Publications by authors named "Achim Goepferich"

84 Publications

Targeted Delivery of Soluble Guanylate Cyclase (sGC) Activator Cinaciguat to Renal Mesangial Cells via Virus-Mimetic Nanoparticles Potentiates Anti-Fibrotic Effects by cGMP-Mediated Suppression of the TGF-β Pathway.

Int J Mol Sci 2021 Mar 4;22(5). Epub 2021 Mar 4.

Department of Pharmaceutical Technology, University of Regensburg, 93053 Regensburg, Germany.

Diabetic nephropathy (DN) ranks among the most detrimental long-term effects of diabetes, affecting more than 30% of all patients. Within the diseased kidney, intraglomerular mesangial cells play a key role in facilitating the pro-fibrotic turnover of extracellular matrix components and a progredient glomerular hyperproliferation. These pathological effects are in part caused by an impaired functionality of soluble guanylate cyclase (sGC) and a consequentially reduced synthesis of anti-fibrotic messenger 3',5'-cyclic guanosine monophosphate (cGMP). Bay 58-2667 (cinaciguat) is able to re-activate defective sGC; however, the drug suffers from poor bioavailability and its systemic administration is linked to adverse events such as severe hypotension, which can hamper the therapeutic effect. In this study, cinaciguat was therefore efficiently encapsulated into virus-mimetic nanoparticles (NPs) that are able to specifically target renal mesangial cells and therefore increase the intracellular drug accumulation. NP-assisted drug delivery thereby increased in vitro potency of cinaciguat-induced sGC stabilization and activation, as well as the related downstream signaling 4- to 5-fold. Additionally, administration of drug-loaded NPs provided a considerable suppression of the non-canonical transforming growth factor β (TGF-β) signaling pathway and the resulting pro-fibrotic remodeling by 50-100%, making the system a promising tool for a more refined therapy of DN and other related kidney pathologies.
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http://dx.doi.org/10.3390/ijms22052557DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7961750PMC
March 2021

Atherosclerosis: Conventional intake of cardiovascular drugs versus delivery using nanotechnology - A new chance for causative therapy?

J Control Release 2021 May 29;333:536-559. Epub 2021 Mar 29.

Department of Pharmaceutical Technology, University of Regensburg, Universitätsstraße 31, 93053 Regensburg, Germany. Electronic address:

Atherosclerosis is the leading cause of death in developed countries. The pathogenetic mechanism relies on a macrophage-based immune reaction to low density lipoprotein (LDL) deposition in blood vessels with dysfunctional endothelia. Thus, atherosclerosis is defined as a chronic inflammatory disease. A plethora of cardiovascular drugs have been developed and are on the market, but the major shortcoming of standard medications is that they do not address the root cause of the disease. Statins and thiazolidinediones that have recently been recognized to exert specific anti-atherosclerotic effects represent a potential breakthrough on the horizon. But their whole potential cannot be realized due to insufficient availability at the pathological site and severe off-target effects. The focus of this review will be to elaborate how both groups of drugs could immensely profit from nanoparticulate carriers. This delivery principle would allow for their accumulation in target macrophages and endothelial cells of the atherosclerotic plaque, increasing bioavailability where it is needed most. Based on the analyzed literature we conclude design criteria for the delivery of statins and thiazolidinediones with nanoparticles for anti-atherosclerotic therapy. Nanoparticles need to be below a diameter of 100 nm to accumulate in the atherosclerotic plaque and should be fabricated using biodegradable materials. Further, the thiazolidinediones or statins must be encapsulated into the particle core, because especially for thiazolidindiones the uptake into cells is prerequisite for their mechanism of action. For optimal uptake into targeted macrophages and endothelial cells, the ideal particle should present ligands on its surface which bind specifically to scavenger receptors. The impact of statins on the lectin-type oxidized LDL receptor 1 (LOX1) seems particularly promising because of its outstanding role in the inflammatory process. Using this pioneering concept, it will be possible to promote the impact of statins and thiazolidinediones on macrophages and endothelial cells and significantly enhance their anti-atherosclerotic therapeutic potential.
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http://dx.doi.org/10.1016/j.jconrel.2021.03.034DOI Listing
May 2021

Fluorescent Nanoparticles Coated with a Somatostatin Analogue Target Blood Monocyte for Efficient Leukaemia Treatment.

Pharm Res 2020 Oct 9;37(11):217. Epub 2020 Oct 9.

Department of Pharmaceutical Technology Department, Faculty of Chemistry and Pharmacy, Regensburg University, 93040, Regensburg, Germany.

Background: Leukaemia is the most prevalent form of cancer-causing death in a large number of populations and needs prompt and effective treatment. Chemotherapeutics can be used to treat leukaemia, but their pronounced killing effects to other living cells is still an issue. Active targeting to certain specific receptors in leukaemic cells is the best way to avoid damage to other living cells. Leukaemic cells can be targeted using novel nanoparticles (NPs) coated with a specific ligand, such as octreotide (OCD), to target somatostatin receptor type 2 (SSTR), which is expressed in leukaemic cells.

Methods: Amino-PEGylated quantum dots (QDs) were chosen as model NPs. The QDs were first succinylated using succinic anhydride and then coated with OCD. The reactivity and selectivity of the formulated QDs-OCD were studied in cell lines with well-expressed SSTR, while fluorescence was detected using confocal laser scanning microscopy (CLSM) and flow cytometry (FACS). Conclusively, QD-OCD targeting to blood cells was studied in vivo in mice and detected using inductively coupled plasma mass spectrometry and CLSM in tissues.

Results: Highly stable QDs coated with OCD were prepared. FACS and CLSM showed highly definite interactions with overexpressed SSTR in the investigated cell lines. Moreover, the in vivo results revealed a higher concentration of QDs-OCD in blood cells. The fluorescence intensity of the QDs-OCD was highly accumulated in blood cells, while the unmodified QDs did not accumulate significantly in blood cells.

