New retinoblastoma (RB) drug delivery approaches: anti-tumor effect of atrial natriuretic peptide (ANP)-conjugated hyaluronic-acid-coated gold nanoparticles for intraocular treatment of chemoresistant RB.

Intraocular drug delivery is a promising approach for treatment of ocular diseases. Chemotherapeutic drugs used in retinoblastoma (RB) treatment often lead to side effects and drug resistances. Therefore, new adjuvant therapies are needed to treat chemoresistant RBs. Biocompatible gold nanoparticles (GNPs) have unique antiangiogenic properties and can inhibit cancer progression. The combination of gold and low-molecular-weight hyaluronan (HA) enhances the stability of GNPs and promotes the distribution across ocular barriers. Attached to HA-GNPs, the atrial natriuretic peptide (ANP), which diminishes neovascularization in the eye, is a promising new therapeutic agent for RB treatment. In the study presented, we established ANP-coupled HA-GNPs and investigated their effect on the tumor formation potential of chemoresistant RB cells in an in ovo chicken chorioallantoic membrane model and an orthotopic in vivo RB rat eye model. Treatment of etoposide-resistant RB cells with ANP-HA-GNPs in ovo resulted in significantly reduced tumor growth and angiogenesis compared with controls. The antitumorigenic effect could be verified in the rat eye model, including a noninvasive application form via eye drops. Our data suggest that ANP-HA-GNPs represent a new minimally invasive, adjuvant treatment option for RB.

Airway Delivery of Hydrogel-Encapsulated Niclosamide for the Treatment of Inflammatory Airway Disease.

Repurposing of the anthelminthic drug niclosamide was proposed as an effective treatment for inflammatory airway diseases such as asthma, cystic fibrosis, and chronic obstructive pulmonary disease. Niclosamide may also be effective for the treatment of viral respiratory infections, such as SARS-CoV-2, respiratory syncytial virus, and influenza. While systemic application of niclosamide may lead to unwanted side effects, local administration via aerosol may circumvent these problems, particularly when the drug is encapsulated into small polyethylene glycol (PEG) hydrospheres. In the present study, we examined whether PEG-encapsulated niclosamide inhibits the production of mucus and affects the pro-inflammatory mediator CLCA1 in mouse airways in vivo, while effects on mucociliary clearance were assessed in excised mouse tracheas. The potential of encapsulated niclosamide to inhibit TMEM16A whole-cell Cl- currents and intracellular Ca2+ signalling was assessed in airway epithelial cells in vitro. We achieved encapsulation of niclosamide in PEG-microspheres and PEG-nanospheres (Niclo-spheres). When applied to asthmatic mice via intratracheal instillation, Niclo-spheres strongly attenuated overproduction of mucus, inhibited secretion of the major proinflammatory mediator CLCA1, and improved mucociliary clearance in tracheas ex vivo. These effects were comparable for niclosamide encapsulated in PEG-nanospheres and PEG-microspheres. Niclo-spheres inhibited the Ca2+ activated Cl- channel TMEM16A and attenuated mucus production in CFBE and Calu-3 human airway epithelial cells. Both inhibitory effects were explained by a pronounced inhibition of intracellular Ca2+ signals. The data indicate that poorly dissolvable compounds such as niclosamide can be encapsulated in PEG-microspheres/nanospheres and deposited locally on the airway epithelium as encapsulated drugs, which may be advantageous over systemic application.

Monitoring the degradation of reduction-sensitive gene carriers with fluorescence spectroscopy and flow cytometry.

Polycations like poly(ethylene imine) (PEI) or poly(L-lysine) (pLL) form nanometer-sized complexes with nucleic acids (polyplexes) which can be used for gene delivery. It is known that the properties of these -carriers can be greatly improved by introducing disulfide bridges on the polymers, thus making them reduction sensitive. However, little is known about how such modified carriers behave intracellularly. Here, we describe a method that uses the reduction-sensitive fluorescent dye BODIPY FL L-cystine to label PEI and pLL. Our probe is activated under reductive conditions leading to strongly increased fluorescence intensity. Subsequently, we show how the intracellular route of polyplexes made from these labeled polymers can be monitored by flow cytometry.

