Steerable intravitreal inserts for drug delivery: in vitro and ex vivo mobility experiments

The progress of wet age-related macular degeneration can now be controlled by intravitreal drug injection. This approach requires repeated injections, which could be avoided by delivering the drug to the retina. Intraocular implants are a promising solution for drug delivery near the retina. Currently, their accurate placement is challenging, and they can only be removed after a vitrectomy. In this paper, we introduce an approach for minimally invasive retinal drug delivery using magnetic intraocular inserts. We briefly discuss the electromagnetic-control system for magnetic implants and then focus on evaluating their ability to move in the vitreous humor. The mobility of magnetic intraocular implants is estimated in vitro with synthesized vitreous humors, and ex vivo with experiments on cadaver porcine eyes. Preliminary results show that with such magnetic implants a vitrectomy can be avoided.

Thermoreversible hyaluronan-based hydrogel supports in vitro and ex vivo disc-like differentiation of human mesenchymal stem cells

BACKGROUND CONTEXT The fate of human mesenchymal stem cells (hMSCs) supplied to the degenerating intervertebral disc (IVD) is still not fully understood and can be negatively affected by low oxygen, pH, and glucose concentration of the IVD environment. The hMSC survival and yield upon injection of compromised IVD could be improved by the use of an appropriate carrier and/or by predifferentiation of hMSCs before injection. PURPOSE To optimize hMSC culture conditions in thermoreversible hyaluronan-based hydrogel, hyaluronan-poly(N-isopropylacrylamide) (HA-pNIPAM), to achieve differentiation toward the disc phenotype in vitro, and evaluate whether preconditioning contributes to a better hMSC response ex vivo. STUDY DESIGN In vitro and ex vivo whole-organ culture of hMSCs. METHODS In vitro cultures of hMSCs were conducted in HA-pNIPAM and alginate for 1 week under hypoxia in chondropermissive medium alone and with the supplementation of transforming growth factor β1 or growth and differentiation factor 5 (GDF-5). Ex vivo, hMSCs were either suspended in HA-pNIPAM and directly supplied to the IVDs or predifferentiated with GDF-5 for 1 week in HA-pNIPAM and then supplied to the IVDs. Cell viability was evaluated by Live-Dead assay, and DNA, glycosaminoglycan (GAG), and gene expression profiles were used to assess hMSC differentiation toward the disc phenotype. RESULTS The HA-pNIPAM induced hMSC differentiation toward the disc phenotype more effectively than alginate: in vitro, higher GAG/DNA ratio and higher collagen type II, SOX9, cytokeratin-19, cluster of differentiation 24, and forkhead box protein F1 expressions were found for hMSCs cultured in HA-pNIPAM compared with those cultured in alginate, regardless of the addition of growth factors. Ex vivo, direct combination of HA-pNIPAM with the disc environment induced a stronger disc-like differentiation of hMSCs than predifferentiation of hMSCs followed by their delivery to the discs. CONCLUSIONS Hyaluronan-based thermoreversible hydrogel supports hMSC differentiation toward the disc phenotype without the need for growth factor supplementation in vitro and ex vivo. Further in vivo studies are required to confirm the suitability of this hydrogel as an effective stem cell carrier for the treatment of IVD degeneration.

Porcine extrahepatic vascular endothelial asialoglycoprotein receptor 1 mediates xenogeneic platelet phagocytosis in vitro and in human-to-pig ex vivo xenoperfusion.