Conclusion: The formulated novel QDs-OCD can target SSTR overexpressed in blood cells with great potential for treating blood cancer.
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http://dx.doi.org/10.1007/s11095-020-02938-1DOI Listing
October 2020

Biomedical nanoparticle design: What we can learn from viruses.

J Control Release 2021 Jan 30;329:552-569. Epub 2020 Sep 30.

Department of Pharmaceutical Technology, University of Regensburg, Universitaetsstrasse 31, 93053 Regensburg, Germany. Electronic address:

Viruses are nanomaterials with a number of properties that surpass those of many synthetic nanoparticles (NPs) for biomedical applications. They possess a rigorously ordered structure, come in a variety of shapes, and present unique surface elements, such as spikes. These attributes facilitate propitious biodistribution, the crossing of complex biological barriers and a minutely coordinated interaction with cells. Due to the orchestrated sequence of interactions of their stringently arranged particle corona with cellular surface receptors they effectively identify and infect their host cells with utmost specificity, while evading the immune system at the same time. Furthermore, their efficacy is enhanced by their response to stimuli and the ability to spread from cell to cell. Over the years, great efforts have been made to mimic distinct viral traits to improve biomedical nanomaterial performance. However, a closer look at the literature reveals that no comprehensive evaluation of the benefit of virus-mimetic material design on the targeting efficiency of nanomaterials exists. In this review we, therefore, elucidate the impact that viral properties had on fundamental advances in outfitting nanomaterials with the ability to interact specifically with their target cells. We give a comprehensive overview of the diverse design strategies and identify critical steps on the way to reducing them to practice. More so, we discuss the advantages and future perspectives of a virus-mimetic nanomaterial design and try to elucidate if viral mimicry holds the key for better NP targeting.
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http://dx.doi.org/10.1016/j.jconrel.2020.09.045DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7525328PMC
January 2021

Adenovirus-Mimetic Nanoparticles: Sequential Ligand-Receptor Interplay as a Universal Tool for Enhanced / Cell Identification.

ACS Appl Mater Interfaces 2020 Aug 22;12(31):34689-34702. Epub 2020 Jul 22.

Department of Pharmaceutical Technology, University of Regensburg, Universitaetsstrasse 31, 93053 Regensburg, Germany.

Viral infection patterns often rely on precisely coordinated sequences of distinct ligand-receptor interactions, leading in many cases to an outstanding target cell specificity. A successful mimicry of viral targeting strategies to create more site-specific nanoparticles (NPs) would therefore require particle-cell interactions to also be adequately controllable. In the present study, hetero-multivalent block-copolymer NPs present their attached ligands in a sterically controlled manner to create a sequential NP-cell interaction similar to the cell infiltration strategy of human adenovirus type 2. Targeting renal mesangial cells, particles therefore initially bind angiotensin II receptor type 1 (AT1r) on the cell surface via a structurally flexible AT1r antagonist. After a mandatory spatial approach, particle endocytosis is realized via binding of immobile αβ integrins with a previously concealed secondary ligand, thereby creating a stepwise particle-cell interplay of primary NP attachment and subsequent uptake. Manufactured adenovirus-mimetic NPs show great avidity for both target motifs , leading to a substantial binding as well as subsequent cell uptake into target mesangial cells. Additionally, steric shielding of secondary ligand visibility leads to a highly controllable, sequential ligand-receptor interaction, whereby hetero-functional NPs activate mesangial cell surface integrins only after a successful prior binding to the AT1r. This stepwise cell identification significantly enhances mesangial cell specificity in co-culture assays with different off-target cells. Additionally, described NPs display excellent robustness by efficiently accumulating in the mesangium upon injection, thereby opening new paths for possible drug delivery applications.
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http://dx.doi.org/10.1021/acsami.0c10057DOI Listing
August 2020

Laser-induced graphene interdigitated electrodes for label-free or nanolabel-enhanced highly sensitive capacitive aptamer-based biosensors.

Biosens Bioelectron 2020 Sep 21;164:112272. Epub 2020 May 21.

Institute of Analytical Chemistry, Chemo-and Biosensors, Faculty of Chemistry and Pharmacy, University of Regensburg, 31, 93053, Regensburg, Germany. Electronic address:

Highly porous laser-induced graphene (LIG) is easily generated in complex electrode configurations such as interdigitated electrodes (IDEs). Here, we demonstrate that their superior capacitive response at low frequencies can be exploited in affinity biosensors using thrombin aptamers as model biorecognition elements. Of specific interest was the effect of electrode surface area on capacitance detection, and the comparison between a label-free format and enhancement strategies afforded by carboxy group bearing polymeric nanoparticles or liposomes. Electrochemical impedance spectroscopy (EIS) was used to investigate the LIG performance and optimize the biosensor design. Interestingly, the label-free strategy performed extremely well and additional labels decreased the limit of detection or increased the sensitivity only minimally. It is assumed that the highly porous nature of the LIG structures dominates the capacitive response so that labels removed from the surface have only limited influence Also, while slight performance changes can be observed for smaller vs. larger electrode structures, the performance of a LIG IDE is reasonably independent of its size. In the end, a dynamic range of 5 orders of magnitude was obtained (0.01 nM-1000 nM) with a limit of detection as low as 0.12 pM. When measured in serum, this increased to 1.3 pM. The good reproducibility (relative standard deviation (RSD), 4.90%) and repeatability (RSD, 2.59%) and good long-term stability (>7 weeks at 4 °C) prove that a LIG-based capacitance sensor is an excellent choice for affinity-based biosensor. The ease-of-production, the simplicity of modification and the superior performance even in a label-free format indicate that LIG-based biosensors should be considered in point-of-care diagnostics in the future.
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http://dx.doi.org/10.1016/j.bios.2020.112272DOI Listing
September 2020

Nanoparticles Mimicking Viral Cell Recognition Strategies Are Superior Transporters into Mesangial Cells.