Two different techniques to facilitate reconstruction of the long incus process with bone cement: a feasibility study in human cadaveric temporal bones.

The purpose of this feasibility study was to evaluate two novel techniques facilitating bone cement repair of ossicular discontinuity between the incus and stapes. An isolated damage of the long incus process can be repaired using bone cement. However, bridging of a large gap between incus remnant and stapes head with bone cement is difficult, since viscous cement is not stable and the wet cement bridge may collapse. Ten fresh-frozen cadaveric human temporal bones were used. The long process of the incus was subtotally resected. A novel instrument and polylactide acid (PLA) scaffolds were applied to support ossicular reconstruction with bone cement. Stability of cement bridging was tested by checking for a round window reflex or motion of the stapes by palpating the malleus handle. Both the instrument as well as the PLA scaffolds were relatively easy to insert into the middle ear. However, bone cement adhered to the instrument irrespective of cement viscosity and contact time of the instrument with the ossicles. The bone cement plug had to be detached and sculptured. By contrast, PLA scaffolds could be used in a standardized manner and generated stable cement reconstructions. Curved PLA scaffolds were superior to straight ones. Initial results in cadaveric human temporal bones suggest that implantable PLA scaffolds might be suitable to support bone cement repair, even in very large defects of the long incus process.

Tumor accumulation of NIR fluorescent PEG-PLA nanoparticles: impact of particle size and human xenograft tumor model.

Cancer therapies are often terminated due to serious side effects of the drugs. The cause is the nonspecific distribution of chemotherapeutic agents to both cancerous and normal cells. Therefore, drug carriers which deliver their toxic cargo specific to cancer cells are needed. Size is one key parameter for the nanoparticle accumulation in tumor tissues. In the present study the influence of the size of biodegradable nanoparticles was investigated in detail, combining in vivo and ex vivo analysis with comprehensive particle size characterizations. Polyethylene glycol-polyesters poly(lactide) block polymers were synthesized and used for the production of three defined, stable, and nontoxic near-infrared (NIR) dye-loaded nanoparticle batches. Size analysis based on asymmetrical field flow field fractionation coupled with multiangle laser light scattering and photon correlation spectroscopy (PCS) revealed narrow size distribution and permitted accurate size evaluations. Furthermore, this study demonstrates the constraints of particle size data only obtained by PCS. By the multispectral analysis of the Maestro in vivo imaging system the in vivo fate of the nanoparticles next to their accumulation in special red fluorescent DsRed2 expressing HT29 xenografts could be followed. This simultaneous imaging in addition to confocal microscopy studies revealed information about the accumulation characteristics of nanoparticles inside the tumor tissues. This knowledge was further combined with extended size-dependent fluorescence imaging studies at two different xenograft tumor types, the HT29 (colorectal carcinoma) and the A2780 (ovarian carcinoma) cell lines. The combination of two different size measurement methods allowed the characterization of the dependence of nanoparticle accumulation in the tumor on even rather small differences in the nanoparticle size. While two nanoparticle batches (111 and 141 nm in diameter) accumulated efficiently in the human xenograft tumor tissue, the slightly bigger nanoparticles (diameter 166 nm) were rapidly eliminated by the liver.

How stealthy are PEG-PLA nanoparticles? An NIR in vivo study combined with detailed size measurements.

