 32°C, reversible lower critical solution temperature phase transition (LCST)
• Swollen hydrated state to a shrunken dehydrated state, losing about 90% of its mass.
• 3D-dimensional hydrogel when crosslinked with N,N’-methylene-bis-acrylamide
(MBAm) or N,N’-cystamine-bis-acrylamide (CBAm).
• PNIPAm expels its liquid contents at a temperature near that of the human body
• PNIPAm has been investigated by many researchers for possible applications in tissue
engineering and controlled drug delivery.
3a. Poly(N-isopropylacrylamide) – “PNIPAAm or PNIPAm”
LCST = Lower Critical Solution Temperature
3a. Poly(N-isopropylacrylamide) – “PNIPAAm or PNIPAm”
• lower critical solution temperature (LCST) at 32°C
• soluble below its LCST, but precipitates above the LCST
• Reversible formation (below LCST) and cleavage (above LCST) of the hydrogen bonds
between –NH and C=O groups of pNIPAAm chains and the surrounding water molecules.
• Pentagonal water structure that is generated among the water molecules adjacent to the
hydrophobic molecular groups
Poly(N-isopropylacrylamide) – “PNIPAAm or PNIPAm”
3a. Poly(N-isopropylacrylamide) – “PNIPAAm or PNIPAm”
3a. Poly(N-isopropylacrylamide) (PNIPAAm or PNIPAm) Application: Controlling Cell Adhesion
3a. Poly(N-isopropylacrylamide) (PNIPAAm or PNIPAm) Application: Controlling Size and Surface Texture
3a. Poly(N-isopropylacrylamide) (PNIPAAm or PNIPAm) Application: Controlled Drug Delivery
Slow drug release Rapid Drug Release
3a. Changes in the Physical Properties of PNIPAM with External Stimuli
Materials exhibit shape-memory properties if they are able to fix a temporary
shape and recover back to their “remembered” permanent shape when exposed
to an external stimulus
3b. Shape Memory Polymers
Large deformation can be induced and recovered through
temperature or stress changes (pseudoelasticity)
Shape Memory Polymers (SMP)
• “Memorize” a macroscopic (permanent) shape
• Fixed to a temporary and shape under specific conditions of temperature and stress
• Relax to the original, stress-free condition under thermal, electrical, or environmental
command.
• This relaxation is associated with elastic deformation stored during prior
manipulation
3b. Shape Memory Polymers
3b. Elastic Polymer
Example: Rubber
Two distinct types of cross-linking:
(1) nonreversible cross-link (which can be either a covalent or a physical
cross-link) used to fix the permanent shape.
(2) Reversible “cross-link” (usually in the form of a thermal transition such as
Tg, Tm, or clearing point of a liquid crystalline material) responsible for
holding the temporary shape
3b. Crosslinking is Essential
Example: Rubber
29
3b. Non-Reversible Cross-Links
Physical and Chemical Cross Links for restoring permanent shape
30
3b. Glass State
• Liquid
Chains move freely
• Amorphous state
Below the critical temperature, long distance movements are frozen (transition to
the amorphous state)
Glass Temperature Tg
Crystalline and semi-crystalline polymers have up to two thermal phase transitions
(melting of the crystalline domains or glass transition)
Glas-like, hard Rubber-like, soft
cooling heating
3b. Thermal Shape Memory Polymers
• A rubbery compound (elastomer)
• Can be amorphous thermoplastics (covalently cross-linked) with Tg below room
temperature to allow full chain mobility- the restoring force in entropy
• Shape memory polymers morph by the glass transition or melting transition from a
hard to a soft phase which is responsible for the shape memory effect.
31
3b. Heating/Cooling Cycle
2002 Wiley-VCH
3b. Recovery Cycle
• Strain recovery of a cross-linked, castable shape-memory polymer upon rapid
exposure to a water bath at T = 80 °C
• (a) UV light is absorbed by the ligand complexes and converted to localized heat, which
disrupts the phase separation;
• (b) the material can then be deformed;
• (c) removal of the light while the material is deformed allows the metal ligand complexes to
reform and lock in the temporary shape
• (d) additional exposure to and subsequent removal of UVlight allows for a return to the
permanent shape.