Adv Sci (Weinh) 2020 Jun 22;7(11):1903204. Epub 2020 Apr 22.

Department of Pharmaceutical Technology University of Regensburg Regensburg 93053 Germany.

Poor drug availability in the tissue of interest is a frequent cause of therapy failure. While nanotechnology has developed a plethora of nanocarriers for drug transport, their ability to unequivocally identify cells of interest remains moderate. Viruses are the ideal nanosized carriers as they are able to address their embedded nucleic acids with high specificity to their host cells. Here, it is reported that particles endowed with a virus-like ability to identify cells by three consecutive checks have a superior ability to recognize mesangial cells (MCs) in vivo compared to conventional nanoparticles. Mimicking the initial viral attachment followed by a stepwise target cell recognition process leads to a 5- to 15-fold higher accumulation in the kidney mesangium and extensive cell uptake compared to particles lacking one or both of the viral traits. These results highlight the relevance that the viral cell identification process has on specificity and its application on the targeting strategies of nanomaterials. More so, these findings pave the way for transporting drugs into the mesangium, a tissue that is pivotal in the development of diabetic nephropathy and for which currently no efficient pharmacotherapy exists.
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http://dx.doi.org/10.1002/advs.201903204DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7284201PMC
June 2020

Design of dye and superparamagnetic iron oxide nanoparticle loaded lipid nanocapsules with dual detectability in vitro and in vivo.

Int J Pharm 2020 Jul 21;585:119433. Epub 2020 May 21.

Department of Pharmaceutical Technology, University of Regensburg, 93053 Regensburg, Germany. Electronic address:

Lipid nanocapsules are treasured nanoparticulate systems, although they lack detectability in biological environments. To overcome this, we designed LNCs loaded simultaneously with fluorescent dye and superparamagnetic iron oxide nanoparticles (Dual LNCs). The introduction of both labels did not alter nanoparticle characteristics such as size (50 nm), size distribution (polydispersity index < 0.1) or surface modifications, including the effectiveness of targeting ligands. Furthermore, the colloidal stability, particle integrity and biocompatibility of the nanoparticles were not negatively affected by label incorporation. These Dual LNCs are concomitantly visualizable via fluorescence and transmitted light imaging after either the internalization by cells or systemic administration to mice. Importantly, they are detectable in liver sections of mice using transmission electron microscopy without additional enhancement. The iron content of 0.24% (m/m) is sufficiently high for precise quantification of nanoparticle concentrations via inductively coupled plasma optical emission spectroscopy. Dual LNCs are precious tools for the investigation of in vitro and in vivo performances of lipid nanocapsule formulations, since they allow for the use of complementary imaging methods for broad range detectability.
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http://dx.doi.org/10.1016/j.ijpharm.2020.119433DOI Listing
July 2020

Thermodynamic, Spatial and Methodological Considerations for the Manufacturing of Therapeutic Polymer Nanoparticles.

Pharm Res 2020 Feb 24;37(3):59. Epub 2020 Feb 24.

Department of Pharmaceutical Technology, University of Regensburg, Universitaetsstrasse 31, 93053, Regensburg, Germany.

Purpose: Evaluate fundamental parameters that dictate the effectiveness of drug loading.

Methods: A model water-soluble drug lacking ionizable groups, pirfenidone (PFD), was encapsulated through nanoprecipitation in poly(ethylene glycol)-poly(lactic acid) (PEG-PLA)-poly(lactic-co-glycolic acid) (PLGA) NPs. Firstly, the thermodynamic parameters predicting drug-polymer miscibility were determined to assess the system's suitability. Then, the encapsulation was evaluated experimentally by two different techniques, bulk and microfluidic (MF) nanoprecipitation. Additionally, the number of molecules that fit in a particle core were calculated and the loading determined experimentally for different core sizes. Lastly, the effect of co-encapsulation of α-lipoic acid (LA), a drug with complementary therapeutic effects and enhanced lipophilicity, was evaluated.

Results: The thermodynamic miscibility parameters predicted a good suitability of the selected system. MF manufacturing enhanced the encapsulation efficiency by 60-90% and achieved a 2-fold higher NP cellular uptake. Considering spatial constrictions for drug encapsulation and increasing the size of the PLGA core the number of PFD molecules per NP was raised from under 500 to up to 2000. More so, the co-encapsulation of LA increased the number of drug molecules per particle by 96%, with no interference with the release profile.

Conclusions: Thermodynamic, spatial and methodological parameters should be considered to optimize drug encapsulation.
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http://dx.doi.org/10.1007/s11095-020-2783-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7040083PMC
February 2020

The Effect of Ligand Mobility on the Cellular Interaction of Multivalent Nanoparticles.

Macromol Biosci 2020 04 20;20(4):e1900427. Epub 2020 Feb 20.

Department of Pharmaceutical Technology, University of Regensburg, Regensburg, Universitaetsstrasse 31, 93053, Germany.

Multivalent nanoparticle binding to cells can be of picomolar avidity making such interactions almost as intense as those seen with antibodies. However, reducing nanoparticle design exclusively to avidity optimization by the choice of ligand and its surface density does not sufficiently account for controlling and understanding cell-particle interactions. Cell uptake, for example, is of paramount significance for a plethora of biomedical applications and does not exclusively depend on the intensity of multivalency. In this study, it is shown that the mobility of ligands tethered to particle surfaces has a substantial impact on particle fate upon binding. Nanoparticles carrying angiotensin-II tethered to highly mobile 5 kDa long poly(ethylene glycol) (PEG) chains separated by ligand-free 2 kDa short PEG chains show a superior accumulation in angiotensin-II receptor type 1 positive cells. In contrast, when ligand mobility is constrained by densely packing the nanoparticle surface with 5 kDa PEG chains only, cell uptake decreases by 50%. Remarkably, irrespective of ligand mobility and density both particle types have similar EC50 values in the 1-3 × 10 m range. These findings demonstrate that ligand mobility on the nanoparticle corona is an indispensable attribute to be considered in particle design to achieve optimal cell uptake via multivalent interactions.
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http://dx.doi.org/10.1002/mabi.201900427DOI Listing
April 2020

Hydrogel microspheres evading alveolar macrophages for sustained pulmonary protein delivery.

