Polymers in Biomedicine Introduction of Polymers ?· Polymers in Biomedicine • Introduction of Polymers…

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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 http://www.youtube.com/watch?feature=endscreen&NR=1&v=TpvNEZCvk84http://www.youtube.com/watch?feature=endscreen&NR=1&v=TpvNEZCvk84http://www.youtube.com/watch?feature=endscreen&NR=1&v=TpvNEZCvk84http://www.youtube.com/watch?feature=endscreen&NR=1&v=TpvNEZCvk84http://www.youtube.com/watch?feature=endscreen&NR=1&v=TpvNEZCvk84http://www.youtube.com/watch?feature=endscreen&NR=1&v=TpvNEZCvk84http://www.youtube.com/watch?feature=endscreen&NR=1&v=TpvNEZCvk84http://www.youtube.com/watch?feature=endscreen&NR=1&v=TpvNEZCvk84http://www.youtube.com/watch?feature=endscreen&NR=1&v=TpvNEZCvk84http://www.youtube.com/watch?feature=endscreen&NR=1&v=TpvNEZCvk84http://www.youtube.com/watch?feature=endscreen&NR=1&v=TpvNEZCvk84http://www.youtube.com/watch?feature=endscreen&NR=1&v=TpvNEZCvk84http://www.youtube.com/watch?v=pxIJdjizQes&feature=relatedhttp://www.youtube.com/watch?v=pxIJdjizQes&feature=relatedhttp://www.youtube.com/watch?v=pxIJdjizQes&feature=relatedhttp://www.youtube.com/watch?v=pxIJdjizQes&feature=relatedhttp://www.youtube.com/watch?v=pxIJdjizQes&feature=relatedhttp://www.youtube.com/watch?v=pxIJdjizQes&feature=relatedhttp://www.youtube.com/watch?v=pxIJdjizQes&feature=relatedhttps://www.youtube.com/watch?v=iBZAwhxwHX0https://www.youtube.com/watch?v=by53LP0Yu4c12/8/2016 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 polymersolvent and polymerpolymer interactions. Good solvent: polymersolvent interactions dominate and the polymer chains are relaxed Poor solvent, the polymer will aggregate due to a restricted chain movement because of increased polymerpolymer 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> 32C, 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 32C 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 40C. (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/296/5573/1673 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, 429445 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 - and -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 wei nicht, ob das Medikament bei einem wirkt und welche Nebenwirkungen zu erwarten sind Welche Limitationen mchte 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 CUrea (5M) Reduction agent NCJ. 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 Erhlt den osmotischen Druck zwischen den Blutgefen und dem Gewebe Transportermolekl Denaturiertes Albumin als Plattform fr 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) NCN CFusing 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 Erhhte Zellaufnahme Gd-DOTA (30-50 Gruppen) Magnetresonanztomographie Polyethylenglykol (16) Pharmakokinetik N CY. 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)EDCPEO2000-NHScHSA-PEO(2000)162-22NHS-DOTA Gd2+cHSA-PEO(2000)16-DOTA2-24cHSA-PEO(2000)16-Gd2-25Gd Polyethylenoxid Gute Wasserlslichkeit Keine Aggregation Hhere Stabilitt PEO-Seitenketten Albumin-Rckgrat Denaturierung Albumin-Hlle PEO-Hlle kovalent konjugiertes DOX Gd-DOTA fr MRI MRI contrast agent Y. Wu, T. Weil, Advanced Healthcare Materials, 2013, 2(6): 884-894. Mageschneiderte Biopolymere fr 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 mol/kg appliziert Normale Dosis: 400 mol/kg Gd-HSA-DOX: Ca. 40-fach verbesserte Detektion des implantierten Tumors Visualisierung des Tumors mittels MRI HSACationized HSA (cHSA)EDCPEO2000-NHScHSA-PEO(2000)162-22NHS-DOTAGd2+cHSA-PEO(2000)16-DOTA2-24 cHSA-PEO(2000)16-Gd2-25Rasche 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 fr den Wirkstofftransport Angewandte Chem. Int. Ed. 55, 23, 6586-6598 (2016) Doxorubicin-Wirkstoff Diamant-Trger Polymer Polypeptid-Hlle Adv. Funct. Mater. 25, 42, 65766585 (2015) Verfolgung von Nanodiamanten und Wirkstoffen in Lebenden Zellen Diamant-Trger Verfolgung des Transports und der Freisetzung von Wirkstoffen in lebenden Zellen Tracking ber lange Zeitrume mglich Adv. Funct. Mater. 25, 42, 65766585 (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, 65766585 (2015) Immunohistologie Adv. Funct. Mater. 25, 42, 65766585 (2015) Immunohistochemische Analyse von Brustkrebs-Xenografts (HE, Hematoxilin- und Eosin- Frfung des gesamten Xenografts, das auf dem CAM gewachsen ist); ursprngliche Vergrerung 50x; Ki-67 Antigen-Frbung der Tumor-Xenografts, braun-rote Kerne deuten auf proliferierende Zelle, ursprngliche Vergrerung 200x. *P

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