Co-reporter:Mira Oswald, Simon Geissler, and Achim Goepferich
Molecular Pharmaceutics July 3, 2017 Volume 14(Issue 7) pp:2177-2177
Publication Date(Web):May 17, 2017
DOI:10.1021/acs.molpharmaceut.7b00158
The transport of drugs across the blood–brain barrier is challenging. The use of peptide sequences derived from viruses with a central nervous system (CNS) tropism is one elegant option. A prominent example is the rabies virus glycopeptide-29 (RVG-29), which is said to enable a targeted brain delivery. Although the entry mechanism of the rabies virus into the CNS is very well characterized, it is unknown whether RVG-29-functionalized drug delivery systems (DDSs) follow this pathway. RVG-29-functionalized DDSs present themselves with modifications of the RVG-29 peptide sequence and different physicochemical properties compared to the rabies virus. To our surprise, the impact of these changes on the functionality is completely neglected. This review explores virus-related CNS-targeting strategies by comparing RVG-29-functionalized DDSs with regard to their peptide modification, physiochemical properties and their behavior in cell culture studies with a special focus on the original pathway of rabies virus entry into the CNS.Keywords: blood−brain barrier; CNS-targeting strategies; rabies virus glycopeptide;
Co-reporter:Michael Backofen, Gregoire Schwach, Wolfgang Koechling, Torsten Weiss, Achim Goepferich
European Journal of Pharmaceutical Sciences 2017 Volume 104(Volume 104) pp:
Publication Date(Web):15 June 2017
DOI:10.1016/j.ejps.2017.03.036
The aim of this study was to investigate the interaction between the positively charged gonadotropin releasing hormone receptor antagonist degarelix and the two polyanions alginate and carboxymethyl cellulose (CMC). Light as well as transmission electron microscopy revealed that complexes formed by simple mixing of the peptide with one of the polymers had a nano-structure consisting of twisted fibers. The remarkable unique process of complex formation could be followed by isothermal titration calorimetry: We found that peptide self-aggregates dissolved upon the addition of polyanion and peptide-polymer-complexes formed thereafter with the anionic polymer as a template. Peptide release from the complexes was tested in vitro and in vivo and compared to the dissolution of drug from self-aggregates. In vitro the release was monitored over a period of three months. We could find only slight differences in the release kinetics for the alginate and the CMC complexes compared to the pure drug. An in vivo study in Sprague Dawley rats showed similar degarelix plasma concentration levels for the complex formulations and an aqueous degarelix solution following subcutaneous injection. Overall, our findings suggest a competition between complex formation and peptide aggregation, which did not increase the availability of free drug.Download high-res image (117KB)Download full-size image
Co-reporter:Robert Hennig, Andreas Ohlmann, Janina Staffel, Klaus Pollinger, Alexandra Haunberger, Miriam Breunig, Frank Schweda, Ernst R. Tamm, Achim Goepferich
Journal of Controlled Release 2015 Volume 220(Part A) pp:265-274
Publication Date(Web):28 December 2015
DOI:10.1016/j.jconrel.2015.10.033
The angiotensin II receptor type 1 (AT1R), which is expressed in blood vessels of the posterior eye, is of paramount significance in the pathogenesis of severe ocular diseases such as diabetic retinopathy and age-related macular degeneration. However, small molecule angiotensin receptor blockers (ARBs) have not proven to be a significant therapeutic success. We report here on a nanoparticle system consisting of ARB molecules presented in a multivalent fashion on the surface of quantum dots (Qdots). As a result of the multivalent receptor binding, nanoparticles targeted cells with high AT1R expression and inhibited their angiotensin receptor signaling with an IC50 of 3.8 nM while showing only minor association to cells with low AT1R expression. After intravenous injection into the tail vein of mice, multivalent nanoparticles accumulated in retinal and choroidal blood vessels of the posterior eye. At the same time, multivalent ligand display doubled the Qdot concentration in the blood vessels compared to non-targeted Qdots. Remarkably, ARB-targeted Qdots showed no pronounced accumulation in AT1R-expressing off-target tissues such as the kidney. Following systemic application, this multivalent targeting approach has the potential to amplify AT1R blockade in the eye and concomitantly deliver a therapeutic payload into ocular lesions.
Co-reporter:Robert Hennig; Anika Veser; Susanne Kirchhof;Achim Goepferich
Molecular Pharmaceutics 2015 Volume 12(Issue 9) pp:3292-3302
Publication Date(Web):August 7, 2015
DOI:10.1021/acs.molpharmaceut.5b00301
The use of angiotensin receptor blockers (ARBs) for treatment of ocular diseases associated with neovascularizations, such as proliferative diabetic retinopathy, shows tremendous promise but is presently limited due to short intravitreal half-life. Conjugation of ARB molecules to branched polymers could vastly augment their therapeutic efficacy. EXP3174, a potent non-peptide ARB, was conjugated to branched poly(ethylene glycol) (PEG) and poly(amido amine) (PAMAM) dendrimers: 7.8 ligand molecules were tethered to each 40 kDa PEG molecule whereas 16.7 ligand molecules were linked to each PAMAM generation 5 dendrimer. The multivalent PEG and PAMAM conjugates blocked AT1R signaling with an IC50 of 224 and 36.3 nM, respectively. The 6-fold higher affinity of the multivalent ligand-conjugated PAMAM dendrimers was due to their unique microarchitecture and ability to suppress polymer–drug interactions. Remarkably, both polymer–drug conjugates exhibited no cytotoxicity, in stark contrast to plain PAMAM dendrimers. With sufficiently long vitreous half-lives, both synthesized polymer–ARB conjugates have the potential to pave a new path for the therapy of ocular diseases accompanied by retinal neovascularizations.
Co-reporter:Nadine Hammer, Ferdinand P. Brandl, Susanne Kirchhof, Achim M. Goepferich
Journal of Controlled Release 2014 Volume 183() pp:67-76
Publication Date(Web):10 June 2014
DOI:10.1016/j.jconrel.2014.03.031
The reversible attachment of proteins to polymers is one potential strategy to control protein release from hydrogels. In this study, we report the reversible attachment of lysozyme to poly(ethylene glycol) (PEG) by degradable carbamate linkers. Phenyl groups with different substituents were used to control the rate of carbamate hydrolysis and the resulting protein release. Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed modification with 1–3 PEG chains per lysozyme molecule. Protein PEGylation and PEG chain elimination occurred without changes in secondary protein structure, as demonstrated by circular dichroism spectroscopy. The lytic activity of lysozyme was restored to 73.4 ± 1.7%–92.5 ± 1.2% during PEG chain elimination. Attached PEG chains were eliminated within 24 h to 28 days, depending on the used linker molecule. When formulated into hydrogels, a maximum of about 60% of the initial dose was released within 7 days to 21 days. Linker elimination occurs ‘traceless’, so that the protein is released in its native, unmodified form. Altogether, we believe that tethering proteins by degradable carbamate linkers is a promising strategy to control their release from hydrogels.
Co-reporter:Susanne Kirchhof, Ferdinand P. Brandl, Nadine Hammer and Achim M. Goepferich
Journal of Materials Chemistry A 2013 vol. 1(Issue 37) pp:4855-4864
Publication Date(Web):29 Jul 2013
DOI:10.1039/C3TB20831A
The Diels–Alder (DA) reaction was investigated as a cross-linking mechanism for poly(ethylene glycol) (PEG) based hydrogels. Two complementary macromonomers were synthesized by functionalizing star-shaped PEG with furyl and maleimide groups. Gel formation occurred in water at 37 °C; the gelation time ranged between 171 ± 25 min and 14 ± 1 min depending on the used hydrogel formulation. The complex shear modulus was dependent on the concentration, branching factor and molecular weight of the macromonomers; values between 2821 ± 1479 Pa and 37097 ± 6698 Pa were observed. Hydrogel swelling and degradation were influenced by the same parameters; the degradation time varied between a few days and several weeks. Gel dissolution was found to occur by retro-DA reaction and subsequent hydrolysis of maleimide groups. Calculations of the network mesh size revealed that the prepared hydrogels would be suitable for the controlled release of therapeutic proteins.
