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[Display omitted] The specific delivery of a drug to its site of action also known as targeted drug delivery is a topic in the field of pharmaceutics studied for decades. One approach extensively investigated in this context is the use ligand functionalized nanoparticles. These particles are modified to carry receptor specific ligands, enabling them to accumulate at a desired target site. However, while this concept initially appears straightforward to implement, in-depth research has revealed several challenges hindering target site specific particle accumulation - some of which remain unresolved to this day. One of these challenges consists in the still incomplete understanding of how nanoparticles interact with biological systems. This knowledge gap significantly compromises the predictability of particle distribution in biological systems, which is critical for therapeutic efficacy. One of the most crucial steps in delivery is the attachment of nanoparticles to cells at the target site. This attachment occurs via the formation of multiple ligand receptor bonds. A process also referred to as multivalent interaction. While multivalency has been described extensively for individual molecules and macromolecules respectively, little is known on the multivalent binding of nanoparticles to cells. Here, we will specifically introduce the concept of avidity as a measure for favorable particle membrane interactions. Also, an overview about nanoparticle and membrane properties affecting avidity will be given. Thereafter, we provide a thorough review on literature investigating the correlation between nanoparticle avidity and success in targeted particle delivery. In particular, we want to analyze the currently uncertain data on the existence and nature of the correlation between particle avidity and biodistribution. [ABSTRACT FROM AUTHOR]

[Display omitted] The development of nanomedical devices has led to a considerable number of clinically applied nanotherapeutics. Yet, the overall poor translation of nanoparticular concepts into marketable systems has not met the initial expectations and led to increasing criticism in recent years. Most novel nano approaches thereby use highly refined formulations including a plethora of active targeting sequences, but ultimately fail to reach their target due to a generally high off-target deposition in organs such as the liver or kidney. In this context, we argue that initial nanoparticle (NP) development should not entirely become set on conventional formulation aspects. In contrast, we propose a change of focus towards a prior analysis of general sites of NP in vivo deposition and an assessment of how accumulation in these organs or tissues can be harnessed to develop therapies for site-related pathologies. We therefore give a comprehensive overview of existing nanotherapeutic targeting strategies for specific cell types within three of the usual suspects, i.e. the liver, kidney and the vascular system. We discuss the physiological surroundings and relevant pathologies of described tissues as well as the implications for NP-mediated drug delivery. Additionally, successful cell-selective NP concepts using active targeting strategies are assessed. By bringing together both (patho)physiological aspects and concepts for cell-selective NP formulations, we hope to show a novel opportunity for the development of more promising nanotherapeutic devices. [ABSTRACT FROM AUTHOR]

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. [Display omitted] [ABSTRACT FROM AUTHOR]

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 pH 7.4; 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. [ABSTRACT FROM AUTHOR]

Neovascular diseases of the posterior eye like age-related macular degeneration, proliferative diabetic retinopathy or retinopathy of prematurity carry a tremendous burden for patient and health care system alike. Although intravitreal injections of anti-VEGF based therapeutics have significantly improved the visual outcome for many patients, current therapeutic options still show significant drawbacks such as the injection-related risk of contracting an infection. Due to their ability to encapsulate drugs with otherwise poor bioavailability, accumulate in areas of increased vascular permeability and control the release of active ingredients over time, nanoparticle systems have been widely researched to enhance current therapeutic strategies and expand the therapeutic arsenal. In this review, emphasis is placed both on the possibilities and drawbacks that a systemic nanoparticle-based therapy could have in the context of neovascular posterior eye diseases. Recent investigations into intravenous and intravitreal administration of nanomaterials and their potential to deliver potent drugs and genes to pathologic lesions will also be presented. Furthermore, we will focus on the exceptional anti-oxidative and anti-angiogenic properties of selected nanoscale systems that carve out new paths for the treatment of these severe posterior eye diseases. [ABSTRACT FROM AUTHOR]

More and more people worldwide are affected by severe eye diseases eventually leading to visual impairment or blindness. In most cases, the treatment involves the application of ophthalmic dosage forms such as eye drops, suspensions or ointments. Unfortunately, some of the therapeutic approaches have major shortcomings, especially in the treatment of the posterior segment of the eye, where many vision-threatening diseases originate. Therefore, research focuses on the development of new materials ( e . g ., for vitreous substitution) and more advanced drug delivery systems. Hydrogels are an extremely versatile class of materials with many potential applications in ophthalmology. They found widespread application as soft contact lenses, foldable intraocular lenses, in situ gelling formulations for ophthalmic drug delivery and ocular adhesives for wound repair; their use as vitreous substitutes and intravitreal drug delivery systems is currently under investigation. In this article, we review the different applications of hydrogels in ophthalmology with special emphasis placed on the used polymers and their suitability as ocular drug delivery systems. [ABSTRACT FROM AUTHOR]