Int J Pharm 2019 Jul 8;566:652-661. Epub 2019 Jun 8.

Department of Pharmaceutical Technology, Faculty of Chemistry and Pharmacy, University of Regensburg, 93040 Regensburg, Germany. Electronic address:

Pulmonary delivery is a highly attractive alternative to injections for biologics such as therapeutic proteins. However, bioavailabilities generally suffer from the presence of phagocytic cells that clear particulate matter entering the lung. In this study, microgel particles were developed using an all-aqueous two-phase system approach and evaluated for their efficacy as an inhalable controlled release system. Norbornene- and thiol-modified four- and eight-armed poly (ethylene glycol) with an average molecular mass of 10,000 Da were prepared as macromonomers for microgel formation. Emulsions of precursor solution droplets containing macromonomers and Irgacure 2959 as photocatalyst were prepared in a dextran solution. Irradiation with UV light was used to covalently crosslink the droplets by triggering the thiol-ene reaction. The resulting microgels were processed to dry powder inhaler formulations, and respirable aerodynamic sizes were assessed in vitro. Microgels were loaded with the model proteins lysozyme and bovine serum albumin, with encapsulation efficiencies of 51.5% and 73.6%, respectively. Depending on the macromonomer type, protein-loaded microgels released their cargo over a 6-14 day period. In an MTT assay, the particles did not show significant cytotoxicity, and their recognition by alveolar macrophages was considerably lower than for polystyrene control particles. This makes the microgels a promising pulmonary delivery system for proteins and other biologics.
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http://dx.doi.org/10.1016/j.ijpharm.2019.06.019DOI Listing
July 2019

Influenza A virus mimetic nanoparticles trigger selective cell uptake.

Proc Natl Acad Sci U S A 2019 05 29;116(20):9831-9836. Epub 2019 Apr 29.

Department of Pharmaceutical Technology, University of Regensburg, 93053 Regensburg, Germany

Poor target cell specificity is currently a major shortcoming of nanoparticles (NPs) used for biomedical applications. It causes significant material loss to off-target sites and poor availability at the intended delivery site. To overcome this limitation, we designed particles that identify cells in a virus-like manner. As a blueprint, we chose a mechanism typical of influenza A virus particles in which ectoenzymatic hemagglutinin activation by target cells is a mandatory prerequisite for binding to a secondary target structure that finally confirms cell identity and allows for uptake of the virus. We developed NPs that probe mesangial cells for the presence of angiotensin-converting enzyme on their surface using angiotensin I (Ang-I) as a proligand. This initial interaction enzymatically transforms Ang-I to a secondary ligand angiotensin II (Ang-II) that has the potential to bind in a second stage to Ang-II type-1 receptor (AT1R). The presence of the receptor confirms the target cell identity and triggers NP uptake via endocytosis. Our virus-mimetic NPs showed outstanding target-cell affinity with picomolar avidities and were able to selectively identify these cells in the presence of 90% off-target cells that carried only the AT1R. Our results demonstrate that the design of virus-mimetic cell interactive NPs is a valuable strategy to enhance NP specificity for therapeutic and diagnostic applications. Our set of primary and secondary targets is particularly suited for the identification of mesangial cells that play a pivotal role in diabetic nephropathy, one of the leading causes of renal failure, for which currently no treatment exists.
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http://dx.doi.org/10.1073/pnas.1902563116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6525500PMC
May 2019

Intracellular availability of poorly soluble drugs from lipid nanocapsules.

Eur J Pharm Biopharm 2019 Jun 7;139:23-32. Epub 2019 Mar 7.

Department of Pharmaceutical Technology, Faculty of Chemistry and Pharmacy, University of Regensburg, 93040 Regensburg, Germany. Electronic address:

Lipid nanocapsules (LNCs) are extensively used as drug carrier systems, due to their small size distribution, biocompatibility and ease of preparation. They are especially useful for lipophilic drugs to overcome physicochemical constraints that limit their efficacy, such as low solubility in aqueous media. The aim of this work was to investigate the relationship between the intracellular availability of poorly soluble drugs delivered via LNCs and their biological efficacy in cells in vitro. Cyclosporin A (CsA) with a logP = 4.3 (Lucangioli et al., 2003) and Itraconazole (It) with a logP = 6.2 (Bhardwaj et al., 2013) served as model lipophilic compounds, as they are highly promising candidates for the treatment of neovascular ocular diseases. Due to their lipophilic properties and the resulting preference for the oily core of LNCs, high encapsulation efficiencies were achieved. Drug-loaded LNCs with particle sizes around 50 nm were grafted with an αvβ3 integrin ligand (RGD) to optimize cellular uptake by human dermal microvascular endothelial cells. Even though RGD-LNCs showed excellent internalization, they exhibited insufficient inhibitory effects in vitro regarding endothelial cell proliferation, vascular endothelial growth factor expression, and tube formation in contrast to free drugs. This loss of efficacy could be explained by negligible intracellular availability of the poorly soluble drugs from LNCs.
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http://dx.doi.org/10.1016/j.ejpb.2019.03.007DOI Listing
June 2019

Interaction of functionalized nanoparticles with serum proteins and its impact on colloidal stability and cargo leaching.

Soft Matter 2019 Jan 9;15(4):709-720. Epub 2019 Jan 9.

Department of Pharmaceutical Technology, University of Regensburg, 93040 Regensburg, Germany.