Co-reporter:Matthias Ferstl;Markus Drechsler;Reinhard Rachel;Matthias Rischer;Jürgen Engel;Michael Backofen;Achim Goepferich
Journal of Pharmaceutical Sciences 2013 Volume 102( Issue 8) pp:2599-2607
Publication Date(Web):
DOI:10.1002/jps.23619
Abstract
We investigated how the structure of nanofibers, resulting from interactions between anionic polyelectrolytes and cationic peptides, relies on the properties of the polyelectrolyte component. By using hyaluronate (H), carboxymethylcellulose (CMC), xanthan (X), and ozarelix (O), a cationic decapeptide, we determined the influence of characteristic polyelectrolyte parameters such as size and charge density on the formation of polyelectrolyte–peptide complexes. Transmission electron microscopy of unstained, frozen hydrated, or negatively stained samples revealed that the interaction between different anionic polyelectrolytes and ozarelix led to the formation of distinctly shaped nanofibers. CMC formed rather flexible structures with alternating thin and thick segments within the nanofibers with diameters ranging from 10 to 16 nm and a length of up to 1 μm. Hyaluronate, a high-molecular-mass molecule, formed extra-long aggregates of more than 5 μm. Individual fibers with a diameter of 8 nm aggregated to bigger strands. The nonlinear polysaccharide xanthan gum led to highly coiled structures. The diameter of the respective nanofibers varied between 15 and 25 nm. Isothermal titration calorimetry was used to determine the binding constants and the thermodynamic parameters of the different polyelectrolyte–peptide complexes. The binding constant, which was of the order of 106 M−1, indicated a strong binding affinity, but also showed differences among the polyelectrolytes. These differences might be useful for prospective applications as drug delivery systems. © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 102:2599–2607, 2013
Co-reporter:Daniel Schweizer, Karin Schönhammer, Michael Jahn, and Achim Göpferich
Biomacromolecules 2013 Volume 14(Issue 1) pp:
Publication Date(Web):November 19, 2012
DOI:10.1021/bm301352x
The objective of the present study was to investigate ionic interactions between alginate and a monoclonal antibody (mAb1) and to utilize those interactions for the sustained release of mAb1. The existence of ionic interactions between alginate and mAb1 was strongly reflected by their rheological behavior. A 3–4 times increase in storage modulus (G′) was observed by addition of 30 mg/mL mAb1 to a 20 mg/mL alginate solution. This increase was strongly dependent on pH and ionic strength. In vitro release studies revealed a marked pH-dependence of release rates and the reversibility of alginate-mAb1 complexation under physiological conditions. Two alginate-mAb1 sustained release formulations were developed by an internal gelation technique using CaCO3 and CaHPO4 as calcium sources for physical cross-linking. The CaCO3 formulation provided a stable pH-environment, optimally suited for pH-sensitive proteins. CaHPO4 led to a lower pH and stronger alginate-mAb1 interactions. The CaHPO4 cross-linked alginate released mAb1 over a period of 10–15 days. The long release period and changes in viscoelastic properties of alginate, when being mixed with mAb1, indicate the incorporation of mAb1 molecules into a mixed network with alginate. The results of this study demonstrate that ionic interactions between polyanions and mAb1 are present and that they can be exploited for sustained release delivery of mAb1.
Co-reporter:Robert Hennig;Klaus Pollinger;Joerg Tessmar;Rebecca Wenzel;Miriam Breunig;Achim Goepferich;Rudolf Fuchshofer;Ernst R. Tamm;Andreas Ohlmann
PNAS 2013 Volume 110 (Issue 15 ) pp:6115-6120
Publication Date(Web):2013-04-09
DOI:10.1073/pnas.1220281110
To date, diseases affecting vascular structures in the posterior eye are mostly treated by laser photocoagulation and multiple
intraocular injections, procedures that destroy healthy tissue and can cause vision-threatening complications. To overcome
these drawbacks, we investigate the feasibility of receptor-mediated nanoparticle targeting to capillary endothelial cells
in the retina after i.v. application. Cell-binding studies using microvascular endothelial cells showed receptor-specific
binding and cellular uptake of cyclo(RGDfC)-modified quantum dots via the αvβ3 integrin receptor. Conversely, Mueller cells
and astrocytes, representing off-target cells located in the retina, revealed only negligible interaction with nanoparticles.
In vivo experiments, using nude mice as the model organism, demonstrated a strong binding of the ligand-modified quantum dots
in the choriocapillaris and intraretinal capillaries upon i.v. injection and 1-h circulation time. Nontargeted nanoparticles,
in contrast, did not accumulate to a significant amount in the target tissue. The presented strategy of targeting integrin
receptors in the retina could be of utmost value for future intervention in pathologies of the posterior eye, which are to
date only accessible with difficulty.
Co-reporter:Tobias Miller;Reinhard Rachel;Ahmed Besheer;Senta Uezguen
Pharmaceutical Research 2012 Volume 29( Issue 2) pp:448-459
Publication Date(Web):2012 February
DOI:10.1007/s11095-011-0555-x
Stability of polymeric micelles upon injection is essential for a drug delivery system but is not fully understood. We optimized an analytical test allowing quantification of micellar stability in biofluids and applied it to a variety of block copolymer micelles with different hydrophobic block architechtures.Polymeric micelles were prepared from four different polymers and investigated via encapsulation of two fluorescent dyes. Samples were incubated in human serum; changes in Foerster Resonance Energy Transfer (FRET) were recorded as a function of time. This fluorescence-based approach was supported semi-quantitatively by results from Asymmetrical Flow Field-Flow-Fractionation (AF4).After incubation experiments, micellar stability was determined by calculation of two stability-indicating parameters: residual micellar fractions (RMFs) and in vitro serum half-lives. Both parameters showed that PEG-PVPy micelles rapidly destabilized after 3 h (RMF < 45%), whereas PEG-PLA, PEG-PLGA and PEG-PCL micelles were far more stable (RMFs 65 to 98%).This FRET-based assay is a valuable tool in evaluating and screening serum stability of polymeric micelles and revealed low serum stability of PEG-PVPy micelles compared to polyester-based micelles.
Co-reporter:Shaaban K. Osman, Ferdinand P. Brandl, Gamal M. Zayed, Jörg K. Teßmar, Achim M. Göpferich
Polymer 2011 Volume 52(Issue 21) pp:4806-4812
Publication Date(Web):29 September 2011
DOI:10.1016/j.polymer.2011.07.059
Inclusion complex formation between β-cyclodextrin (β-CD) and suitable guest molecules has frequently been exploited to design self-assembled polymer networks. In this paper, we report on hydrogels composed of eight-armed poly(ethylene glycol) (PEG) grafted with β-CD (8armPEG20k-CD), adamantane (8armPEG20k-ad) or cholesterol (8armPEG20k-chol). Mixtures of 8armPEG20k-CD and 8armPEG20k-ad showed viscous behavior (G″ > G′); 8armPEG20k-CD and 8armPEG20k-chol formed elastic hydrogels (G′ > G″). To study the interaction of adamantane and cholesterol grafted PEG with β-CD, linear model compounds (mPEG5k-ad and mPEG5k-chol) were synthesized. Isothermal titration calorimetry (ITC) experiments showed a higher association constant for inclusion complexes formed with mPEG5k-chol (47,000 ± 1650) than for those formed with mPEG5k-ad (30,000 ± 900). Fluorescence spectroscopy and dynamic light scattering (DLS) measurements further showed the ability of mPEG5k-chol to self-assemble into micelles. Self-assembly of 8armPEG20k-chol further increased the strength of the formed hydrogels. Altogether, this study contributes to a better understanding of cyclodextrin based biomaterials.