Abstract: In reconstructive surgery, there is a tremendous clinical need for adequate implants to repair soft tissue defects resulting from traumatic injury, tumor resection, or congenital anomalies. Adipose tissue engineering holds the promise to provide answers to this still increasing demand. The current approaches to adipose tissue engineering are comprehensively reviewed detailing the different cell carriers under investigation. A special focus is put on the applied cells. The delivered mesenchymal stem cells act in a dual role as building block of the new tissue and modulators of the host response. The conditioning of the cells in vitro prior to implantation decisively influences the tissue development and long-term survival in vivo. The special role of vascularization in adipose engineering is discussed. In all parts, key messages are defined providing the base for future advances in the generation of fat substitutes. [Copyright &y& Elsevier]

AbstractA major shortcoming in the treatment of mesangial cell-associated diseases such as IgA nephropathy, diabetic nephropathy, or lupus nephritis, which frequently progress to end-stage renal disease, is poor drug availability in the glomerular mesangium. Drug delivery via active targeting of nanoparticles, using ligands attached to the particle surface for target cell recognition to increase the biodistribution to the mesangium, is a promising strategy to overcome this hurdle. However, although several glomerular tissue targeting approaches have been described, so far no study has demonstrated the particles’ ability to deliver sufficient drug amounts combined with an appropriate nanoparticle target retention time to trigger relevant biological effects in the mesangium. In our study, we encapsulated erastin, a ferroptosis-inducing model compound, into adenovirus-mimetic, mesangial cell-targeting nanoparticles, enabling the direct visualisation of biological effects through ferroptosis-dependent histological changes. By intravital microscopy and analysis of histological sections, we were not only able to localise the injected particles over 10 days within the target cells but also to demonstrate biological activity in the renal glomeruli. In conclusion, we have characterised adenovirus-mimetic nanoparticles as a highly suitable drug delivery platform for the treatment of mesangial cell-associated diseases and additionally provided the basis for a potential renal disease model. [ABSTRACT FROM AUTHOR]

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. Unlabelled Image • Viruses possess characteristic traits leading to their unique targeting capacity. • Nanoparticles can implement these viral strategies to reach a better efficiency. • Mimicry includes surface design, ligand presentation and stimuli responsiveness. • Combination of all traits into an "artificial virus" can be a promising strategy. [ABSTRACT FROM AUTHOR]

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 Oct = 4.3 (Lucangioli et al., 2003) and Itraconazole (It) with a logP Oct = 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. [ABSTRACT FROM AUTHOR]

Interferon-γ (IFN-γ) is well known to reduce the infectivity of viral pathogens by altering their tissue tropism. This effect is induced by upregulation of cholesterol 25-hydroxylase (CH25H). Given the similarity of viral pathogens and ligand-functionalized nanoparticles in the underlying strategy of receptor-mediated cell recognition, it appears conceivable that IFN-γ exceeds similar effects on nanoparticles. Concretely, IFN-γ-induced activation of CH25H could decrease nanoparticle avidity for target cells via depletion of clathrin-coated pits. We hypothesized that this effect would cause deterioration of target-cell specific accumulation of nanoparticles. To prove our hypothesis, we investigated the cell tropism of angiotensin II functionalized nanoparticles (NP Lys-Ang II) in a co-culture system of angiotensin II subtype 1 receptor (AT 1 R) positive rat mesangial target cells (rMCs) and AT 1 R-negative HeLa off-target cells. In the presence of IFN-γ we observed an up to 5-fold loss of target cell preference for NP Lys-Ang II. Thus, our in vitro results suggest a strong influence of IFN-γ on nanoparticle distribution, which is relevant in the context of nanotherapeutic approaches to cancer treatment, as IFN-γ is strongly expressed in tumors. For the target cell tropism of viruses, our results provide a conclusive hypothesis for the underlying mechanism behind non-directed viral distribution in the presence of IFN-γ. [Display omitted] • IFN-γ impaired performance of ligand-decorated NPs in target/off-target cell co-culture. • Target cell-specific NP uptake, recognition, and directed accumulation were reduced. • Loss in NP performance was correlated with an increase in CH25H concentration. • Unsupervised-learning algorithm found subpopulations in cell co-culture NP distribution. [ABSTRACT FROM AUTHOR]