3b. Photoactive Shape Memory Polymers
Applications for Shape Memory Polymers
• Intravenous cannula
• Self-adjusting orthodontic wires
• Pliable tools for small scale surgical procedures where currently metal-based shape
memory alloys such as Nitinol are widely used.
• Minimally invasive implantation of a device in its small temporary shape which
after activating the shape memory by e.g. temperature increase assumes its
permanent (and mostly bulkier) shape.
From top to bottom:
Knot tightened in 20 sec when heated to 40°C.
(a) A smart surgical suture self-tightening at elevated
temperatures (left).
(b) A thermoplastic shape-memory polymer fiber was
programmed by stretching to about 200% at a high
temperature and fixing the temporary shape by
cooling.
(c) After forming a loose knot, both ends of the suture
were fixed.
Temperature-induced Self-Tightening Knot
(a) Degradable shape-memory suture for wound
closure
(b) The photo series from the animal experiment
shows (top to bottom) the shrinkage of the fiber
while the temperature increases from 20 to 41 °C.
http://www.sciencemag.org/cgi/content/full/296/55
73/1673
3b. Degradable shape-memory suture for wound closure
3b. Applications: Intravenous Cannula
• Pictures of the shape memory foam deploying in in vitro aneurysm model
• Foam starts in compressed form (upper left) and expands to fill 60% the
aneurysm (lower right). The time from the laser initiation to the final image was
approximately 10 seconds. http://cbst.ucdavis.edu/research/aneurysm-treatment
Aus dem Film „Die Reise ins Ich“ 1987
Polymer Therapeutics & Drug Delivery
3. Drug Delivery
Drug delivery ensures that a pharmacologically active substance arrives at a relevant in
vivo location with minimal side-effects
3. Time Development of Polymer Drug Delivery Vehicles
Nano Lett. 2010, 10, 3223-–3230
3. Polymer Drug Delivery Vehicles
Ideal Systemic Delivery Particle
• Non-toxic vehicle
• Non-immunogenic
• Intracellular delivery
• Specific targeting of cells & intracellular
compartments
• Controlled stability & degradability after release
Challenges
• Drug release profiles
• Stabilization
• Extended circulation
• Plasma protein binding
• Specific targeting
3. Controlled Drug Delivery
Controlled drug delivery
• Site-specific delivery
• Reduced side effects
• Increased bioavailabilty
• Increased therapeutic effectiveness
3. “Nano” Plays an Important Role in the Body
3. Polymer Therapeutics
• A family of new chemical entities composed of polymers (R. Duncan)
• Conjugation of drugs to polymers, nanoparticles etc. (5-100 nm)
• Greater molecular weight = longer blood circulation
• In addition: Stabilization, improved solubility
Nanowirkstoffe
• Nano-sized
• Different pharmakokinetics
• Higher drug loading
• Space for cell targeting groups
• More challenging degradation
• Toxic metabolites
Approved Anti-Tumor Drug Doxorubicin
• Small molecule
• No space for attaching new functions
3. Polymer Therapeutics
Approved
Late development phases
3. “Size Matters” - The EPR-Effect ‘‘Enhanced Permeability and Retention Effect’’
Peer, D, et al. Nature Nanotechnology 2007, 2, 751-760
Duncan, R. Nature Reviews Cancer 2006, 6, 688-701
Peer, D, et al. Nature Nanotechnology 2007, 2, 751-760
Duncan, R. Nature Reviews Cancer 2006, 6, 688-701
EPR effect (passive targeting)
• Decreased systemic drug
elimination.
• Enhanced retention of the drug-
carrier complex in the tumor as
compared to the blood
(tumor : blood ratio of >2500).
• Leaky vasculature is
characteristic of solid tumors
and inflamed tissue and allows
nano-sized objects to enter.
3. “Size Matters” - The EPR-Effect ‘‘Enhanced Permeability and Retention Effect’’
3. Polymer Therapeutics - Architectures
3. Drug Delivery Agents
• Drug: Doxorubicin
• Chemotherapeutic against ovarian cancer
• Product name: Doxcil
• Significantly reduced cardiotoxicity of Doxorubicin
Http://www.doxil.com
Doxil®: Liposomal Formulation of Doxorubicin (100 nm size) Approved February, 2005.