The majority of effort in the area of polymeric nanocarriers is aimed at providing controlled drug delivery in vivo. Therefore, it is essential to understand the delicate interplay of polymeric NPs with serum proteins in order to forecast their performance in a biological system. In this study, the interaction of serum proteins with functionalized polymeric colloids as a function of particle charge and hydrophobicity was investigated. Moreover, impact on NP stability and cargo leaching was assessed. The hard protein corona of polymeric NPs with either uncharged methoxy groups (methoxy-NPs), positively charged amine groups (amine-NPs), negatively charged carboxylic acid groups (carboxyl-NPs) or zwitterionic NPs decorated with amine and carboxylic acid groups (zwitterion-NPs) was quantitatively and qualitatively analyzed and correlated with the respective colloidal stability using fluorescence resonance energy transfer. Positively charged amine-NPs displayed an enhanced interaction with serum proteins via electrostatic interactions resulting in a hard corona consisting of diverse protein components. As revealed by FRET and agarose gel electrophoresis, the enhanced adsorption of proteins onto the colloidal surface significantly altered the NP identity and severely impaired the colloidal integrity as the lipophilic cargo was continuously leached out of the hydrophobic NP core. These results highlight the importance of generating a profound knowledge of the bio-nano interface as adherence of biomolecules can severely compromise the performance of a colloidal drug delivery system by changing its identity and integrity.
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http://dx.doi.org/10.1039/c8sm02189aDOI Listing
January 2019

Ligand Density and Linker Length are Critical Factors for Multivalent Nanoparticle-Receptor Interactions.

ACS Appl Mater Interfaces 2019 Jan 20;11(1):1311-1320. Epub 2018 Dec 20.

Department of Pharmaceutical Technology, Faculty of Chemistry and Pharmacy , University of Regensburg , 93040 Regensburg , Germany.

Although there are a large number of studies available for the evaluation of the therapeutic efficacy of targeted polymeric nanoparticles, little is known about the critical attributes that can further influence their uptake into target cells. In this study, varying cRGD ligand densities (0-100% surface functionalization) were combined with different poly(ethylene glycol) (PEG) spacer lengths (2/3.5/5 kDa), and the specific receptor binding of targeted core-shell structured poly(lactic- co-glycolic acid)/poly(lactic acid)-PEG nanoparticles was evaluated using αβ integrin-overexpressing U87MG glioblastoma cells. Nanoparticles with 100% surface functionalization and short PEG2k linkers displayed a high propensity to form colloidal clusters, allowing for the cooperative binding to integrin receptors on the cellular membrane. In contrast, the high flexibility of longer PEG chains enhanced the chance of ligand entanglement and shrouding, decreasing the number of ligand-receptor binding events. As a result, the combination of short PEG2k linkers and a high cRGD surface modification synergistically increased the uptake of nanoparticles into target cells. Even though to date, the nanoparticle size and its degree of functionalization are considered to be the major determinants for controlling the uptake efficiency of targeted colloids, these results strongly suggest that the role of the linker length should be carefully taken into consideration for the design of targeted drug delivery formulations to maximize the therapeutic efficacy and minimize adverse side effects.
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http://dx.doi.org/10.1021/acsami.8b18843DOI Listing
January 2019

Nanoparticles Targeting Retinal and Choroidal Capillaries In Vivo.

Methods Mol Biol 2019 ;1834:391-404

Department of Pharmaceutical Technology, University of Regensburg, Regensburg, Germany.

The functionalization of nanoparticles with specific receptor ligands enables their accumulation in targeted tissues and can be used therapeutically to transport drugs or for diagnostic purposes (Parveen et al., Nanomedicine 8:147-166, 2012). We could recently show that targeting endothelial cells in retinal and choroidal capillaries can be realized even under physiological conditions using quantum dots as model nanoparticles functionalized with an integrin-binding peptide (Pollinger et al., Proc Natl Acad Sci 110:6115-6120, 2013). Even though the chemistry that we used was well described in the literature and may be considered standard for the purpose, there are a number of preparation steps that are delicate and deserve special attention. It is, therefore, our intention to describe step by step the critical methods of ligand immobilization on quantum dot surfaces to facilitate the reader to reproduce our work. Here we describe the chemical modification of quantum dots with c(RGDfC) as targeting peptide that allows the resulting modified nanoparticles to adhere to endothelial cells also in the retinal tissue. We illustrate the properties of the resulting particles by showing some of the in vitro results from our previous studies. Doing so, we concomitantly encourage the reader to check particles intended for targeting cells in vivo first by extensive in vitro analysis of particle interaction with cells by the means of flow cytometry and confocal microscopy to confirm the successful functionalization. Only then the application of functionalized quantum dots into the systemic circulation of mice led to the desired localization of nanoparticles in the retinal and choroidal blood vessels (Pollinger et al., Proc Natl Acad Sci 110:6115-6120, 2013).
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http://dx.doi.org/10.1007/978-1-4939-8669-9_25DOI Listing
April 2019

Fabrication of antibody-loaded microgels using microfluidics and thiol-ene photoclick chemistry.

Eur J Pharm Biopharm 2018 Jun 19;127:194-203. Epub 2018 Feb 19.