Hydrogel-based wound dressings are one of the most promising materials in wound care, fulfilling important dressing requirements, including: (a) keeping the wound moist whilst absorbing extensive exudate, (b) adhesion-free coverage of sensitive underlying tissue, (c) pain reduction through cooling and (d) a potential for active intervention in the wound healing process. Although numerous hydrogel-based dressings are already on the market, new wound care treatment options are urgently required to address the growing number of severe, acute and chronic wounds experienced by today’s aging society. High cost, safety concerns surrounding the use of drugs and allogeneic/xenogeneic materials, and issues specific to hydrogels, such as lack of mechanical stability, are impeding clinical implementation of many novel approaches. To meet clinical needs, the development of new hydrogel wound dressings that overcome these drawbacks would be desirable. This review provides a broad overview of recent progress – commercial and scientific – in the field of bioactive hydrogel wound dressings. A variety of approaches to bioactive wound healing, such as bioactive dressing materials, use of antimicrobial agents, inclusion of biological signaling molecules like growth factors, and incorporation of seeded stem cells, are considered and critically evaluated. [ABSTRACT FROM AUTHOR]

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 2000 -Mal) during the preparation of liposomes via two common preparation methods, which can be distinguished by the insertion of DSPE-PEG 2000 -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. [ABSTRACT FROM AUTHOR]

• Most retinal diseases underly multifactorial pathomechanisms. • The retinal pigment epithelium (RPE) plays a key part in these pathomechanisms. • Targeted RPE therapy can be used to efficiently treat retinal diseases. • Intravitreal and systemic nanoparticle systems can reach the RPE. • Guidance for the development of RPE-specific nanotherapeutics is provided. The retinal pigment epithelium (RPE) plays a crucial part in sight-threatening diseases. In this review, we shed light on the pivotal implication of the RPE in age-related macular degeneration, diabetic retinopathy and retinopathy of prematurity; and explain why a paradigm shift toward targeted RPE therapy is needed to efficiently fight these retinal diseases. We provide guidance for the development of RPE-specific nanotherapeutics by giving a comprehensive overview of the possibilities and challenges of drug delivery to the RPE and highlight successful nanotherapeutic approaches targeting the RPE. [ABSTRACT FROM AUTHOR]

The local administration of antibodies can represent in many cases a significant improvement for antibody-based therapies. The benefits of local delivery include high drug concentrations at the target site, the possibility of lower drug dosing and less systemic drug exposure. Currently, the most relevant delivery sites for therapeutic antibodies are the posterior segments of the eye, mucosal surfaces, the articular joints and the central nervous system (CNS). In addition, the oral and pulmonary route may enable non-invasive systemic antibody delivery. However, local antibody delivery to these sites is characterized by short drug residence times and a low compliance of administration. Controlled release (CR) systems can address these limitations and, thereby, enable and improve local delivery applications by achieving long lasting local drug concentrations, improved efficacy-dosing ratios and reduced treatment-associated side effects. The requirements for CR antibody formulations are more complex compared to conventional CR systems for small molecules, and their development poses an enormous technical challenge. Therefore, the review highlights experiences and challenges gathered in the development of the different CR systems for antibodies to date. Additionally, the unmet technological needs encountered in the field are described. This includes a critical evaluation of the limited capability of various CR systems to preserve antibody stability, delivery site specific considerations, as well as the processability of a CR system with a particular focus on drug loading and injectability. We believe that the success of CR and local delivery approaches could create an enormous added value for patients in the future. [ABSTRACT FROM AUTHOR]