PURPOSE: Detailed in vivo and ex vivo analysis of nanoparticle distribution, accumulation and elimination processes were combined with comprehensive particle size characterizations. METHODS: The in vivo fate of near infrared (NIR) nanoparticles in nude mice was carried out using the Maestro™ in vivo fluorescence imaging system. Asymmetrical field flow field fractionation (AF4) coupled with multi-angle laser light scattering (MALLS), photon correlation spectroscopy (PCS) and transmission electron microscopy (TEM) were employed for detailed in vitro characterization. RESULTS: PEG-PLA block polymers were synthesized and used for the production of defined, stable, nontoxic nanoparticles. Nanoparticle analysis revealed narrow size distribution; AF4/MALLS permitted further accurate size evaluation. Multispectral fluorescence imaging made it possible to follow the in vivo fate non-invasively even in deep tissues over several days. Detailed fluorescence ex vivo imaging studies were performed and allowed to establish a calculation method to compare nanoparticle batches with varying fluorescence intensities. CONCLUSION: We combined narrow-size distributed nanoparticle batches with detailed in vitro characterization and the understanding of their in vivo fate using fluorescence imaging, confirming the wide possibilities of the non-invasive technique and presenting the basis to evaluate future size-dependent passive tumor accumulation studies.

Enzymatically degradable poly(ethylene glycol) based hydrogels for adipose tissue engineering.

Adipose tissue engineering requires biomaterials that promote the differentiation of seeded adipocytes. Here, we report on the development and characterization of in situ forming, poly(ethylene glycol) (PEG) based hydrogels for soft tissue augmentation. Branched PEG-amines were modified with collagenase-sensitive peptides and cross-linked with branched PEG-succinimidyl propionates without the use of free-radical initiators (enzymatically degradable hydrogels). Alanine-modified PEG-amines were used for the preparation of non-degradable gels. Depending on the used polymer concentration, the strength of degradable gels after swelling ranged from 1708 to 7412 Pa; the strength of non-degradable hydrogels varied between 1496 and 7686 Pa. Enzyme mediated gel degradation occurred within 10, 16, and 19 days (5%, 10%, and 15% initial polymer content). To evaluate their suitability as scaffold materials for adipose tissue engineering, the hydrogels were functionalized with the laminin-derived adhesion peptide YIGSR, and seeded with 3T3-L1 preadipocytes. Compared to a standard two-dimensional cell culture model, the developed hydrogels significantly enhanced the intracellular triglyceride accumulation of encapsulated adipocytes. Functionalization with YIGSR further enhanced lipid synthesis within differentiating adipocytes. Long-term studies suggested that enzymatically degradable hydrogels furthermore promote the formation of coherent adipose tissue-like structures featuring many mature unilocular fat cells.

Effects of a dexamethasone-releasing stent on osteoneogenesis in a rabbit model.

BACKGROUND: This study is an evaluation of wound healing in an animal model for surgery of frontal sinusitis and treatment effect of topically released dexamethasone using a drug-releasing stent with special emphasis of osteoneogenesis. METHODS: A prospective, controlled, randomized, double-blinded animal study was performed. Nineteen New Zealand white rabbits were subjected to surgery via an external approach, a 4-mm circular wound was created on the medial side of the maxillary sinus and the underlying bone was denuded of periosteum. The wound was covered in a randomized fashion with either a silicone foil or a new dexamethasone-releasing stent system. Twelve to 30 days later, the animals were killed and a histological examination was performed. RESULTS: In comparison with the baseline bony thickness (40 micrometer) obtained in one animal, osteoneogenesis occurred on both paranasal sides but was significantly less if a dexamethasone-releasing stent was applied (117 [95% CI, 116-128]; 52 [95% CI, 43-64]; p < 0.001). Maximal bony thickness was observed in both treatment groups between days 20 and 25 with a tendency toward a higher percentage decrease in the dexamethasone-treated sides (p < 0.08). Using a visual analog scale (0-5) a significantly smoother bony surface was observed for dexamethasone (2 [95% CI, 1.1-1.9]; 2 [95% CI, 1.8-2.2]; p < 0.01). CONCLUSION: Using a new drug-releasing stent system, dexamethasone efficiently decreases postoperative osteoneogenesis in a standardized animal wound model for endoscopic sinus surgery. Therefore, the use of this system may be of value to decrease restenosis rates using corticosteroids in selected patients after frontal sinus surgery, especially the endoscopic modified Lothrop procedure.