Co-reporter:Matthias Ferstl, Andrea Strasser, Hans-Joachim Wittmann, Markus Drechsler, Matthias Rischer, Jürgen Engel, and Achim Goepferich
Langmuir 2011 Volume 27(Issue 23) pp:14450-14459
Publication Date(Web):October 17, 2011
DOI:10.1021/la202252m
We investigated whether cationic peptides that contain hydrophobic side chains were able to stabilize themselves via hydrophobic interactions between neighboring peptide molecules upon electrostatic binding to oppositely charged polyelectrolytes. The interaction mechanism was examined through a model system consisting of the anionic polyelectrolyte alginate and the cationic decapeptide ozarelix. The interaction resulted in the formation of highly ordered complexes that were noticeable upon visual inspection. These complexes were then investigated by microscopic techniques and shown to exhibit a branched network structure. Cryogenic-temperature transmission electron microscopy (cryo-TEM) and negative staining TEM revealed that the molecular interactions between alginate and ozarelix led to the formation of nanofibers. The rodlike nanofibers had a diameter distribution of 4–8 nm. Isothermal titration calorimetry was used to determine the thermodynamic parameters of the alginate–ozarelix interaction. The binding constant was found to be on the order of 106 M–1, indicating a high binding affinity. The interaction of the peptide with the polyelectrolyte triggered profound changes in the conformation of ozarelix, which was confirmed by UV spectroscopy and circular dichroism. On the basis of these experimental results, a theoretical modeling study of the alginate–ozarelix interaction was conducted to gain a better molecular-level understanding of the complex structure. It revealed that, upon binding of ozarelix to alginate, new intermolecular and intramolecular aromatic interactions between the ozarelix molecules occurred. These interactions changed the conformation of the peptide, a modification in which the aromatic side chains played a major role. Our results indicate that the cationic peptides interact with the polyanions via electrostatic interactions, but are additionally stabilized via hydrophobic interactions. This binding mode may serve as a powerful tool to extend the duration of drug release in hydrogel drug delivery systems.
Co-reporter:A. Zaky, A. Elbakry, A. Ehmer, M. Breunig, A. Goepferich
Journal of Controlled Release 2010 Volume 147(Issue 2) pp:202-210
Publication Date(Web):15 October 2010
DOI:10.1016/j.jconrel.2010.07.110
The purpose of this study was to reveal factors that have an impact on the protein release kinetics from triglyceride microspheres prepared by spray congealing. We investigated the effect of protein particle size, morphology and distribution on protein release from microspheres by confocal laser scanning microscopy (CLSM). The microspheres were loaded with three types of model particles made of FITC-labeled bovine serum albumin: freeze dried protein, spherical particles obtained by precipitation in the presence of PEG and micronized material. Investigation by light microscopy and laser light diffraction revealed that the freeze dried material consisted mainly of app. 29 μm elongated shaped particles. The precipitated BSA consisted mainly of 9.0 μm diameter spherically shaped particles while the micronized protein prepared by jet milling consisted of 4.9 μm sized rounded particles of high uniformity. Microspheres were embedded into a cold-curing resin and cut with a microtome. Subsequent investigation by CLSM revealed major differences of distribution of the polydisperse protein particles inside the microsphere sections depending on the type of BSA that was used. Particles of micronized and precipitated protein were distributed almost throughout the microsphere cross section. The protein distribution had a marked impact on the release kinetics in phosphate buffer. Large protein particles led to a considerably faster release than small ones. By staining the release medium we demonstrated that in all three cases there was a strong correlation between protein release and buffer intrusion.Scanning confocal microscopy images of microtome sections reveal that the release of BSA (FITC-labeled, green) from lipid microspheres and the intrusion of water (Sulforhodamine stained, red) are correlated.
Co-reporter:Ferdinand Brandl, Fritz Kastner, Ruth M. Gschwind, Torsten Blunk, Jörg Teßmar, Achim Göpferich
Journal of Controlled Release 2010 Volume 142(Issue 2) pp:221-228
Publication Date(Web):3 March 2010
DOI:10.1016/j.jconrel.2009.10.030
Hydrogels are extensively studied as matrices for the controlled release of macromolecules. To evaluate the mobility of embedded molecules, these drug delivery systems are usually characterized by release studies. However, these experiments are time-consuming and their reliability is often poor. In this study, gels were prepared by step-growth polymerization of poly(ethylene glycol) (PEG) and loaded with fluoresceine isothiocyanate (FITC) labeled dextrans. Mechanical testing and swelling studies allowed prediction of the expected FITC-dextran diffusivity. The translational diffusion coefficients (D) of the incorporated FITC-dextrans were measured by fluorescence recovery after photobleaching (FRAP) and pulsed field gradient NMR spectroscopy. Because the determined values of D agreed well with those obtained from release studies, mechanical testing, FRAP, and pulsed field gradient NMR spectroscopy are proposed as alternatives to release experiments. The applied methods complemented each other and represented the relative differences between the tested samples correctly. Measuring D can therefore be used to rapidly evaluate the potential of newly developed drug delivery systems.Correlation of diffusion coefficients (rapidly determined by mechanical testing, fluorescence recovery after photobleaching, and NMR spectroscopy) and long-term release profiles of hydrogel-based drug delivery systems.
Co-reporter:Karin Schoenhammer;Julie Boisclair;Helmut Schuetz;Holger Petersen;Achim Goepferich
Journal of Pharmaceutical Sciences 2010 Volume 99( Issue 10) pp:4390-4399
Publication Date(Web):
DOI:10.1002/jps.22149
Abstract
Poly(ethyleneglycol) 500 dimethylether (PEG500DME) was tested as a novel solvent for the manufacture of an injectable in situ forming depot (ISFD) containing poly(D,L-lactide-co-glycolide) (PLGA). The sustained release of pasireotide from the ISFD was evaluated in vitro and in vivo. Furthermore, the local tolerability of the delivery system using PEG500DME was investigated in subcutaneous (s.c.) tissue over 48 days. A flow-through cell was used to determine the in vitro drug release from the ISFD in comparison to a peptide suspension without polymer. The biocompatibility as well as the pharmacokinetic profile of the ISFD was investigated in rabbits. A prolonged peptide release over at least 48 days with an initial burst lower than 1% was observed in vitro for the ISFD compared to the suspension without polymer. A similar tissue response as it was observed for other common PLGA delivery systems was found upon histopathological examination of tissue from the administration site in rabbits. A sustained release of at least 48 days in vivo confirmed the in vitro observation including the low initial plasma concentration levels. Two ISFDs with different peptide loads were used to correlate the in vitro and in vivo data (IVIVC). Overall, the functionality of the ISFD containing PEG500DME as a novel solvent was demonstrated in vitro and in vivo. In addition, the local tolerability of the system confirmed the biocompatibility of PEG500DME in parenteral depots. © 2010 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 99:4390–4399, 2010
Co-reporter:Ferdinand Brandl, Nadine Hammer, Torsten Blunk, Joerg Tessmar and Achim Goepferich
Biomacromolecules 2010 Volume 11(Issue 2) pp:
Publication Date(Web):January 22, 2010
DOI:10.1021/bm901235g
Tethering drug substances to a gel network is an effective way of controlling the release kinetics of hydrogel-based drug delivery systems. Here, we report on in situ forming, biodegradable hydrogels that allow for the covalent attachment of peptides or proteins. Hydrogels were prepared by step-growth polymerization of branched poly(ethylene glycol). The gel strength ranged from 1075 to 2435 Pa; the degradation time varied between 24 and 120 h. Fluorescence recovery after photobleaching showed that fluorescently labeled bovine serum albumin (FITC-BSA) was successfully bound to the gel network during gel formation. Within 168 h, the mobility of the tethered molecules gradually increased due to polymer degradation. Using FITC-BSA and lysozyme as model proteins, we showed the potential of the developed hydrogels for time-controlled release. The obtained release profiles had a sigmoidal shape and matched the degradation profile very well; protein release was complete after 96 h.