[Display omitted] Immunization is a straightforward concept but remains for some pathogens like HIV-1 a challenge. Thus, new approaches towards increasing the efficacy of vaccines are required to turn the tide. There is increasing evidence that antigen exposure over several days to weeks induces a much stronger and more sustained immune response compared to traditional bolus injection, which usually leads to antigen elimination from the body within a couple of days. Therefore, we developed a poly(ethylene) glycol (PEG) hydrogel platform to investigate the principal feasibility of a sustained release of antigens to mimic natural infection kinetics. Eight-and four-armed PEG macromonomers of different MWs (10, 20, and 40 kDa) were end-group functionalized to allow for hydrogel formation via covalent cross-linking. An HIV-1 envelope (Env) antigen in its trimeric (Env tri) or monomeric (Env mono) form was applied. The soluble Env antigen was compared to a formulation of Env attached to silica nanoparticles (Env-SiNPs). The latter are known to have a higher immunogenicity compared to their soluble counterparts. Hydrogels were tunable regarding the rheological behavior allowing for different degradation times and release timeframes of Env-SiNPs over two to up to 50 days. Affinity measurements of the VCR01 antibody which specifically recognizes the CD4 binding site of Env, revealed that neither the integrity nor the functionality of Env mono -SiNPs (K d = 2.1 ± 0.9 nM) and Env tri -SiNPs (K d = 1.5 ± 1.3 nM), respectively, were impaired after release from the hydrogel (K d before release: 2.1 ± 0.1 and 7.8 ± 5.3 nM, respectively). Finally, soluble Env and Env-SiNPs which are two physico-chemically distinct compounds, were co-delivered and shown to be sequentially released from one hydrogel which could be beneficial in terms of heterologous immunization or single dose vaccination. In summary, this study presents a tunable, versatile applicable, and effective delivery platform that could improve vaccination effectiveness also for other infectious diseases than HIV-1. [ABSTRACT FROM AUTHOR]

Abstract: Mitochondria are of an increasing interest in pharmaceutical and medical research since it has been reported that dysfunction of these organelles contributes to several diseases with a great diversity of clinical appearance. By the fact that mitochondria are located inside the cell and, in turn, origins of mitochondrial diseases or targets of drugs are located inside mitochondria, a drug molecule has to cross several barriers. This is a severe drawback for the selective accumulation of drug molecules in mitochondria. Therefore, targeting strategies such as direct drug modification or encapsulation into nanocarriers have to be applied to achieve an accumulation of drug molecules in these organelles. In this review, it will be demonstrated how properties and dysfunctions of mitochondria are generating a need for the development of mitochondria specific therapies. Furthermore, intracellular targets of mitochondrial diseases, strategies to utilize mitochondrial specificities and targeting approaches will be discussed. Finally, techniques to investigate mitochondrial characteristics and functionality are reviewed. [Copyright &y& Elsevier]

Abstract: Hydrogels play an important role in drug delivery and tissue engineering applications due to their excellent biocompatibility and their variable mechanical and physical properties, which allow their optimization for many different aspects of the intended use. In this study, we examined the suitability of poly(ethylene glycol) (PEG)-based hydrogels as release systems for nanometer-sized drugs or drug carriers, like nanoparticles, using the radically cross-linkable oligo(poly(ethylene glycol)fumarate) (OPF) together with two cross-linking agents. Different fluorescent nanoparticulate probes with respect to size and physical structure were incorporated in the cross-linked hydrogels, and the obtained release profiles were correlated with the physical properties and the chemical structure of the gels, indicating a strong dependence of the release on the chosen PEG prepolymers. The prepared hydrogels were characterized by oscillatory rheometry and swelling experiments. Release experiments as well as diffusion measurements using fluorescence recovery after photobleaching showed the great potential of this type of hydrogels for the preparation of adjustable release systems by altering the molecular weights of the used PEG molecules. [Copyright &y& Elsevier]

Abstract Purpose  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). Methods  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. Results  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. Conclusions  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. [ABSTRACT FROM AUTHOR]

Abstract: When designing suitable biomaterials for tissue-engineering applications, biological and chemical parameters are frequently taken into account, while the equally important physical design variables have often been neglected. For a rational design of biomaterials, however, all variables influencing cell function and tissue morphogenesis have to be considered. This review will stress the development of cross-linked hydrogels and outline the impact of their physical properties on cell function and tissue morphogenesis. In the first part, the principles of cellular mechanosensitivity, as well as the influence of substrate mechanics on cell behavior, will be discussed. Afterwards, methods to characterize the mechanical properties of biomaterials will be presented. The subsequent chapters will address hydrogels that allow for the control of their physical qualities followed by a discussion of their use in tissue-engineering applications. [Copyright &y& Elsevier]

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 (SSTR2), 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 SSTR2, 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 SSTR2 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 SSTR2 overexpressed in blood cells with great potential for treating blood cancer. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