Nanomedicine: Delivery of Doxorubicin
• Chemotherapeutic against breast cancer
• Product name: Abraxane® • Approved 2005 ($134 turnover /
year)* • Drug: Paclitaxel
Protein Nanocarriers: Serum Albumin (Abraxane)
Http://www.abraxisbio.com
*Data from Small Times
• Nanoparticles (diameter ~130 nm) of an albumin shell surrounding the paclitaxel drugs
• Uptake albumin-paclitaxel nanoparticles by the EPR effect
Kratz et al, J. Control. Release (2012) 161, 2, 429–445
a) Chemical structure of mPEG45-b-PCL80-b-PPEEA10 and schematic illustration of the formation of micellar nanoparticles and the loading with paclitaxel and siRNA
3. Polymeric Drug - Gene Transporter
Challenges Associated with Stability
Ideal: drug is
retained in the blood
& released in tumor
cells
Premature release of drugs while
still circulating in the blood Reduction of burst release
• Low CMC: Polymeric micelles lower
CMC, higher stability
• Cross-linking
• Onion-type multilayered structures,
additional diffusion barriers
Onion-type structure
additional diffusion barriers
Q. Sun et al., J. Control. Release (2012)
Stability in the Blood Stream upon iv Application,
• Micelle decomposition in the bloodstream due to α- and β-
globulins (protein adsorption, drug extraction)
Burst release of drug molecules
• Local or systemic toxicity, lowered drug availability to the tumor
and reduced therapeutic efficacy
Q. Sun et al. , Journal of Controlled Release (2012)
3. Transport of Polymer Drugs into Diseases Tissue
1. Transport via blood circulation 2. Transport into tumor tissue
3. Transport into tumor cells
Challenges:
• Drug should 1) circulate and get release in 2) and in 3).
• Drug should be inert in blood stream but sticky in tumor tissue
Blutkompartimente
3. “Active” Drug Targeting
• Specific binding to membrane proteins on
cancer cells
• Integrins, CXCR4, folate receptors
H. Ringsdorf, J. Polym. Sci. Polym. Symp. 1975, 51, 135.
3. “Active” Drug Targeting
3. Synthesis of a Polymer with Active Targeting Capabilities
3. Synthesis of a Polymer with Active Targeting Capabilities
• Peptide “WSC02” actively targets CXCR4 receptors that play an important role in
• cell migration and HIV infection
3. Implementing Cell Uptake via a pH Switch
• Comparison of drug release from pH-responsive PAMA-DMMA nanogels and PAMA-SA nanogels nonsensitive to pH change.
Intracellular transport of nanoparticles. After internalization, the nanoparticle is trafficked along the endolysosomal network in vesicles with the aid of motor proteins and cytoskeletal structures. ER, endoplastic reticulum; ERC, endocytic recycling compartment; MTOC, microtubule-organizing center; MVB, multivesicular bodies.
• The pathway is mainly determined by the size and surface properties of the nanoparticles, as well as the type (e.g., macrophages vs. endothelial cells) and activation status of the cells.
• Despite the significant progress in recent years, the details of uptake routes for some nanoparticles remain elusive.
• CNT, carbon nanotube; MSN, mesoporous silica nanoparticle; SPION, superparamagnetic iron oxide nanoparticle.
3. Challenges Associated with Release
Efficient Intracellular Release
• Only free intracellular drugs that bind to their targets are therapeutically effective
(effective cytosolic drug concentration / overall therapeutic efficacy)
• Intra-lysosome release upon pH changes
• Amine-containing polymers: endosomal membrane-disruption activity by a “proton
sponge” mechanism
Q. Sun et al., J. Control. Release (2012) asap
• Intra-cytosol release: Cytosolic
signals for faster drug release
(GSH cleave the disulfide bonds
to release conjugated drugs).
Cleavable linkers that have been used for stimuli-responsive drug release. The dotted line in each molecule indicates the bond that will be broken upon activation by the corresponding stimulus (indicated in parentheses).