Co-reporter:Ferdinand P. Brandl, Anna K. Seitz, Jörg K.V. Teßmar, Torsten Blunk, Achim M. Göpferich
Biomaterials 2010 31(14) pp: 3957-3966
Publication Date(Web):
DOI:10.1016/j.biomaterials.2010.01.128
Co-reporter:Wolfgang Hild;Klaus Pollinger;Chiara Cabrele;Andrea Caporale;Max Keller;Nicola Pluym;Armin Buschauer;Reinhard Rachel;Joerg Tessmar;Miriam Breunig;Achim Goepferich
PNAS 2010 Volume 107 (Issue 23 ) pp:10667-10672
Publication Date(Web):2010-06-08
DOI:10.1073/pnas.0912782107
More selective interactions of nanoparticles with cells would substantially increase their potential for diagnostic and therapeutic
applications. Thus, it would not only be highly desirable that nanoparticles can be addressed to any cell with high target
specificity and affinity, but that we could unequivocally define whether they rest immobilized on the cell surface as a diagnostic
tag, or if they are internalized to serve as a delivery vehicle for drugs. To date no class of targets is known that would
allow direction of nanoparticle interactions with cells alternatively into one of these mutually exclusive events. Using MCF-7
breast cancer cells expressing the human Y1-receptor, we demonstrate that G protein-coupled receptors provide us with this option. We show that quantum dots carrying
a surface-immobilized antagonist remain with nanomolar affinity on the cell surface, and particles carrying an agonist are
internalized upon receptor binding. The receptor functions like a logic “and-gate” that grants cell access only to those particles
that carry a receptor ligand “and” where the ligand is an agonist. We found that agonist- and antagonist-modified nanoparticles
bind to several receptor molecules at a time. This multiligand binding leads to five orders of magnitude increased-receptor
affinities, compared with free ligand, in displacement studies. More than 800 G protein-coupled receptors in humans provide
us with the paramount advantage that targeting of a plethora of cells is possible, and that switching from cell recognition
to cell uptake is simply a matter of nanoparticle surface modification with the appropriate choice of ligand type.
Co-reporter:Sonja Bauhuber;Constantin Hozsa;Miriam Breunig ;Achim Göpferich
Advanced Materials 2009 Volume 21( Issue 32-33) pp:3286-3306
Publication Date(Web):
DOI:10.1002/adma.200802453
Abstract
Nucleic acids are not only expected to assume a pivotal position as “drugs” in the treatment of genetic and acquired diseases, but could also act as molecular cues to control the microenvironment during tissue regeneration. Despite this promise, the efficient delivery of nucleic acids to their side of action is still the major hurdle. One among many prerequisites for a successful carrier system for nucleic acids is high stability in the extracellular environment, accompanied by an efficient release of the cargo in the intracellular compartment. A promising strategy to create such an interactive delivery system is to exploit the redox gradient between the extra- and intracellular compartments. In this review, emphasis is placed on the biological rationale for the synthesis of redox sensitive, disulfide-based carrier systems, as well as the extra- and intracellular processing of macromolecules containing disulfide bonds. Moreover, the basic synthetic approaches for introducing disulfide bonds into carrier molecules, together with examples that demonstrate the benefit of disulfides at the individual stages of nucleic acid delivery, will be presented.
Co-reporter:Asmaa Elbakry, Alaa Zaky, Renate Liebl, Reinhard Rachel, Achim Goepferich and Miriam Breunig
Nano Letters 2009 Volume 9(Issue 5) pp:2059-2064
Publication Date(Web):March 30, 2009
DOI:10.1021/nl9003865
Although uptake into cells is highly complex and regulated, heterogeneous particle collectives are usually employed to deliver small interfering RNA (siRNA) to cells. Within these collectives, it is difficult to accurately identify the active species, and a decrease in efficacy is inherent to such preparations. Here, we demonstrate the manufacture of uniform nanoparticles with the deposition of siRNA on gold in a layer-by-layer approach, and we further report on the cellular delivery and siRNA activity as functions of surface properties.
Co-reporter:Karin Schoenhammer;Holger Petersen;Frank Guethlein
Pharmaceutical Research 2009 Volume 26( Issue 12) pp:
Publication Date(Web):2009 December
DOI:10.1007/s11095-009-9969-0
Poly(D,L-lactide-co-glycolide) (PLGA) solutions in poly(ethyleneglycol)600 (PEG600), N-methyl-2-pyrrolidone (NMP) and poly(ethyleneglycol)500dimethylether (PEG500DME) as a novel solvent, were investigated as suitable for use in injectable in situ forming depots (ISFD).The hemolytic potential of the solvents was investigated. Viscosimetry was used to determine rheological properties of solvents and PLGA solutions. DSC was used to evaluate the stability of the PLGA solutions through investigation of the melting behavior of semicrystalline PEGs which depended on tempering and glass transition temperature of the PLGA. Phase separation was studied to determine ternary phase diagrams. In vitro release kinetics of the solvents and the surrogate methylene blue were investigated.Significantly less hemolysis was observed for PEG500DME compared to PEG600 and NMP. Newtonian fluid properties were found for all polymer solutions. A melting point depression of the solvents was detected in presence of PLGA. The duration of tempering of the polymer solutions showed no impact on their melting behavior. The initial in vitro release of methylene blue was according to the solvent diffusion kinetics.Low hemolytic potential, suitable viscosity for injection, stability of PLGA solutions in PEG500DME and the correlation between phase separation and in vitro release confirmed the potential of PEG500DME as a promising solvent for ISFD.
Co-reporter:Miriam Breunig, Constantin Hozsa, Uta Lungwitz, Kazuo Watanabe, Isao Umeda, Hiroyuki Kato, Achim Goepferich
Journal of Controlled Release 2008 Volume 130(Issue 1) pp:57-63
Publication Date(Web):25 August 2008
DOI:10.1016/j.jconrel.2008.05.016
Poly(ethylene imine) (PEI) has gained increasing attention in the delivery of small interfering RNAs (siRNAs) into cells. In order to further optimize PEI for this application, the first goal of this study was to examine particular steps of siRNA delivery with various PEI derivatives as carriers. Furthermore, the hypothesis that disulfide cleavable carrier systems are favorable for the release of siRNA into the cell cytoplasm was investigated. Flow cytometry and confocal microscopy were used to assess the cellular uptake and intracellular distribution of siRNA, which were then related to gene silencing efficacy. We observed a strong correlation between cellular uptake and RNAi activity. The cellular uptake of siRNA was more efficient with increasing branching of the polymer, i.e. linear PEI (lPEI) 5 kDa < lPEI cross-linked via disulfide bonds (ssPEI) < branched PEI (bPEI) 25 kDa. However, it was also evident that the siRNA release from the carrier, which was promoted by ssPEI, played an important role in the accessibility of siRNA for the gene silencing complex. Therefore, we suggest that a combination of a high branching density and reductively cleavable bonds within the PEI-based carrier system could be one possible step towards improving siRNA delivery.