[Display omitted] • MLN-NP showed avidities with picomolar K d values for soluble ACE2 and target cells. • A fluorogenic ACE2 activity assay revealed an IC 50 value of the NPs of 2.88 nM. • Target cell selectivity was confirmed in mono- and coculture experiments. • CLSM proved the interaction of nanoparticles with the target enzyme. • Targeting ectoenzymes is beneficial regarding adverse drug effects. Pharmacotherapy is often limited by undesired side effects while insufficient drug reaches the site of action. Active-targeted nanotherapy should provide a solution for this problem, by using ligands in the nanoparticle corona for the identification of receptors on the target-cell surface. However, since receptor binding is directly associated with pharmacological responses, today's targeting concepts must be critically evaluated. We hypothesized that addressing ectoenzymes would help to overcome this problem, but it was not clear if particles would show sufficiently high avidity to provide us with a viable alternative to classical ligand-receptor concepts. We scrutinized this aspect by immobilizing the highly selective angiotensin-converting enzyme 2 (ACE2) inhibitor MLN-4760 in the corona of block-copolymer nanoparticles and investigated enzyme binding via microscale thermophoresis and flow cytometry. Excellent avidities with K d values as low as 243 pM for soluble ACE2 and 306 pM for ACE2-positive cells were obtained. In addition, the inhibitory activity had an IC 50 value of 2.88 nM. Reliable target cell identification could be proven in coculture experiments. High avidity is the basis for minimizing material loss to off-target sites and paves the way for a paradigm shift in nanoparticle targeting which does not trigger unintended side effects following target cell identification. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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% offtarget 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. [ABSTRACT FROM AUTHOR]

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 . [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 αvβ3 and αvβ5 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. [ABSTRACT FROM AUTHOR]

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-PEG 2000 ). 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. [ABSTRACT FROM AUTHOR]

The angiotensin II receptor type 1 (AT 1 R), 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 AT 1 R expression and inhibited their angiotensin receptor signaling with an IC 50 of 3.8 nM while showing only minor association to cells with low AT 1 R 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 AT 1 R-expressing off-target tissues such as the kidney. Following systemic application, this multivalent targeting approach has the potential to amplify AT 1 R blockade in the eye and concomitantly deliver a therapeutic payload into ocular lesions. [ABSTRACT FROM AUTHOR]

Eight-armed PEG was functionalized with furyl and maleimide groups (8armPEG20k-Fur and 8armPEG20k-Mal); degradable hydrogels were obtained by cross-linking via Diels–Alder chemistry. To increase the stability to degradation, the macromonomers were modified by introducing a hydrophobic 6-aminohexanoic acid spacer between PEG and the reactive end-groups (8armPEG20k-Ahx-Fur and 8armPEG20k-Ahx-Mal). In an alternative approach, the number of reactive groups per macromonomer was increased by branching the terminal ends of eight-armed PEG with lysine (Lys) and Ahx residues (8armPEG20k-Lys-Ahx-Fur 2 and 8armPEG20k-Lys-Ahx-Mal 2 ). The hydrolytic resistance of the synthesized macromonomers was determined by UV spectroscopy; the obtained hydrogels were characterized by rheology and degradation studies. The degradation time of 5% (w/v) 8armPEG20k-Ahx hydrogels (28 days) was twice as long as the degradation time of 5% (w/v) 8armPEG20k hydrogels (14 days); this is explained by increased hydrolytic resistance of the maleimide group. Using dendritic 8armPEG20k-Lys-Ahx macromonomers substantially increased the stability of the resulting hydrogels; degradation of 5% (w/v) 8armPEG20k-Lys-Ahx hydrogels occurred after 34 weeks. 8armPEG20k hydrogels had the largest mesh size of all tested hydrogels, while hydrogels made from dendritic 8armPEG20k-Lys-Ahx macromonomers showed the smallest value. To evaluate their potential for the controlled release of therapeutic antibodies, the hydrogels were loaded with bevacizumab. The incorporated bevacizumab was released over 10 days (8armPEG20k) and 42 days (8armPEG20k-Ahx), respectively; release from 8armPEG20k-Lys-Ahx hydrogels was not completed after 105 days. In summary, we believe that 8armPEG20k-Ahx or 8armPEG20k-Lys-Ahx hydrogels could serve as controlled release system for therapeutic antibodies such as bevacizumab. [ABSTRACT FROM AUTHOR]