Department of Pharmaceutical Technology, Faculty of Chemistry and Pharmacy, University of Regensburg, 93040 Regensburg, Germany. Electronic address:

Reducing burst effects, providing controlled release, and safeguarding biologics against degradation are a few of several highly attractive applications for microgels in the field of controlled release. However, the incorporation of proteins into microgels without impairing stability is highly challenging. In this proof of concept study, the combination of microfluidics and thiol-ene photoclick chemistry was evaluated for the fabrication of antibody-loaded microgels with narrow size distribution. Norbornene-modified eight-armed poly(ethylene glycol) with an average molecular mass of 10,000 Da, 20,000 Da, or 40,000 Da were prepared as macromonomers for microgel formation. For functionalization, either hydrolytically cleavable ester or stable amide bonds were used. A microfluidic system was employed to generate precursor solution droplets containing macromonomers, the cross-linker dithiothreitol and the initiator Eosin-Y. Irradiation with visible light was used to trigger thiol-ene reactions which covalently cross-linked the droplets. For all bond-types, molecular masses, and concentrations gelation was very rapid (<20 s) and a plateau for the complex shear modulus was reached after only 5 min. The generated microgels had a rod-like shape and did not show considerable cellular toxicity. Stress conditions during the fabrication process were simulated and it could be shown that fabrication did not impair the activity of the model proteins lysozyme and bevacizumab. It was confirmed that the average hydrogel network mesh size was similar or smaller than the hydrodynamic diameter of bevacizumab which is a crucial factor for restricting diffusion and delaying release. Finally, microgels were loaded with bevacizumab and a sustained release over a period of 30 ± 4 and 47 ± 7 days could be achieved in vitro.
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http://dx.doi.org/10.1016/j.ejpb.2018.02.024DOI Listing
June 2018

Alkaline poly(ethylene glycol)-based hydrogels for a potential use as bioactive wound dressings.

J Biomed Mater Res A 2017 Dec 20;105(12):3360-3368. Epub 2017 Sep 20.

Department of Pharmaceutical Technology, University of Regensburg, Regensburg, 93053, Germany.

The number of patients with chronic wounds is increasing constantly in today's aging society. However, little work is done so far tackling the associated disadvantageous shift of the wound pH. In our study, we developed two different approaches on pH-modulating wound dressing materials, namely, bioactive interpenetrating polymer network hydrogels based on poly(ethylene glycol) diacrylate/N-vinylimidazole/alginate (named VI ) and poly(ethylene glycol) diacrylate/2-dimethylaminoethyl methacrylate/N-carboxyethylchitosan (named DMAEMA ). Both formulations showed a good cytocompatibility and wound healing capacity in vitro. The developed dressing materials significantly increased the cell ingrowth in wounded human skin constructs; by 364% and 313% for the VI and the DMAEMA hydrogel formulation, respectively. Additionally, VI hydrogels were found to be suitable scaffolds for superficial cell attachment. Our research on the material properties suggests that ionic interactions and hydrogen bonds are the driving forces for the mechanical and swelling properties of the examined hydrogels. High amounts of positively charged amino groups in DMAEMA hydrogels caused increased liquid uptake (around 190%), whereas VI hydrogels showed a 10-fold higher maximum compressive stress in comparison to hydrogels without ionizable functional groups. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 3360-3368, 2017.
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http://dx.doi.org/10.1002/jbm.a.36177DOI Listing
December 2017

Controlled Antibody Release from Degradable Thermoresponsive Hydrogels Cross-Linked by Diels-Alder Chemistry.

Biomacromolecules 2017 Aug 6;18(8):2410-2418. Epub 2017 Jul 6.

Department of Pharmaceutical Technology, Faculty of Chemistry and Pharmacy, University of Regensburg , 93040 Regensburg, Germany.

Amine-modified four- and eight-armed poloxamines were prepared and subsequently functionalized with maleimide or furyl groups. Aqueous solutions of these polymers exhibited an immediate gelation at a temperature above 37 °C. Concomitantly, Diels-Alder reactions gradually cross-linked and cured the gels. Different ratios between four- and eight-armed macromonomers were used to tune hydrogel stability and mechanical properties. In this way, hydrogel stability could be precisely controlled in the range of 14 to 329 days. Controlled release of the model antibody bevacizumab was achieved over a period of 7, 21, and 115 days. Release profiles were triphasic with a low burst; approximately 87% of the released antibody was intact and displayed functional binding. The hydrogels presented in this study are degradable, nontoxic, rapidly gelling, stable, and provide controlled antibody release. They can be tailored to match the demands of various applications and present an attractive platform for antibody delivery.
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http://dx.doi.org/10.1021/acs.biomac.7b00587DOI Listing
August 2017

Targeting the Central Nervous System (CNS): A Review of Rabies Virus-Targeting Strategies.

Mol Pharm 2017 07 16;14(7):2177-2196. Epub 2017 Jun 16.

Department for Pharmaceutical Technology, University of Regensburg , Universitätsstraße 31, 94030 Regensburg, Germany.

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.
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http://dx.doi.org/10.1021/acs.molpharmaceut.7b00158DOI Listing
July 2017

Polyanion based controlled release system for the GnRH-receptor antagonist degarelix.

Eur J Pharm Sci 2017 Jun 25;104:65-71. Epub 2017 Mar 25.

Department of Pharmaceutical Technology, University of Regensburg, 93053 Regensburg, Germany; University of Regensburg, Universitätsstraße 31, 93053 Regensburg, Germany. Electronic address:

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.
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http://dx.doi.org/10.1016/j.ejps.2017.03.036DOI Listing
June 2017

pH-Modulating Poly(ethylene glycol)/Alginate Hydrogel Dressings for the Treatment of Chronic Wounds.

Macromol Biosci 2017 05 20;17(5). Epub 2016 Dec 20.

Department of Pharmaceutical Technology, University of Regensburg, Universitätsstr. 31, 93053, Regensburg, Germany.

The development of chronic wounds has been frequently associated with alkaline pH values. The application of pH-modulating wound dressings can, therefore, be a promising treatment option to promote normal wound healing. This study reports on the development and characterization of acidic hydrogel dressings based on interpenetrating poly(ethylene glycol) diacrylate/acrylic acid/alginate networks. The incorporation of ionizable carboxylic acid groups results in high liquid uptake up to 500%. The combination of two separate polymer networks significantly improves the tensile and compressive stability. In a 2D cell migration assay, the application of hydrogels (0% to 1.5% acrylic acid) results in complete "wound" closure; hydrogels with 0.25% acrylic acid significantly increase the cell migration velocity to 19.8 ± 1.9 µm h . The most promising formulation (hydrogels with 0.25% acrylic acid) is tested on 3D human skin constructs, increasing keratinocyte ingrowth into the wound by 164%.
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http://dx.doi.org/10.1002/mabi.201600369DOI Listing
May 2017

Determination of the activity of maleimide-functionalized phospholipids during preparation of liposomes.