BACKGROUND Asialoglycoprotein receptor-1 (ASGR1) mediates capture and phagocytosis of platelets in pig-to-primate liver xenotransplantation. However, thrombocytopenia is also observed in xenotransplantation or xenoperfusion of other porcine organs than liver. We therefore assessed ASGR1 expression as well as ASGR1-mediated xenogeneic platelet phagocytosis in vitro and ex vivo on porcine aortic, femoral arterial, and liver sinusoidal endothelial cells (PAEC/PFAEC/PLSEC). METHODS Porcine forelimbs were perfused with whole, heparinized human or autologous pig blood. Platelets were counted at regular intervals. Pig limb muscle and liver, as well as PAEC/PFAEC/PLSEC, were characterized for ASGR1 expression. In vitro, PAEC cultured on microcarrier beads and incubated with non-anticoagulated human blood were used to study binding of human platelets and platelet-white blood cell aggregation. Carboxyfluorescein diacetate succinimidyl ester-labeled human platelets were exposed to PAEC/PFAEC/PLSEC and analyzed for ASGR1-mediated phagocytosis. RESULTS Human platelet numbers decreased from 102 ± 33 at beginning to 13 ± 6 × 10/μL (P < 0.0001) after 10 minutes of perfusion, whereas no significant decrease of platelets was seen during autologous perfusions (171 ± 26 to 122 ± 95 × 10/μL). The PAEC, PFAEC, and PLSEC all showed similar ASGR1 expression. In vitro, no correlation was found between reduction in platelet count and platelet-white blood cell aggregation. Phagocytosis of human carboxyfluorescein diacetate succinimidyl ester-labeled platelets by PAEC/PFAEC/PLSEC peaked at 15 minutes and was inhibited (P < 0.05 to P < 0.0001) by rabbit anti-ASGR1 antibody and asialofetuin. CONCLUSIONS The ASGR1 expressed on aortic and limb arterial pig vascular endothelium plays a role in binding and phagocytosis of human platelets. Therefore, ASGR1 may represent a novel therapeutic target to overcome thrombocytopenia associated with vascularized pig-to-primate xenotransplantation.

Effect on infection resistance of a local antiseptic and antibiotic coating on osteosynthesis implants: an in vitro and in vivo study

The purpose of this study was to acquire information about the effect of an antibacterial and biodegradable poly-L-lactide (PLLA) coated titanium plate osteosynthesis on local infection resistance. For our in vitro and in vivo experiments, we used six-hole AO DC minifragment titanium plates. The implants were coated with biodegradable, semiamorphous PLLA (coating about 30 microm thick). This acted as a carrier substance to which either antibiotics or antiseptics were added. The antibiotic we applied was a combination of Rifampicin and fusidic acid; the antiseptic was a combination of Octenidin and Irgasan. This produced the following groups: Group I: six-hole AO DC minifragment titanium plate without PLLA; Group II: six-hole AO DC minifragment titanium plate with PLLA without antibiotics/antiseptics; Group III: six-hole AO DC minifragment titanium plate with PLLA + 3% Rifampicin and 7% fusidic acid; Group IV: six-hole AO DC minifragment titanium plate with PLLA + 2% Octenidin and 8% Irgasan. In vitro, we investigated the degradation and the release of the PLLA coating over a period of 6 weeks, the bactericidal efficacy of antibiotics/antiseptics after their release from the coating and the bacterial adhesion of Staphylococcus aureus to the implants. In vivo, we compared the infection rates in white New Zealand rabbits after titanium plate osteosynthesis of the tibia with or without antibacterial coating after local percutaneous bacterial inoculations at different concentrations (2 x 10(5)-2 x 10(8)): The plate, the contaminated soft tissues and the underlying bone were removed under sterile conditions after 28 days and quantitatively evaluated for bacterial growth. A stepwise experimental design with an "up-and-down" dosage technique was used to adjust the bacterial challenge in the area of the ID50 (50% infection dose). Statistical evaluation of the differences between the infection rates of both groups was performed using the two-sided Fisher exact test (p < 0.05). Over a period of 6 weeks, a continuous degradation of the PLLA coating of 13%, on average, was seen in vitro in 0.9% NaCl solution. The elution tests on titanium implants with antibiotic or antiseptic coatings produced average release values of 60% of the incorporated antibiotic or 62% of the incorporated antiseptic within the first 60 min. This was followed by a much slower, but nevertheless continuous, release of the incorporated antibiotic and antiseptic over days and weeks. At the end of the test period of 42 days, 20% of the incorporated antibiotic and 15% of the incorporated antiseptic had not yet been released from the coating. The antibacterial effect of the antibiotic/antiseptic is not lost by integrating it into the PLLA coating. The overall infection rate in the in vivo investigation was 50%. For Groups I and II the infection rate was both 83% (10 of 12 animals). In Groups III and IV with antibacterial coating, the infection rate was both 17% (2 of 12 animals). The ID50 in the antibacterial coated Groups III and IV was recorded as 1 x 10(8) CFU, whereas the ID50 values in the Groups I and II without antibacterial coating were a hundred times lower at 1 x 10(6) CFU, respectively. The difference between the groups with and without antibacterial coating was statistically significant (p = 0.033). Using an antibacterial biodegradable PLLA coating on titanium plates, a significant reduction of infection rate in an in vitro and in vivo investigation could be demonstrated. For the first time, to our knowledge, we were able to show, under standardized and reproducible conditions, that an antiseptic coating leads to the same reduction in infection rate as an antibiotic coating. Taking the problem of antibiotic-induced bacterial resistance into consideration, we thus regard the antiseptic coating, which shows the same level of effectiveness, as advantageous.