The angiotensin II receptor type 1 (AT1R) is a G protein-coupled receptor of paramount significance since it is overexpressed in a number of diseased tissues that are highly attractive for nanoparticle targeting. However, it is also expressed at physiological levels in healthy tissue. Multivalent interactions mediated by multiple AT1R-binding moieties per nanoparticle could promote a high binding avidity to AT1R overexpressing cells and concomitantly spare off-target tissue. To investigate the feasibility of this approach, angiotensin II was thiolated and conjugated to PEGylated quantum dots. Nanoparticle binding, uptake and affinity to several cell lines was investigated in detail. The colloids were rapidly taken up by clathrin-mediated endocytosis into AT1R-expressing cells and showed no interaction with receptor negative cells. The EC50of the thiolated angiotensin II was determined to be 261 nM, whereas the ligand-conjugated Qdots activated the receptor with an EC50of 8.9 nM. This 30-fold higher affinity of the nanoparticles compared to the unconjugated peptide clearly demonstrated the presence of multivalent effects when using agonist-targeted nanoparticles. Our study provides compelling evidence that, despite being immediately endocytosed, Ang II-coupled nanoparticles exert potent multivalent ligand–receptor interactions that can be used to establish high affinities to an AT1R overexpressing cell and tissue. [ABSTRACT FROM PUBLISHER]

Liver sinusoidal endothelial cells (LSEC) are characterized by the presence of fenestrations that are not bridged by a diaphragm. The molecular mechanisms that control the formation of the fenestrations are largely unclear. Here we report that mice, which are deficient in plasmalemma vesicle-associated protein (PLVAP), develop a distinct phenotype that is caused by the lack of sinusoidal fenestrations. Fenestrations with a diaphragm were not observed in mouse LSEC at three weeks of age, but were present during embryonic life starting from embryonic day 12.5. PLVAP was expressed in LSEC of wild-type mice, but not in that of Plvap-deficient littermates. Plvap-/- LSEC showed a pronounced and highly significant reduction in the number of fenestrations, a finding, which was seen both by transmission and scanning electron microscopy. The lack of fenestrations was associated with an impaired passage of macromolecules such as FITC-dextran and quantum dot nanoparticles from the sinusoidal lumen into Disse's space. Plvap-deficient mice suffered from a pronounced hyperlipoproteinemia as evidenced by milky plasma and the presence of lipid granules that occluded kidney and liver capillaries. By NMR spectroscopy of plasma, the nature of hyperlipoproteinemia was identified as massive accumulation of chylomicron remnants. Plasma levels of low density lipoproteins (LDL) were also significantly increased as were those of cholesterol and triglycerides. In contrast, plasma levels of high density lipoproteins (HDL), albumin and total protein were reduced. At around three weeks of life, Plvap-deficient livers developed extensive multivesicular steatosis, steatohepatitis, and fibrosis. PLVAP is critically required for the formation of fenestrations in LSEC. Lack of fenestrations caused by PLVAP deficiency substantially impairs the passage of chylomicron remnants between liver sinusoids and hepatocytes, and finally leads to liver damage. [ABSTRACT FROM AUTHOR]

The conjugation of receptor ligands to shielded nanoparticles is a widely used strategy to precisely control nanoparticle–cell interactions. However, it is often overlooked that a ligand's affinity can be severely impaired by its attachment to the polyethylene glycol (PEG) chains that are frequently used to protect colloids from serum protein adsorption. Using the model ligand EXP3174, a small-molecule antagonist for the angiotensin II receptor type 1 (AT 1 R), we investigated the ligand's affinity before and after its PEGylation and when attached to PEGylated nanoparticles. The PEGylated ligand displayed a 580-fold decreased receptor affinity compared to the native ligand. Due to their multivalency, the nanoparticles regained a low nanomolar receptor affinity, which is in the range of the affinity of the native ligand. Moreover, a four orders of magnitude higher concentration of free ligand was required to displace PEGylated nanoparticles carrying EXP3174 from the receptor. On average, one nanoparticle was decorated with 11.2 ligand molecules, which led to a multivalent enhancement factor of 22.5 compared to the monovalent PEGylated ligand. The targeted nanoparticles specifically bound the AT 1 R and showed no interaction to receptor negative cells. Our study shows that the attachment of a small-molecule ligand to a PEG chain can severely affect its receptor affinity. Concomitantly, when the ligand is tethered to nanoparticles, the immense avidity greatly increases the ligand–receptor interaction. Based on our results, we highly recommend the affinity testing of receptor ligands before and after PEGylation to identify potent molecules for active nanoparticle targeting. [ABSTRACT FROM AUTHOR]

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 24h to 28days, depending on the used linker molecule. When formulated into hydrogels, a maximum of about 60% of the initial dose was released within 7days to 21days. 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. [ABSTRACT FROM AUTHOR]