Co-reporter:Florian Sommer;Klaus Pollinger;Barbara Weiser;Ferdinand Brandl;Jörg Teßmar;Torsten Blunk;Achim Göpferich
Graefe's Archive for Clinical and Experimental Ophthalmology 2008 Volume 246( Issue 9) pp:1275-1284
Publication Date(Web):2008/09/01
DOI:10.1007/s00417-008-0846-z
In cases of severe retinal diseases, the vitreous body has to be removed and replaced by a suitable biomaterial. Currently, however, no satisfying long-term vitreous substitute is in clinical use. A novel therapeutic concept represents the combination of hyalocytes with suitable biomaterials. The goal of the present study was to evaluate the potential of bFGF and TGF-β1 as tools to control hyalocyte proliferation and the accumulation of extracellular matrix (ECM).In vitro investigation on the influence of different concentrations of bFGF and TGF-β1 on hyalocyte morphology, proliferation and ECM production.Both growth factors affected hyalocyte morphology; small, round cells could be observed after bFGF supplementation, whereas the cells appeared more completely spread when cultured with TGF-β1. Hyalocyte proliferation was increased 3-fold by 10 ng/ml bFGF; 1 ng/ml TGF-β1 in contrast reduced cell proliferation to about 40% of the control. Converse effects of the growth factors could also be observed on the ECM accumulation of hyalocytes; whereas bFGF halved ECM accumulation, TGF-β1 enhanced the ECM production up to 3-fold. Precultivation of hyalocytes with bFGF for two passages had no influence on their subsequent accumulation of glycosaminoglycans (GAG). However, cells precultivated with bFGF exhibited a doubled accumulation of collagen compared to controls.The observed opposite effects of bFGF and TGF-β1 on hyalocyte proliferation and ECM accumulation may allow for the control of hyaloycte properties. Therefore, these two growth factors seem to be valuable tools towards the development of a cell-based vitreous substitute.
Co-reporter:S. Koennings, A. Sapin, T. Blunk, P. Menei, A. Goepferich
Journal of Controlled Release 2007 Volume 119(Issue 2) pp:163-172
Publication Date(Web):1 June 2007
DOI:10.1016/j.jconrel.2007.02.005
It was the aim of this study to establish triglyceride matrices as potential carriers for long-term release of brain-derived neurotrophic factor (BDNF), a potential therapeutic for Huntington's disease. First, four different manufacturing strategies were investigated with lysozyme as a model substance: either lyophilized protein was mixed with lipid powder, or suspended in organic solution thereof (s/o). Or else, an aqueous protein solution was dispersed by w/o emulsion in organic lipid solution. Alternatively, a PEG co-lyophilization was performed prior to dispersing solid protein microparticles in organic lipid solution. After removal of the solvent(s), the resulting powder formulations were compressed at 250 N to form mini-cylinders of 2 mm diameter, 2.2 mm height and 7 mg weight. Protein integrity after formulation and release was evaluated from an enzyme activity assay and SDS-PAGE. Confocal microscopy revealed that the resulting distribution of FITC-lysozyme within the matrices depended strongly on the manufacturing method, which had an important impact on matrix performance: matrices with a very fine and homogeneous protein distribution (PEG co-lyophilization) continually released protein for 2 months. The other methods did not guarantee a homogeneous distribution and either failed in sustaining release for more than 1 week (powder mixture), completely liberating the loading (s/o dispersion) or preserving protein activity during manufacturing (w/o emulsion, formation of aggregates and 25% activity loss). Based on these results, miniature-sized implants of 1 mm diameter, 0.8 mm height and 1 mg weight were successfully loaded by the PEG co-lyophilization method with 2% BDNF and 2% PEG. Release studies in phosphate buffer pH 7.4 at 4 and 37 °C revealed a controlled release of either 20 or 60% intact protein over one month as determined by ELISA. SDS-PAGE detected only minor aggregates in the matrix during release at higher temperature. In vivo evaluation of lipid cylinders in the striatum of rat brains revealed a biocompatibility comparable to silicone reference cylinders.
Co-reporter:S. Koennings, A. Berié, J. Tessmar, T. Blunk, A. Goepferich
Journal of Controlled Release 2007 Volume 119(Issue 2) pp:173-181
Publication Date(Web):1 June 2007
DOI:10.1016/j.jconrel.2007.02.008
The aim of this study was to investigate the role of matrix and drug properties on controlled release from triglyceride matrices. Mini-cylinders of 2 mm diameter, 2.2 mm height and 7 mg weight were produced by compression of lipid powder obtained by using a polyethylene glycol (PEG) co-lyophilization method for the model substances lysozyme and FITC-dextran (Mw 4000 Da). Lysozyme was released with decreasing velocity from glyceryl trilaurate, -myristate, -palmitate and -stearate for more than 14 months. Release correlated well with triglyceride lipophilicity defined by the chain length of the fatty acids. Contact angle measurements and the analysis of buffer penetration visualized by confocal microscopy emphasized the role of matrix wettability as a prerequisite for release. A comparison with FITC-dextran revealed that the protein itself enhances matrix wettability and hence its release due to its surface active properties. FITC-dextran remained trapped within the matrix only to be released at lower compression force or after the addition of surfactant. Protein added externally to the release buffer at 0.1% (w/v) was not efficient in lowering the contact angle and increasing the release rate of FITC-dextran. Tween®20 and 81 could be used in different concentrations (0.1, 0.01 and 0.001% (w/v)) to alter lysozyme and FITC-dextran release profiles: resulting release rates showed a close dependence on the contact angle of the respective release medium and triglyceride matrix material. However, both Tweens® seem to act not only by reducing the release medium contact angle but also by moderately affecting interparticulate adhesion of the matrix material.
Co-reporter:F. Brl;M. Henke;S. Rothschenk;R. Gschwind;M. Breunig;T. Blunk;J. Tessmar;A. Göpferich
Advanced Engineering Materials 2007 Volume 9(Issue 12) pp:
Publication Date(Web):19 DEC 2007
DOI:10.1002/adem.200700221
Hydrogels are attractive materials for biomedical applications due to their versatility and excellent biocompatibility. In this study, we report the preparation of poly(ethylene glycol) (PEG) based hydrogels for intraocular applications. We synthesized branched PEG-succinimidyl propionates (10 kDa molecular weight) and different types of PEG-amines (linear and branched, 2 and 10 kDa molecular weight). Transparent hydrogels were formed in situ upon chemical reaction of these macromers. The gels were characterized by oscillatory rheometry and NMR experiments. By varying the concentration of macromers, the functionality of the PEG-amine, and the conditions during cross-linking, gels with adequate gelation times of approx 5–10 min and gel strengths of approx 350–1500 Pa were obtained. The cross-linked hydrogels showed no cytotoxic effects and may be used as vitreous substitutes or intraocular drug release systems.
Co-reporter:Miriam Breunig;Uta Lungwitz;Renate Liebl;Achim Goepferich;
Proceedings of the National Academy of Sciences 2007 104(36) pp:14454-14459
Publication Date(Web):August 28, 2007
DOI:10.1073/pnas.0703882104
Nonviral nucleic acid delivery to cells and tissues is considered a standard tool in life science research. However, although
an ideal delivery system should have high efficacy and minimal toxicity, existing materials fall short, most of them being
either too toxic or little effective. We hypothesized that disulfide cross-linked low-molecular-weight (MW) linear poly(ethylene
imine) (MW <4.6 kDa) would overcome this limitation. Investigations with these materials revealed that the extracellular high
MW provided outstandingly high transfection efficacies (up to 69.62 ± 4.18% in HEK cells). We confirmed that the intracellular
reductive degradation produced mainly nontoxic fragments (cell survival 98.69 ± 4.79%). When we compared the polymers in >1,400
individual experiments to seven commercial transfection reagents in seven different cell lines, we found highly superior transfection
efficacies and substantially lower toxicities. This renders reductive degradation a highly promising tool for the design of
new transfection materials.