Effects of topically applied dexamethasone on mucosal wound healing using a drug-releasing stent.

OBJECTIVE/HYPOTHESIS: Evaluation of the impact of continuously topically released dexamethasone using a drug-releasing stent on quality of regenerated mucosa after full thickness injury in the paranasal sinuses. STUDY DESIGN: Prospective, controlled, randomized, double-blinded animal study. METHODS: Nineteen New Zealand white rabbits were subjected to surgery: via an external approach, a 4 mm circular wound was created on the medial side of the maxillary sinus. The wound was covered in a randomized fashion with either a silicone foil or a new drug releasing stent system. Twelve to 30 days later, the animals were killed and histology and electron microscopy were performed. One animal was used for baseline comparisons at day 0. RESULTS: No animals were lost due to infection or dislocation of the stent, leaving 18 animals for evaluation of postoperative healing quality. According to macroscopic examination, extent of granulations was smaller in the treatment group (dexamethasone: median 0 [95% confidence interval: 0.1-0.6]) than the silicone group (2 [1.5-2.3]; P < or = .05). Epithelial wound healing was complete in all specimens, whereas the stroma was significantly thinner in the dexamethasone-group (44 [37-60]; 178 [148-214]). Improved healing quality was achieved significantly more often on the treatment, than on the control side. Scanning electron microscopy revealed no difference between both groups. CONCLUSIONS: Using a new drug-releasing stent system, dexamethasone efficiently decreases granulation formation and stroma thickness without impeding epithelial differentiation. Therefore, the use of this system may be of value to decrease restenosis rates in selected patients after frontal sinus surgery.

Soluble signalling factors derived from differentiated cartilage tissue affect chondrogenic differentiation of rat adult marrow stromal cells.

BACKGROUND: Chondral defects show lack of proper regeneration whereas osteochondral lesions display limited regeneration capacity. Latter is probably due to immigration of chondroprogenitor cells from the subchondral bone. Known chondroprogenitor cells for cartilage tissues are multi-potent adult marrow stromal or mesenchymal stem cells (MSCs). In vitro chondrogenic differentiation of these precursor cells usually require cues from growth and signalling factors provided in vivo by surrounding tissues and cells. We hypothesise that signalling factors secreted by differentiated cartilage tissue can initiate and maintain chondrogenic differentiation status of MSCs. METHODS: To study such paracrine communication between allogenic rat articular cartilage and rat MSCs embedded in alginate beads a novel coculture system without addition of external growth factors has been established. RESULTS: Impact of cartilage on differentiating MSCs was observed at two different time points. Firstly, sustained expression of Sox9 was observed at an early stage which indicated induction of chondrogenic differentiation. Secondly, late stage repression of collagen X indicated pre-hypertrophic arrest of differentiation. In the culture supernatant we have identified vascular endothelial growth factor alpha (VEGF-164 alpha), matrix metalloproteinase (MMP) -13 and tissue inhibitors of MMPs (TIMP-1 and TIMP-2) which could be traced back either to the cartilage explant or to the MSCs under the influence of cartilage. CONCLUSION: The identified factors might be involved in regulation of collagen X gene and protein expression and therefore, may have an impact on the control and regulation of MSCs differentiation.

Measuring cell adhesion on RGD-modified, self-assembled PEG monolayers using the quartz crystal microbalance technique.