Abstract: The sustained and localized delivery of monoclonal antibodies has become highly relevant, because of the increasing number of investigated local delivery applications in recent years. As the local delivery of antibodies is associated with high technological hurdles, very few successful approaches have been reported in the literature so far. Alginate-based delivery systems were previously described as promising sustained release formulations for monoclonal antibodies (mAbs). In order to further investigate their applicability, a single-dose animal study was conducted to compare the biocompatibility, the pharmacokinetics and the bioavailability of a human monoclonal antibody liquid formulation with two alginate-based sustained delivery systems after subcutaneous administration in rats. 28days after injection, the depot systems were still found in the subcutis of the animals. A calcium cross-linked alginate formulation, which was injected as a hydrogel, was present as multiple compartments separated by subcutaneous tissue. An in situ forming alginate formulation was recovered as a single compact and cohesive structure. It can be assumed that the multiple compartments of the hydrogel formulation led to almost identical pharmacokinetic profiles for all tested animals, whereas the compact nature of the in situ forming system resulted in large interindividual variations in pharmacokinetics. As compared to the liquid formulation the hydrogel formulations led to lower mAb serum levels, and the in situ forming system to a shift in the time to reach the maximum mAb serum concentration (Tmax) from 2 to 4days. Importantly, it was shown that after 28days only marginal amounts of residual mAb were present in the alginate matrix and in the tissue at the injection site indicating nearly complete release. In line with this finding, systemic drug bioavailability was not affected by using the controlled release systems. This study successfully demonstrates the suitability and underlines the potential of polyanionic systems for local and controlled mAb delivery. [Copyright &y& Elsevier]

With about 50–60million 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±5nm, a polydispersity index (PdI) of 0.23±0.03 and a neutral zeta potential of −0.07±0.05mV. After single administration to porcine eyes in vitro, 3165±597ngCsA/gcornea were detected in corneal tissue, while the levels of Restasis® a commercial formulation were, with 545±137ngCsA/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. [ABSTRACT FROM AUTHOR]

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 [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract: Based on the enhanced permeability and retention (EPR) effect, nanoparticles are believed to accumulate in tumors. In this conjunction, the stability of drug encapsulation is assumed to be sufficient. For clarification purposes, PEGylated poly-(d,l-lactic acid) (PEG-PDLLA) micelles which incorporated the hydrophobic model drug dechloro-4-iodo-fenofibrate (IFF) were investigated. H2N-PEG-PDLLA was synthesized, coupled to 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) and labeled with 111-indium. From this polymeric species, mixed micelles with H3CO-PEG-PDLLA were prepared which encapsulated the 125-iodine or 131-iodine labeled drug IFF. Bioimaging and biodistribution experiments in healthy and AR42J-tumor bearing mice were carried out to quantify the uptake of the drug and its carrier in single organs. As a result, upon injection of this system, a rapid dissociation of the polymeric carrier and the incorporated drug (<10min post inj.) was revealed. Regardless of the premature release, the drug showed an enhanced tumor accumulation compared to the polymeric carrier. In conclusion, the self-assembling system allowed for successful solubilization of the hydrophobic drug by physical incorporation into micelles whereas the tumor targeting properties of the drug delivery system could not be sufficiently shown. [Copyright &y& Elsevier]

Purpose: To gain mechanistic insights into drug loading and lyophilization of polymeric micelles. Methods: PEGylated poly-4-(vinylpyridine) micelles were loaded with dexamethasone. Three different methods were applied and compared: O/W emulsion, direct dialysis, cosolvent evaporation. Micellar dispersions with the highest drug load were lyophilized with varying lyoprotectors: sucrose, trehalose, maltose, a polyvinylpyrrolidine derivative, and β-cyclodextrin derivatives. For comparison, other PEGylated block copolymer micelles (PEGylated polylactic acid, polylactic acid-co-glycolic acid, polycaprolactone) were freeze-dried. Results: Drug loading via direct dialysis from acetone was a less effective loading method which led to dexamethasone loads <2% w/w. O/W emulsion technique from dichlormethane increased drug load up to ~13% w/w; optimized cosolvent evaporation increased load up to ~19% w/w. An important step for cosolvent evaporation was solubility screen of the drug prior to preparation. Loading was maintained upon lyophilization with β-cyclodextrins which proved to be versatile stabilizers for other block copolymer micelles. Conclusion: Careful solvent selection prior to cosolvent evaporation was a beneficial approach to load hydrophobic drugs into polymeric micelles. Moreover, β-cyclodextrins could be used as versatile lyoprotectors for these micelles. [ABSTRACT FROM AUTHOR]