Co-reporter:Stephanie Koennings;Joerg Tessmar;Torsten Blunk
Pharmaceutical Research 2007 Volume 24( Issue 7) pp:1325-1335
Publication Date(Web):2007 July
DOI:10.1007/s11095-007-9258-8
It was the aim of this study to identify the governing mechanisms during protein release from cylindrical lipid matrices by visualizing mass transport and correlating the data with in vitro dissolution testing.Glyceryl trimyristate cylinders of 2 mm diameter, 2.2 mm height and 7 mg weight were manufactured by compression of a protein–lipid powder mixture prepared by a polyethylene glycol (PEG) co-lyophilization technique. BSA was fluorescence-labeled and the distribution visualized and quantified at different stages of the release process by confocal microscopy in parallel to the quantification in the release buffer. The impact of matrix loading and protein molecular weight was assessed with the model proteins lysozyme, BSA, alcohol dehydrogenase and thyroglobulin.Buffer penetration and protein release occurred simultaneously from the outer regions of the cylinder progressing towards the center. Release from the top and bottom of the matrix was not negligible but much slower than penetration from the side, probably due to an oriented arrangement of lipid flakes during compression. The different quantification strategies were found to yield identical results. At 6% protein loading, buffer penetration was complete after 4 days, while only 60% of the protein was liberated in that time and release continued up to day 63. Protein release kinetics could be described using the power law equation Mt /M∞= ktn with an average time exponent n of 0.45 (±0.04) for loadings varying between 1 and 8%. A percolation threshold at 5% pure protein loading and 3–4% mixed loading (PEG and protein at a 1:1 mass ratio) could be identified. Release rate was found to decrease with increasing molecular weight.Protein release from lipid-based matrices is a purely diffusion controlled mechanism. Potential protein stabilization approaches should address the time span between complete buffer penetration of the matrix and 100% release of the remaining loading, which would be exposed to an aqueous environment before leaving the matrix.
Co-reporter:Robert Knerr;Barbara Weiser;Achim Göpferich;Claudia Steinem;Sigrid Drotleff
Macromolecular Bioscience 2006 Volume 6(Issue 10) pp:827-838
Publication Date(Web):13 OCT 2006
DOI:10.1002/mabi.200600106
Summary: In this study, the suitability of a flow-through quartz crystal microbalance system for the detection of the adhesion of rMSCs and 3T3-L1 fibroblasts on different surfaces is demonstrated. Frequency shifts for rMSCs of −6.7 mHz · cell−1 and −2.0 mHz · cell−1 for 3T3-L1 cells could be detected on non-modified gold sensors, revealing that the frequency shift per cell is comparable to that of a static setup. Modifying the sensor surface with SAMs of thioalkylated ω-amine-terminated PEG derivatives led to cell-adhesion-resistant surfaces. Total frequency shifts of only −20 ± 7 Hz showed that protein adsorption was also significantly reduced. Attaching 35 pmol · mm−2 of the GRGDS cell adhesion motif to the SAMs induced specific cell adhesion due to RGD-integrin interactions; the resonance frequency dropped by 3.4 mHz · cell−1. Furthermore, the kinetics of cell detachment could be determined. The corresponding processes were completed after 10 min for trypsin, and not before 90 min with GRGDS. Moreover, the detectability of cell adhesion was shown to increase after the addition of manganese cations. The total decrease in the resonance frequency was almost 80 Hz in the presence of Mn2+ (6.4 mHz · cell−1).
Co-reporter:Mohamed Abbas Ibrahim, Ahmed Ismail, Mohamed Ihab Fetouh, Achim Göpferich
Journal of Controlled Release 2005 Volume 106(Issue 3) pp:241-252
Publication Date(Web):2 September 2005
DOI:10.1016/j.jconrel.2005.02.025
In recent years, the acylation of peptides during the erosion of poly(lactic acid) and poly(lactic-co-glycolic acid) microspheres has been described in the literature. To investigate whether insulin is prone to the covalent attachment of lactic or glycolic acid, insulin-loaded PLA and PLGA microspheres containing 5% bovine insulin were manufactured using a w/o/w multiple emulsion-solvent evaporation technique. Microspheres were characterized for their insulin encapsulation efficiency and release characteristics in phosphate-buffered saline (PBS) at pH 7.4 and 37 °C. Moreover, the stability of the peptide during 18 days of release was evaluated using HPLC and HPLC-MS techniques. The results showed that the insulin loading efficiencies of PLA and PLGA microspheres were 75.18% and 79.63%, respectively. The microspheres were spherical with relatively porous surfaces with an average diameter of 40 and 53 μm, respectively. Insulin release from the microspheres was characterized by an initial burst, which was attributed to the amount of protein located on or close to the microsphere surface. The total ion chromatogram (TIC) of insulin samples extracted after 18 days of erosion in phosphate buffer pH 7.4 at 37 °C revealed that deamidation was the major mechanism of instability. Surprisingly, no acylation products were found. Control experiments in concentrated lactic acid solutions confirmed a minimal reactivity of the peptide under these conditions.
Co-reporter:E. Lieb, M. Hacker, J. Tessmar, L.A. Kunz-Schughart, J. Fiedler, C. Dahmen, U. Hersel, H. Kessler, M.B. Schulz, A. Göpferich
Biomaterials 2005 Volume 26(Issue 15) pp:2333-2341
Publication Date(Web):May 2005
DOI:10.1016/j.biomaterials.2004.07.010
One promising strategy to control the interactions between biomaterial surfaces and attaching cells involves the covalent grafting of adhesion peptides to polymers on which protein adsorption, which mediates unspecific cell adhesion, is essentially suppressed. This study demonstrates a surface modification concept for the covalent anchoring of RGD peptides to reactive diblock copolymers based on monoamine poly(ethylene glycol)-block-poly(D,L-lactic acid) (H2N-PEG-PLA). Films of both the amine-reactive (ST-NH-PEG2PLA20) and the thiol-reactive derivative (MP-NH-PEG2PLA40) were modified with cyclic αvβ3/αvβ5 integrin subtype specific RGD peptides simply by incubation of the films with buffered solutions of the peptides. Human osteoblasts known to express these integrins were used to determine cell–polymer interactions. The adhesion experiments revealed significantly increased cell numbers and cell spreading on the RGD-modified surfaces mediated by RGD–integrin-interactions.
Co-reporter:Helmut Reithmeier, Joachim Herrmann, Achim Göpferich
Journal of Controlled Release 2001 Volume 73(2–3) pp:339-350
Publication Date(Web):15 June 2001
DOI:10.1016/S0168-3659(01)00354-6
To investigate the potential of physiological lipids as an alternative to synthetic polymeric materials such as poly(lactide–co-glycolide), peptide-containing glyceryl tripalmitate microparticles were prepared. A modified solvent evaporation method and a melt dispersion technique without the use of organic solvent were employed. Thymocartin (TP-4), an immunomodulating tetrapeptide and insulin were chosen as model peptides and incorporated as a solid or dissolved in 100 μl aqueous solution. The resulting microparticles were characterized with respect to particle size and morphology, biocompatibility, drug content (encapsulation efficiency) and in vitro release behavior. Electron spectroscopy for chemical analysis was used to investigate the adsorption of the model peptides to the lipid matrix material. The modified solvent evaporation as well as the melt dispersion method were suitable for the preparation of microparticles in the size range of 20–150 μm with an acceptable yield. The biocompatibility of the glyceryl tripalmitate microparticles after implantation into NMRI-mice was comparable to poly(lactide–co-glycolide) microparticles. The encapsulation efficiency for both model peptides was high (>80%) even at high theoretical loadings when the peptide was incorporated as a solution with the melt dispersion technique. The in vitro release behavior was substantially influenced by the physicochemical properties of the model peptides used in this study.