Int J Pharm 2016 Nov;514(1):93-102

University of Regensburg, Dept. for Pharmaceutical Technology, Universitätsstraße 31, 94030 Regensburg, Germany. Electronic address:

Numerous examples exist in the literature for the use of maleimide-thiol-reactions in the area of functionalized nanoparticles. Although the hydrolysis tendency of maleimides is well-known, qualitative and quantitative information on the stability and reactivity of maleimide groups during preparation and in final formulations are missing. This is surprising, since hydrolysis of maleimides prevents nanoparticle functionalization and results in an increase of negative surface charge due to the hydrolysis product maleic acid. In this study we investigated the stability of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide-2000] (DSPE-PEG-Mal) during the preparation of liposomes via two common preparation methods, which can be distinguished by the insertion of DSPE-PEG-Mal during or after the liposome formation process (pre-insertion and post-insertion process). The liposomes prepared by the pre-insertion method had 63% active maleimide groups remaining on their surface. The activity decreased dramatically during the purification process down to 32%. The preparation by post-insertion showed minimal effects with regard to maleimide activity. 76% of maleimide groups were active and therefore available for coupling reaction. By identifying active maleimide groups on the surface of the final formulations, the presented work revealed the dramatic impact of preparation methods on the activity of maleimide groups.
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http://dx.doi.org/10.1016/j.ijpharm.2016.06.116DOI Listing
November 2016

Polyanions effectively prevent protein conjugation and activity loss during hydrogel cross-linking.

J Control Release 2016 09 21;238:92-102. Epub 2016 Jul 21.

Department of Pharmaceutical Technology, Faculty of Chemistry and Pharmacy, University of Regensburg, 93040 Regensburg, Germany. Electronic address:

In situ encapsulation is a frequently used method to prepare hydrogels loaded with high quantities of therapeutic proteins. However, many cross-linking reactions, such as Michael-type addition or Diels-Alder (DA) reaction are not tolerant toward nucleophiles; therefore, side-reactions with proteins can occur during cross-linking. This may lead to undesired protein conjugation, activity loss and incomplete protein release. In this study, a number of polyanions, namely alginate, dextran sulfate, hyaluronic acid, heparin, and poly(acrylic acid), were screened for their capability to protect proteins during covalent cross-linking. To this end, lysozyme was incubated with furyl- and maleimide-substituted methoxy poly(ethylene glycol); different pH values were tested. The degree of PEGylation and the residual activity of lysozyme were investigated. Without polyanions, 61.1% of the total lysozyme amount was PEGylated at pH7.4; the residual activity was 20.3% of the initial activity. With the most effective polyanion (dextran sulfate), PEGylation could be completely suppressed; the residual activity was 98.4%. The protective effect of polyanions was attributed to electrostatic interactions with proteins; the "shielding" could be reversed by adding high salt concentrations. Furthermore, the protective effect was dependent on the concentration and molecular mass of the polyanion, but almost independent of the protein concentration. As a proof of concept, hydrogels were loaded with lysozyme and bevacizumab during cross-linking via DA reaction. Without polyanions, a large fraction of the protein was covalently bound to the polymer network resulting in degradation-controlled release; the residual activity of lysozyme was 50.0%. With polyanions, the protein molecules were mobile and their release was diffusion-controlled. The residual activity of lysozyme was 88.9%; the released bevacizumab was structurally intact. Polyanions can, therefore, be used as protective additive to prevent chemical protein modification during hydrogel cross-linking.
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http://dx.doi.org/10.1016/j.jconrel.2016.07.030DOI Listing
September 2016

Design of hydrogels for delayed antibody release utilizing hydrophobic association and Diels-Alder chemistry in tandem.

J Mater Chem B 2016 May 3;4(19):3398-3408. Epub 2016 May 3.

Department of Pharmaceutical Technology, Faculty of Chemistry and Pharmacy, University of Regensburg, 93040 Regensburg, Germany.

Biodegradable hydrogels were prepared from furan- and maleimide-functionalized eight-armed poly(ethylene glycol) with an average molecular mass of 40 000 Da (8armPEG40k-furan and 8armPEG40k-maleimide) using the Diels-Alder (DA) reaction as a cross-linking mechanism. Hydrophobic 6-aminohexanoic acid (C) and 12-aminododecanoic acid (C) spacers were introduced between the polymer backbone and the functional end-groups; the influence on the gel properties was studied. Modification with C and C spacers induced hydrophobic interactions between the macromonomers leading to association and increased viscosity of the polymer solutions; both effects were influenced by the spacer length. In combination with DA cross-linking, hydrophobic derivatives of 8armPEG40k-furan and 8armPEG40k-maleimide led to hydrogels with improved properties. Upon introduction of C spacers, gelation of 8armPEG40k hydrogels occurred twice as fast. Interestingly, no effect was observed when only one of the two components had been modified. Our experiments suggest that the association of macromonomers by hydrophobic interactions facilitates chemical cross-linking via DA chemistry. This hypothesis is supported by calculations of the network mesh size and the Young's modulus of compression, which showed an increased cross-linking density of hydrophobically modified hydrogels. As a consequence of the increased cross-linking density, the degradation stability of C-modified hydrogels increased by a factor of 4. Moreover, hydrophobic modification improved the hydrolytic resistance of maleimides; this also contributes to gel stability. The in vitro release of bevacizumab, which served as a model antibody, could be delayed for almost 60 days using modification with C. Similar trends were observed for C-modified 8armPEG40k hydrogels; however, the effects were considerably weaker. In summary, utilizing hydrophobic association and chemical cross-linking in tandem is a promising approach to create biodegradable hydrogels for delayed antibody release.
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http://dx.doi.org/10.1039/c6tb00223dDOI Listing
May 2016

Cyclic RGD peptides target human trabecular meshwork cells while ameliorating connective tissue growth factor-induced fibrosis.