Purpose: Insulin is a commonly used additive in chondrogenic media for differentiating mesenchymal stem cells (MSCs). The indispensability of other bioactive factors like TGF-β or dexamethasone in these medium formulations has been shown, but the role of insulin is unclear. The purpose of this study was to investigate whether insulin is essential for MSC chondrogenesis and if there is a dose-dependent effect of insulin on MSC chondrogenesis. Methods: We cultivated human MSCs in pellet culture in serum-free chondrogenic medium with insulin concentrations between 0 and 50 μg/ml and assessed the grade of chondrogenic differentiation by histological evaluation and determination of glycosaminoglycan (GAG), total collagen and DNA content. We further tested whether insulin can be delivered in an amount sufficient for MSC chondrogenesis via a drug delivery system in insulin-free medium. Results: Chondrogenesis was not induced by standard chondrogenic medium without insulin and the expression of cartilage differentiation markers was dose-dependent at insulin concentrations between 0 and 10 μg/ml. An insulin concentration of 50 μg/ml had no additional effect compared with 10 μg/ml. Insulin was delivered by a release system into the cell culture under insulin-free conditions in an amount sufficient to induce chondrogenesis. Conclusions: Insulin is essential for MSC chondrogenesis in this system and chondrogenic differentiation is influenced by insulin in a dose-dependent manner. Insulin can be provided in a sufficient amount by a drug delivery system. Therefore, insulin is a suitable and inexpensive indicator substance for testing drug release systems in vitro. [ABSTRACT FROM AUTHOR]

Abstract: A library of 39 strictly linear poly(ethylene glycol)–poly(ethylene imine) (PEG-PEI) diblock copolymers was synthesized for the delivery of plasmid DNA using PEG of 2, 5, or 10kDa in combination with linear PEI with a molecular weight (MW) ranging from 1.5 to 10.8kDa. In contrast to other approaches, the copolymers demonstrated a clear separation between the hydrophilic PEG and the nucleic acid condensing PEI moieties. Hence, the hypothesis was that PEG may not sterically counteract the interaction between the nucleic acid and PEI and that consequently, the copolymers are perfectly suited to build small and stable polyplexes. Analysis of the polyplexes revealed structure–function relationships and the general guideline was that the PEG domain had a greater influence on the physicochemical properties of the polyplexes than PEI. A PEG content higher than 50% led to small (<150nm), nearly neutral polyplexes with favorable stability. The transfection efficacy of these polyplexes was significantly reduced compared to the PEI homopolymer, but was restored by the application of the corresponding degradable copolymer, which involved a redox triggerable PEG domain. In conclusion, valuable design criteria for the optimization of gene delivery carriers, which is only possible through the screening of such a large library, were gained. [Copyright &y& Elsevier]

Abstract: We have investigated the ability of optical oxygen sensors incorporated in a microplate to determine the respiratory activity of cell fractions. Different cell fractions were monitored, in particular to evaluate the long term functionality of isolated mitochondria. It is possible to continuously sense respiratory activity of isolated mitochondria over time. We found that they are functional for three hours but stop respiring at a critical limit of 20% air saturation in the system. Furthermore, inhibition and enhancement of respiratory activity were detected. In conclusion, oxygen sensors are a powerful tool to evaluate the functionality of isolated mitochondria. [Copyright &y& Elsevier]

Polymeric micelles are ideal carriers for solubilization and targeting applications using hydrophobic drugs. Stability of colloidal aggregates upon injection into the bloodstream is mandatory to maintain the drugs' targeting potential and to influence pharmacokinetics. In this review we analyzed and discussed the most relevant stress mechanisms that polymeric micelles and related colloidal carriers encounter upon injection, including (1) dilution, (2) interactions with blood components, and (3) immunological responses of the body. In detail we analyzed the opsonin–dysopsonin hypothesis that points at a connection between a particles' protein-corona and its tissue accumulation by the enhanced permeability and retention (EPR) effect. In the established theory, size is seen as a necessary condition to reach nanoparticle accumulation in disease modified tissue. There is, however, mounting evidence of other sufficient conditions (e.g., particle charge, receptor recognition of proteins adsorbed onto particle surfaces) triggering nanoparticle extravasation by active mechanisms. In conclusion, the analyzed stress mechanisms are directly responsible for in vivo success or failure of the site-specific delivery with colloidal carrier systems. [ABSTRACT FROM AUTHOR]

Purpose: 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. Methods: 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). Results: 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%). Conclusion: 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. [ABSTRACT FROM AUTHOR]