Co-reporter:W Vogelhuber, P Rotunno, E Magni, A Gazzaniga, T Spruß, G Bernhardt, A Buschauer, A Göpferich
Journal of Controlled Release 2001 Volume 73(Issue 1) pp:75-88
Publication Date(Web):18 May 2001
DOI:10.1016/S0168-3659(01)00282-6
Pulsatile release implants were developed that release substances up to 58 days post implantation. With a cylindrical size of 2 mm diameter and 1.8 mm height the matrices can carry as much as 1 mg of drug and allow even for intracranial implantation into a rodent model. The matrices are made of materials that have been used for parenteral applications in humans before such as surface eroding polyanhydrides and bulk eroding poly(d,l-lactic acid) or poly(d,l-lactic acid–co-glycolic acid). The onset of drug release is controlled by the degradation of bulk eroding polymers which are known to exhibit a certain stability over a defined period of time and which start eroding after they reach a critical degree of degradation. The time of drug release onset was found to depend on the molecular weight and the chemical state of the carboxylic acid end of the polymer chain. For testing the onset of release in vivo a nude mouse model was developed where the release of Evan’s blue could be observed visually after subcutaneous application. By combining individual matrices with different release onset, a therapeutic system can be composed that releases drugs after implantation at predetermined time points in a preprogrammed way. Potential applications for such matrices is vaccination and local tumor therapy.
Co-reporter:Claudia Fischbach, Jörg Tessmar, Andrea Lucke, Edith Schnell, Georg Schmeer, Torsten Blunk, Achim Göpferich
Surface Science 2001 Volume 491(Issue 3) pp:333-345
Publication Date(Web):1 October 2001
DOI:10.1016/S0039-6028(01)01297-3
For most tissue engineering approaches aiming at the repair or generation of living tissues the interaction of cells and polymeric biomaterials is of paramount importance. Prior to contact with cells or tissues, biomaterials have to be sterilized. However, many sterilization procedures such as steam autoclave or heat sterilization are known to strongly affect polymer properties. UV irradiation is used as an alternative sterilization method in many tissue engineering laboratories on a routine basis, however, potential alterations of polymer properties have not been extensively considered.In this study we investigated the effects of UV irradiation on spin-cast films made from biodegradable poly(d,l-lactic acid)–poly(ethylene glycol)–monomethyl ether diblock copolymers (Me.PEG–PLA) which have recently been developed for controlled cell-biomaterial interaction. After 2 h of UV irradiation, which is sufficient for sterilization, no alterations in cell adhesion to polymer films were detected, as demonstrated with 3T3-L1 preadipocytes. This correlated with unchanged film topography and molecular weight distribution. However, extended UV irradiation for 5–24 h elicited drastic responses regarding Me.PEG–PLA polymer properties and interactions with biological elements: Large increases in unspecific protein adsorption and subsequent cell adhesion were observed. Changes in polymer surface properties could be correlated with the observed alterations in cell/protein–polymer interactions. Atomic force microscopy analysis of polymer films revealed a marked “smoothing” of the polymer surface after UV irradiation. Investigations using GPC, -NMR, mass spectrometry, and a PEG-specific colorimetric assay demonstrated that polymer film composition was time-dependently affected by exposure to UV irradiation, i.e., that large amounts of PEG were lost from the copolymer surface.The data indicate that sterilization using UV irradiation for 2 h is an appropriate technique for the recently synthesized Me.PEG–PLA diblock copolymers. However, the study also serves as an example that it is indispensable to control the duration of exposure to UV irradiation for a given biomaterial in order not to compromise polymer properties relevant to tissue engineering purposes.
Co-reporter:Andrea Lucke, Jörg Teßmar, Edith Schnell, Georg Schmeer, Achim Göpferich
Biomaterials 2000 Volume 21(Issue 23) pp:2361-2370
Publication Date(Web):1 December 2000
DOI:10.1016/S0142-9612(00)00103-4
To obtain biodegradable polymers with variable surface properties for tissue culture applications, poly(ethylene glycol) blocks were attached to poly(lactic acid) blocks in a variety of combinations. The resulting poly(d,l-lactic acid)-poly(ethylene glycol)-monomethyl ether (Me.PEG-PLA) diblock copolymers were subject to comprehensive investigations concerning their bulk microstructure and surface properties to evaluate their suitability for drug delivery applications as well as for the manufacture of scaffolds in tissue engineering. Results obtained from 1H-NMR, gel permeation chromatography, wide angle X-ray diffraction and modulated differential scanning calorimetry revealed that the polymer bulk microstructure contains poly(ethylene glycol)-monomethyl ether (Me.PEG) domains segregated from poly(d,l-lactic acid) (PLA) domains varying with the composition of the diblock copolymers. Analysis of the surface of polymer films with atomic force microscopy and X-ray photoelectron spectroscopy indicated that there is a variable amount of Me.PEG chains present on the polymer surface, depending on the polymer composition. It could be shown that the presence of Me.PEG chains in the polymer surface had a suppressive effect on the adsorption of two model peptides (salmon calcitonin and human atrial natriuretic peptide). The possibility to modify polymer bulk microstructure as well as surface properties by variation of the copolymer composition is a prerequisite for their efficient use in the fields of drug delivery and tissue engineering.
Co-reporter:S. Joschek, B. Nies, R. Krotz, A. Göpferich
Biomaterials 2000 Volume 21(Issue 16) pp:1645-1658
Publication Date(Web):August 2000
DOI:10.1016/S0142-9612(00)00036-3
The properties of a porous hydroxyapatite ceramic produced by sintering of bovine bone were investigated by using a number of physicochemical methods such as scanning electron microscopy (SEM), SEM in combination with energy dispersive X-ray spectroscopy (SEM-EDX), mercury intrusion porosimetry, krypton-adsorption, contact angle measurements, wide angle X-ray diffraction, Fourier transform infrared spectroscopy, thermal analysis, inductively coupled plasma optical atom emissions spectroscopy and flame atomic absorption spectroscopy. The results indicate that there are considerable differences between the ceramic and native bone. However, the most important properties with respect to the use of such ceramics as a biomaterial for filling bone defects namely the high porosity (⩾57±2%) and the interconnecting pore system are maintained. While macropores with an average diameter of approx. 300 μm contribute 97% to porosity, micropores with an average diameter of 1.3 μm account for only 3% of the total porosity. The surface area was found to be approx. 0.1 m2/g. The contact angles of water (44.6±15.4°, n=5) and tetrahydrofurane (10°) allow the processing of the ceramic to a drug carrier by incubation with aqueous or organic drug solutions. The ceramic is highly crystalline with crystal sizes of 1–7 μm and contains crystal bridges. The investigation of its chemical composition revealed small amounts of other inorganic compounds such as Ca4O(PO4)2, NaCaPO4, Ca3(PO4)2, CaO, and MgO. Besides trace amounts of aluminum, iron, magnesium, potassium, silica, sodium, vanadium and zinc it contains probably carbonated apatite.