In this study, the suitability of a flow-through quartz crystal microbalance system for the detection of the adhesion of rMSCs and 3T3-L1 fibroblasts on different surfaces is demonstrated. Frequency shifts for rMSCs of -6.7 mHz x cell(-1) and -2.0 mHz x cell(-1) for 3T3-L1 cells could be detected on non-modified gold sensors, revealing that the frequency shift per cell is comparable to that of a static setup. Modifying the sensor surface with SAMs of thioalkylated omega-amine-terminated PEG derivatives led to cell-adhesion-resistant surfaces. Total frequency shifts of only -20 +/- 7 Hz showed that protein adsorption was also significantly reduced. Attaching 35 pmol x mm(-2) of the GRGDS cell adhesion motif to the SAMs induced specific cell adhesion due to RGD-integrin interactions; the resonance frequency dropped by 3.4 mHz x cell(-1). Furthermore, the kinetics of cell detachment could be determined. The corresponding processes were completed after 10 min for trypsin, and not before 90 min with GRGDS. Moreover, the detectability of cell adhesion was shown to increase after the addition of manganese cations. The total decrease in the resonance frequency was almost 80 Hz in the presence of Mn(2+) (6.4 mHz x cell(-1)). [image: see text] Staining the cytoskeleton of the rMSCs shows that the GRGDS-modified surfaces are almost completely covered with well-spread cells.

The use of poly(ethylene glycol)-block-poly(lactic acid) derived copolymers for the rapid creation of biomimetic surfaces.

For many tissue engineering applications biomimetic or bioactive polymers would allow for a more precise control of cell behavior in growing tissues than has so far been possible. For this application recently developed amine reactive diblock copolymers (N-succinimidyl tartrate monoamine poly(ethylene glycol)-block-poly(D,L-lactic acid) [ST-NH-PEGxPLAy]) were investigated concerning their reactivity in binding model substances. Their ability to covalently immobilize proteins on their surfaces was examined using polymer films with amine reactive surfaces. Furthermore, thiol reactive polymers were obtained by attaching N-succinimidyl 3-maleinimido propionate, a thiol reactive linker to monoamine poly(ethylene glycol)-block-poly(D,L-lactic acid) [H2N-PEGxPLAy]. This allowed the immobilization of proteins carrying free thiol groups. The amine and thiol reactive polymers were characterized by 1H-NMR spectroscopy and gel permeation chromatography (GPC). Investigation of glass transitions temperatures using modulated differential scanning calorimetry proved suitability for the fabrication of polymeric scaffolds for tissue engineering applications. The functionality of the polymers was demonstrated by investigating their ability to bind model amines, like the fluorescent dye EDANS. Moreover, insulin and somatostatin were covalently attached to the active linker groups via amine and thiol groups. The polymers will permit covalently attaching different bioactive molecules, such as growth and differentiation factors, with fast and gentle procedures securing their biological activity.

Innovative frontal sinus stent acting as a local drug-releasing system.

Contemporary endonasal sinus surgery has given rise to distinct extended procedures focusing on the frontal sinus. However, surgical results sometimes are flawed, with reactive scarring leading to a relapse of insufficiency of drainage and ventilation. Topical application of medicines may offer help, but the hidden operative field around the frontal sinus is not reached by the usual nasal drugs. The effectiveness of an intraoperative insertion of stents is still a subject of debate in the literature. In previous studies we have seen some positive results. Based on this fact we looked for additional modalities to boost the effectiveness of fronto-nasal stents. We present a new device acting both as a stent and also as a local drug-releasing system. The combination of two therapeutic modalities may improve our treatment results in endonasal frontal sinus surgery. The pharmaceutical basics of our device are presented, and the first clinical data are shown. The first clinical trial was completely successful. Modification of the polymer and also of the released pharmaceutical agents may provide future improvements and may allow additional applications of the system in other areas of our surgical specialty.

Acylation of peptides by lactic acid solutions.

