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,