Co-reporter:Susan J. Peter;Lichun Lu;Achim Göpferich;Antonios G. Mikos;Andrea Lucke
Journal of Biomedical Materials Research Part A 1999 Volume 46(Issue 3) pp:390-398
Publication Date(Web):22 JUN 1999
DOI:10.1002/(SICI)1097-4636(19990905)46:3<390::AID-JBM12>3.0.CO;2-N
The adhesion of marrow stromal osteoblasts and the adsorption of fetal bovine serum (FBS) proteins to end-capped poly(D,L-lactic acid) 50:50 (PLA50) of molecular weight 17,000 (PLA5017), non-end-capped PLA50 of molecular weight 11,000 (PLA5011h), and a diblock copolymer made of poly(ethylene glycol)-monomethyl ether of molecular weight 5,000 and PLA50 of molecular weight 20,000 (Me.PEG5–PLA20) were investigated. Cell attachment and proliferation on both PLA50 polymers were equally good. The block copolymer did not allow the proliferation of cells. However, the attached cells were highly differentiated and metabolically active in contrast to the cells on PLA50. Moreover, surface analysis studies using electron spectroscopy revealed that FBS proteins adsorbed well from aqueous solutions to the PLA50 surfaces while they adsorbed substantially less to the block copolymer. These results suggest that Me.PEG-PLA block copolymers may be used to regulate protein adsorption and, therefore, cell adhesion by varying the block composition of the copolymer. © 1999 John Wiley & Sons, Inc. J Biomed Mater Res, 46, 390–398, 1999.
Co-reporter:Matthias Ferstl, Markus Drechsler, Reinhard Rachel, Matthias Rischer, ... Achim Goepferich
Journal of Pharmaceutical Sciences (August 2013) Volume 102(Issue 8) pp:2599-2607
Publication Date(Web):1 August 2013
DOI:10.1002/jps.23619
We investigated how the structure of nanofibers, resulting from interactions between anionic polyelectrolytes and cationic peptides, relies on the properties of the polyelectrolyte component. By using hyaluronate (H), carboxymethylcellulose (CMC), xanthan (X), and ozarelix (O), a cationic decapeptide, we determined the influence of characteristic polyelectrolyte parameters such as size and charge density on the formation of polyelectrolyte-peptide complexes. Transmission electron microscopy of unstained, frozen hydrated, or negatively stained samples revealed that the interaction between different anionic polyelectrolytes and ozarelix led to the formation of distinctly shaped nanofibers. CMC formed rather flexible structures with alternating thin and thick segments within the nanofibers with diameters ranging from 10 to 16 nm and a length of up to 1 μm. Hyaluronate, a high-molecular-mass molecule, formed extra-long aggregates of more than 5 μm. Individual fibers with a diameter of 8 nm aggregated to bigger strands. The nonlinear polysaccharide xanthan gum led to highly coiled structures. The diameter of the respective nanofibers varied between 15 and 25 nm. Isothermal titration calorimetry was used to determine the binding constants and the thermodynamic parameters of the different polyelectrolyte-peptide complexes. The binding constant, which was of the order of 106 M− 1, indicated a strong binding affinity, but also showed differences among the polyelectrolytes. These differences might be useful for prospective applications as drug delivery systems. © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 102:2599–2607, 2013
Co-reporter:Joerg K. Tessmar, Achim M. Göpferich
Advanced Drug Delivery Reviews (30 May 2007) Volume 59(Issues 4–5) pp:274-291
Publication Date(Web):30 May 2007
DOI:10.1016/j.addr.2007.03.020
The tissue engineering of functional tissues depends on the development of suitable scaffolds to support three dimensional cell growth. To improve the properties of the scaffolds, many cell carriers serve dual purposes; in addition to providing cell support, cutting-edge scaffolds biologically interact with adhering and invading cells and effectively guide cellular growth and development by releasing bioactive proteins like growth factors and cytokines.To design controlled release systems for certain applications, it is important to understand the basic principles of protein delivery as well as the stability of each applied biomolecule. To illustrate the enormous progress that has been achieved in the important field of controlled release, some of the recently developed cell carriers with controlled release capacity, including both solid scaffolds and hydrogel-derived scaffolds, are described and possible solutions for unresolved issues are illustrated.
Co-reporter:T. Reintjes, J. Tessmar, A. Göpferich
Journal of Drug Delivery Science and Technology (2008) Volume 18(Issue 1) pp:15-24
Publication Date(Web):1 January 2008
DOI:10.1016/S1773-2247(08)50002-5
This review describes the application of customized surface modifications using biomimetic polymers to attempt to reduce unspecific protein adsorption to the materials or promote specific cell adhesion to implants or tissue engineering scaffolds. Various polymers that are suited to suppress almost all unspecific protein interactions and underlying mechanisms of cell adhesion are presented and discussed. Suitable modifications to the inert polymers that can later favor the adhesion of specific cell types are also described. These modifications involve utilizing the binding characteristics of peptide sequences to promote specific cell attachment, and techniques to incorporate these sequences into the polymers are explained. Finally, it is shown that biomimetic surface modification techniques are also of tremendous importance for drug delivery with nanoparticles, and the opportunities and limits associated with these approaches are demonstrated.
Co-reporter:Christoph Luschmann, Joerg Tessmar, Simon Schoeberl, Olaf Strauss, Carsten Framme, Karl Luschmann, Achim Goepferich
European Journal of Pharmaceutical Sciences (20 November 2013) Volume 50(Issues 3–4) pp:385-392
Publication Date(Web):20 November 2013
DOI:10.1016/j.ejps.2013.07.002
With about 50–60 million cases in the US alone, dry eye disease represents a severe health care problem. Cyclosporin A (CsA) would be a potent candidate for a causal therapy. However, CsA is not sufficiently water soluble to be administrated via simple eye drops. We developed an in situ nanosuspension (INS) as a novel approach towards the administration of CsA to the cornea. It precipitates upon contact with the tear fluid and creates CsA nanoparticles that enter the cornea and release the drug by dissolution. We selected two liquid poly(ethylene glycols) (PEG) that dissolve CsA and create nanoparticles by precipitation of CsA upon water contact. Aqueous solutions of PEG and Solutol, a non-ionic surfactant, were well tolerated by primary human epithelial cells in vitro. To determine the critical water content needed for a precipitation, the solubility of CsA was investigated in quaternary systems of drug, solvent, surfactant and water. The best INS formulation showed a particle size of 505 ± 5 nm, a polydispersity index (PdI) of 0.23 ± 0.03 and a neutral zeta potential of −0.07 ± 0.05 mV. After single administration to porcine eyes in vitro, 3165 ± 597 ngCsA/gcornea were detected in corneal tissue, while the levels of Restasis® a commercial formulation were, with 545 ± 137 ngCsA/gcornea, significantly lower (P < 0.01). These results demonstrate that an INS is a promising, novel approach towards the causal treatment of inflammatory diseases at the anterior eye.A poorly soluble drug, a surface active additive and the maximum possible amount of water that does not induce a precipitation are solved in a non-aqueous, but water miscible solvent to generate a clear solution by gentle stirring. In combination with the tear fluid, the administered droplet exceeds the critical water content and with that the point of precipitation. The drug spontaneously precipitates into nanoparticles and is taken up by the tissue.Download high-res image (184KB)Download full-size image
Co-reporter:Susanne Kirchhof, Ferdinand P. Brandl, Nadine Hammer and Achim M. Goepferich
Journal of Materials Chemistry A 2013 - vol. 1(Issue 37) pp:NaN4864-4864
Publication Date(Web):2013/07/29
DOI:10.1039/C3TB20831A
The Diels–Alder (DA) reaction was investigated as a cross-linking mechanism for poly(ethylene glycol) (PEG) based hydrogels. Two complementary macromonomers were synthesized by functionalizing star-shaped PEG with furyl and maleimide groups. Gel formation occurred in water at 37 °C; the gelation time ranged between 171 ± 25 min and 14 ± 1 min depending on the used hydrogel formulation. The complex shear modulus was dependent on the concentration, branching factor and molecular weight of the macromonomers; values between 2821 ± 1479 Pa and 37097 ± 6698 Pa were observed. Hydrogel swelling and degradation were influenced by the same parameters; the degradation time varied between a few days and several weeks. Gel dissolution was found to occur by retro-DA reaction and subsequent hydrolysis of maleimide groups. Calculations of the network mesh size revealed that the prepared hydrogels would be suitable for the controlled release of therapeutic proteins.
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