To simplify the search for effective mechanisms to suppress peptide acylation inside drug delivery devices made of poly(D,L-lactic acid) (PLA) and poly(lactic-co-glycolic acid), we were looking for a suitable model system that would allow screening of strategies for peptide stabilization. With their low pH and the presence of lactic acid oligomers, diluted lactic acid solutions promised to be a suitable test system that mimics the microclimate in degrading PLA devices. We created solutions of 1-50% (w/w) lactic acid by dilution of concentrated lactic acid. Using high performance liquid chromatography (HPLC) and high performance liquid chromatography coupled with mass spectrometry (HPLC-MS) analysis, oligomer hydrolysis was monitored during the equilibration process of the diluted solutions. Their final oligomer content was determined by titration and by calculations based on HPLC data. HPLC-MS analysis of human atrial natriuretic peptide (ANP) stability in different lactic acid solutions at 37 degrees C for 4 weeks demonstrated that ANP underwent acylation even in diluted solutions containing only 0.05% (w/w) lactic acid oligomers. Purity analysis of lactic acid solutions allowed us to compare the conditions in the solution test-system to the microclimate that prevails inside degrading PLA microspheres.

The effect of poly(ethylene glycol)-poly(D,L-lactic acid) diblock copolymers on peptide acylation.

The combination of poly(ethylene glycol) (PEG) with a biodegradable poly(ester), such as poly(D,L-lactic acid) (PLA), is an approach that has been successfully used for the stabilization of proteins and peptides in several biodegradable delivery devices. The acylation of peptides inside degrading PLA microspheres has been described only recently as another instability mechanism related to the accumulation of polymer degradation products inside eroding PLA. We investigated whether the block copolymerization of PLA with PEG reduces peptide acylation inside degrading microspheres. Diblock copolymers consisting of poly(D,L-lactic acid) covalently bound to poly(ethylene glycol)-monomethyl ether (Me.PEG-PLA) were used for these investigations. Human atrial natriuretic peptide (ANP) was incorporated into microspheres manufactured from Me.PEG5-PLA45, a diblock copolymer with an overall PEG content of 10%. Peptide integrity inside the microspheres was monitored by HPLC-MS analysis during 4 weeks of microsphere degradation in isotonic phosphate buffer (pH 7.4) at 37 degrees C. Inside the degrading Me.PEG5-PLA45 microspheres, acylation products as well as an oxidation product of ANP were formed. The results demonstrate that the combination of PEG with PLA does not necessarily display a favorable effect concerning peptide acylation inside degrading polymer microspheres. However, they also suggested that the acylation reaction is mainly driven by the formation and accumulation of polymer degradation products inside the degrading microspheres.

Peptide acylation by poly(alpha-hydroxy esters).

PURPOSE: Poly(lactic acid) (PLA) and poly(lactic-co-glycolic acid) (PLGA) microspheres were investigated concerning the possible acylation of incorporated peptides. METHODS: Atrial natriuretic peptide (ANP) and salmon calcitonin (sCT) were encapsulated into PLA and PLGA microspheres. Peptide integrity was monitored by HPLC-MS analysis during microsphere degradation for four weeks. sCT fragmentation with endoproteinase Glu-C was used for identifying modified amino acids. Peptide stability in lactic acid solutions was investigated to elucidate possible mechanisms for preventing peptide acylation. RESULTS: Both peptides were acylated by lactic and glycolic acid units inside degrading microspheres in a time-dependent manner. After 21 days, 60% ANP and 7% sCT inside PLA microspheres were acylated. Fragmentation of sCT with endoproteinase Glu-C revealed that besides the N-terminal amine group, lysine, tyrosine or serine are further possible targets to acylation. Stability studies of the peptides in lactic acid solutions suggest that oligomers are the major acylation source and that lower oligomer concentration and higher pH substantially decreased the reaction velocity. CONCLUSIONS: The use of PLA and PLGA for drug delivery needs substantially more circumspection. As, according to FDA standards. the potential hazards of peptide acylation products need to be assessed, our findings may have significant implications for products already on the market. Techniques to minimize the acylation reaction are suggested.