3. Stimulus Responsive Drug Release
c) Protonation (left) or deprotonation (right) results in destruction of the polymer micelle. b) Protonation induces collapse of the polyanion chains, making the liposomal shell leaky and thus promoting efflux of the drug from the liposome. c) Deprotonation leads to swelling of the hydrogel matrix, triggering drug release from the nanosphere.
3. Drug Release from Polymer Micelles & Liposomes
Drug release from two different types of thermosensitive carriers: a) a liposome containing a thermosensitive polymer and b) a nanoparticle coated with a thermosensitive block copolymer. Upon heating, the thermoresponsive component undergoes conformational change, initiating or accelerating the drug release.
3. Drug Release from Polymer Micelles & Liposomes
Photosensitive liposomes constructed through incorporation of light-responsive units into the lipid bilayers with an aim to control the drug release with optical irradiation. The release can be achieved through a) photoisomerization, b) photocleavage, and c) photopolymerization.
3. Drug Release from Polymer Micelles & Liposomes
NanoMEDIZIN
• Wirksamkeit
• Wirkungseintritt oder Pharmakokinetik
• Nebenwirkungen
• Applikationsform (bevorzugt oral)
Man weiß nicht, ob das Medikament bei einem wirkt und welche Nebenwirkungen zu erwarten sind
Welche Limitationen möchte man mit Nanomaterialien adressiere?
Integration of Bioimaging and Therapy in one Plattform – Online Tracking of the Drug inside the Body
Theranostics
S. Mura, P. Couvreur / Advanced Drug Delivery Reviews (2012)
High Need for Innovative Materials for Personalized Medicine
Personalized Medicine
N C
Urea (5M) Reduction agent
N
C
J. Am. Chem. Soc. 132, 14, 5012 (2010) Biomacromolecules 13, 6, 1890 (2012)
Example from our own Research
Humanes Serumalbumin (HSA) Denaturiertes HSA
Humanes Serumalbumin (Protein)
• 55% des Proteinanteils im Blutplasma
• Erhält den osmotischen Druck zwischen
den Blutgefäßen und dem Gewebe
• Transportermolekül
Denaturiertes Albumin als Plattform für die Synthese bioabbaubarer Polymere
Herstellung von Biopolymeren aus denaturierten Proteinen
Polymere basierend auf denaturierten Proteinen
Natives Eiklar (Albumen) Denaturierung Quervernetzung
Irreversible Thermische Protein Denaturierung
J. Am. Chem. Soc. 132, 14, 5012 (2010) Biomaterials 31, 33, 8789-8801 (2010)
N
C
N C
Fusing Polymers & Biomedical Needs – Assoc. Prof. at the National University of Singapore
First demonstration of protein-copolymers
of defined lengths & sequences
Small 8, 22, 3381 (2012) Biomacromolecules 13, 6, 1890 (2012)
Doxorubicin (27-28 Gruppen)