J Drug Target 2016 12 27;24(10):952-959. Epub 2016 Mar 27.

b Department for Human Anatomy and Embryology , University of Regensburg , Regensburg , Germany.

The major risk factor for primary open-angle glaucoma is increased intraocular pressure stemming from elevated outflow resistance in the trabecular meshwork (TM) region. Integrins play a pivotal role in the TM by influencing its biological properties and growth factor signaling. Pathologic changes in the TM are partially mediated by growth factors like connective tissue growth factor (CTGF). Specific targeting of TM cells could play a critical clinical role by increasing the therapeutic efficacy of nanoparticles, e.g. for nonviral gene delivery. Quantum dots with cyclo(RGDfC) covalently immobilized to their surface effectively targeted cultured TM cells and were rapidly and efficiently endocytosed by binding to αβ and αβ integrins. Compared to the integrin-overexpressing U87-MG cell line, the association of RGD-modified nanoparticles with the TM cells was significantly higher. Binding and uptake into TM cells was receptor-mediated and suppressible with free peptide. Soluble cyclic RGD peptides effectively attenuated CTGF-mediated effects and inhibited CTGF signaling. Due to their antagonism for αvβ3 and αvβ5 integrins, these cyclic RGD pentapeptides effectively ameliorated the CTGF-induced effects and strongly promoted specific nanoparticle association. Thus, cyclic RGD peptides are powerful multifunctional ligands for both addressing nanomaterials to the TM and interfering with pathologic CTGF signaling upon arrival.
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http://dx.doi.org/10.3109/1061186X.2016.1163709DOI Listing
December 2016

A life full of life-science polymer research.

Eur J Pharm Biopharm 2015 Nov;97(Pt B):292

APV Focus Group 'Drug Delivery', Germany.

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http://dx.doi.org/10.1016/j.ejpb.2015.07.006DOI Listing
November 2015

HPLC analysis as a tool for assessing targeted liposome composition.

Int J Pharm 2016 Jan 10;497(1-2):293-300. Epub 2015 Nov 10.

University of Regensburg, Department for Pharmaceutical Technology, Universitätsstraße 31, 94030 Regensburg, Germany. Electronic address:

Functionalized phospholipids are indispensable materials for the design of targeted liposomes. Control over the quality and quantity of phospholipids is thereby key in the successful development and manufacture of such formulations. This was also the case for a complex liposomal preparation composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), Cholesterol (CHO), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (DSPE-PEG2000). To this end, an RP-HPLC method was developed. Detection was done via evaporative light scattering (ELS) for liposomal components. The method was validated for linearity, precision, accuracy, sensitivity and robustness. The liposomal compounds had a non-linear quadratic response in the concentration range of 0.012-0.42 mg/ml with a correlation coefficient greater than 0.99 with an accuracy of method confirmed 95-105% of the theoretical concentration. Furthermore, degradation products from the liposomal formulation could be identified. The presented method was successfully implemented as a control tool during the preparation of functionalized liposomes. It underlined the benefit of HPLC analysis of phospholipids during liposome preparation as an easy and rapid control method for the functionalized lipid at each preparation step as well as for the quantification of all components.
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http://dx.doi.org/10.1016/j.ijpharm.2015.11.014DOI Listing
January 2016

Multivalent nanoparticles bind the retinal and choroidal vasculature.

J Control Release 2015 Dec 19;220(Pt A):265-274. Epub 2015 Oct 19.

Department of Pharmaceutical Technology, University of Regensburg, Regensburg, Germany. Electronic address:

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.
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http://dx.doi.org/10.1016/j.jconrel.2015.10.033DOI Listing
December 2015

Diels-Alder Hydrogels for Controlled Antibody Release: Correlation between Mesh Size and Release Rate.

Mol Pharm 2015 Sep 27;12(9):3358-68. Epub 2015 Aug 27.

Department of Pharmaceutical Technology, Faculty of Chemistry and Pharmacy, University of Regensburg , 93040 Regensburg, Germany.

Eight-armed PEG, molecular mass 10 kDa, was functionalized with furyl and maleimide groups, respectively; the obtained macromonomers were cross-linked via Diels-Alder chemistry. The mesh size (ξ) of the prepared hydrogels was determined by swelling studies, rheology, and low field NMR spectroscopy. The in vitro release of fluorescein isothiocyanate labeled dextrans (FDs) and bevacizumab was investigated. The average mesh size (ξavg) increased from 5.8 ± 0.1 nm to 56 ± 13 nm during degradation, as determined by swelling studies. The result of the rheological measurements (8.0 nm) matched the initial value of ξavg. Low field NMR spectroscopy enabled the determination of the mesh size distribution; the most abundant mesh size was found to be 9.2 nm. In combination with the hydrodynamic radius of the molecule (Rh), the time-dependent increase of ξavg was used to predict the release profiles of incorporated FDs applying an obstruction-scaling model. The predicted release profiles matched the experimentally determined release profiles when Rh < ξavg. However, significant deviations from the theoretical predictions were observed when Rh ≥ ξavg, most likely due to the statistical distribution of ξ in real polymer networks. The release profile of bevacizumab differed from those of equivalently sized FDs. The delayed release of bevacizumab was most likely a result of the globular structure and rigidity of the protein. The observed correlation between ξ and the release rate could facilitate the design of controlled release systems for antibodies.
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http://dx.doi.org/10.1021/acs.molpharmaceut.5b00375DOI Listing
September 2015