pH spaltbar, hohe
Wirkstoffbeladung
Positive Ladungen Erhöhte Zellaufnahme
Gd-DOTA (30-50 Gruppen) Magnetresonanztomographie
Polyethylenglykol (16) Pharmakokinetik
N C
Y. Wu, T.W. et al. Biomacromolecules 2012,13, 6, 1890.
Y. Wu, T.W. et al. Adv. Healthcare Mater. 2013,2, 6, 884. Y. Wu, T.W., J. Am. Chem Soc. 2010, 132, 14, 5012.
Y. Wu, T.W. et al. Chem. Commun., 2014, 50,93,14620.
K. Eisele, T.W. et al. Macromol. Rapid Commun. 2010, 31, 1501. Y. Wu, T.W. et al. Small 2012, 8, 22, 3381.
Synthese von bioabbaubaren Polymeren mit vielen Funktionen
HSA Cationized HSA (cHSA)
EDC
PEO2000-NHS
cHSA-PEO(2000)16
2-22
NHS-DOTA Gd2+
cHSA-PEO(2000)16-DOTA
2-24
cHSA-PEO(2000)16-Gd
2-25
Gd
Polyethylenoxid • Gute Wasserlöslichkeit • Keine Aggregation • Höhere Stabilität
PEO-Seitenketten
Albumin-Rückgrat
Denaturierung
Albumin-Hülle
PEO-Hülle
kovalent
konjugiertes DOX
Gd-DOTA für MRI
MRI contrast agent
Y. Wu, T. Weil, Advanced Healthcare Materials, 2013, 2(6): 884-894.
Maßgeschneiderte Biopolymere für die “Theranostik” Portmanteau von Therapie und Diagnostik
Multilayered Architecture for Efficient Drug Transport
• High drug loading content
• Safety: Nontoxic, easy to excrete completely via the liver (into bile) or the kidneys (threshold
for rapid renal excretion: dh of about 5.5 nm)
• Approval: Clear and simple structure, reproducible particle size & distribution known
degradation products, made of FDA-approved building blocks.
PEO = Stabilization
Enzymatic cleavage positive charges for endocytosis
pH cleavable linker for DOX attachment
• Onion-type structure • Covalently linked drugs • Biodegradable
Imaging groups
• Gd-HSA-DOX sichtbare Tumoranreicherung auch nach 24h
• Geringere Anreicherung in Leber und Niere verglichen mit dem freien Wirkstoff DOX
In Vivo Anreicherung?
Herz Leber Milz Lunge Niere Tumor
Freies DOX
Biopolymer-
DOX
Freies DOX
Biopolymer-
DOX
6h
24h
Hoch
Gering
5mg/kg DOX
Fluoreszenz-Detektion von Doxorubicin in verschiedenen Organen
IC50 = 1 nM (72h, MV4-11)
MultiHance®
Kommerzieller Gd-MRI
9 µmol/kg appliziert
Normale Dosis: 400 µmol/kg
Gd-HSA-DOX: Ca. 40-fach verbesserte Detektion des implantierten Tumors
Visualisierung des Tumors mittels MRI
HSACationized HSA (cHSA)
EDCPEO2000-NHS
cHSA-PEO(2000)162-22
NHS-DOTA
Gd2+
cHSA-PEO(2000)16-DOTA2-24 cHSA-PEO(2000)16-Gd
2-25
Rasche
Salz Freies DOX HSA-Gd-DOX
Erste in vivo Ergebnisse • Normales Wachstum • Signifikante Reduktion des
Tumorvolumens
In ersten Studien: Attraktive Wirksamkeit und potentiell geringere Nebenwirkungen
5mg/kg DOX an Tag 0, 3, 7, 10
Verwendung von Nanodiamanten für den Wirkstofftransport
Angewandte Chem. Int. Ed. 55, 23, 6586-6598 (2016)
Doxorubicin-Wirkstoff
Diamant-Träger
Polymer
Polypeptid-Hülle
Adv. Funct. Mater. 25, 42, 6576–6585 (2015)
Verfolgung von Nanodiamanten und Wirkstoffen in Lebenden Zellen
Diamant-Träger
• Verfolgung des Transports und der Freisetzung von Wirkstoffen in lebenden Zellen
• Tracking über lange Zeiträume möglich
Adv. Funct. Mater. 25, 42, 6576–6585 (2015)
• Nanodiamanten reduzieren das
Tumorvolumen in einem CAM-Modell
• Deutliche Reduktion proliferierender
Zellen
Chicken Chorioallantoic Membrane (CAM)
Tumor model
Wirkstofftransport in einem Krebsmodell
Adv. Funct. Mater. 25, 42, 6576–6585 (2015)
Immunohistologie
Adv. Funct. Mater. 25, 42, 6576–6585 (2015)
• Immunohistochemische Analyse von Brustkrebs-Xenografts (HE, Hematoxilin- und Eosin-
Färfung des gesamten Xenografts, das auf dem CAM gewachsen ist); ursprüngliche
Vergrößerung 50x;
• Ki-67 Antigen-Färbung der Tumor-Xenografts, braun-rote Kerne deuten auf proliferierende
Zelle, ursprüngliche Vergrößerung 200x. *P<0.05, **P<0.01,