In Vitro and In Vivo Validation of Human and Goat Chondrocyte Labeling by Green Fluorescent Protein Lentivirus Transduction

We investigated whether human articular chondrocytes can be labeled efficiently and for long-term with a green fluorescent protein (GFP) lentivirus and whether the viral transduction would influence cell proliferation and tissue-forming capacity. The method was then applied to track goat articular chondrocytes after autologous implantation in cartilage defects. Expression of GFP in transduced chondrocytes was detected cytofluorimetrically and immunohistochemically. Chondrogenic capacity of chondrocytes was assessed by Safranin-O staining, immunostaining for type II collagen, and glycosaminoglycan content. Human articular chondrocytes were efficiently transduced with GFP lentivirus (73.4 +/- 0.5% at passage 1) and maintained the expression of GFP up to 22 weeks of in vitro culture after transduction. Upon implantation in nude mice, 12 weeks after transduction, the percentage of labeled cells (73.6 +/- 3.3%) was similar to the initial one. Importantly, viral transduction of chondrocytes did not affect the cell proliferation rate, chondrogenic differentiation, or tissue-forming capacity, either in vitro or in vivo. Goat articular chondrocytes were also efficiently transduced with GFP lentivirus (78.3 +/- 3.2%) and maintained the expression of GFP in the reparative tissue after orthotopic implantation. This study demonstrates the feasibility of efficient and relatively long-term labeling of human chondrocytes for co-culture on integration studies, and indicates the potential of this stable labeling technique for tracking animal chondrocytes for in cartilage repair studies.

Sodium/hydrogen exchanger NHA2 in osteoclasts: subcellular localization and role in vitro and in vivo

NHA2 was recently identified as a novel sodium/hydrogen exchanger which is strongly upregulated during RANKL-induced osteoclast differentiation. Previous in vitro studies suggested that NHA2 is a mitochondrial transporter required for osteoclast differentiation and bone resorption. Due to the lack of suitable antibodies, NHA2 was studied only on RNA level thus far. To define the protein's role in osteoclasts in vitro and in vivo, we generated NHA2-deficient mice and raised several specific NHA2 antibodies. By confocal microscopy and subcellular fractionation studies, NHA2 was found to co-localize with the late endosomal and lysosomal marker LAMP1 and the V-ATPase a3 subunit, but not with mitochondrial markers. Immunofluorescence studies and surface biotinylation experiments further revealed that NHA2 was highly enriched in the plasma membrane of osteoclasts, localizing to the basolateral membrane of polarized osteoclasts. Despite strong upregulation of NHA2 during RANKL-induced osteoclast differentiation, however, structural parameters of bone, quantified by high-resolution microcomputed tomography, were not different in NHA2-deficient mice compared to wild-type littermates. In addition, in vitro RANKL stimulation of bone marrow cells isolated from wild-type and NHA2-deficient mice yielded no differences in osteoclast development and activity. Taken together, we show that NHA2 is a RANKL-induced plasmalemmal sodium/hydrogen exchanger in osteoclasts. However, our data from NHA2-deficient mice suggest that NHA2 is dispensable for osteoclast differentiation and bone resorption both in vitro and in vivo.

Preclinical in vitro and in vivo characterization of the fully human monoclonal IgM antibody KBPA101 specific for Pseudomonas aeruginosa serotype IATS-O11

Pseudomonas aeruginosa infection in ventilator-associated pneumonia is a serious and often life-threatening complication in intensive care unit patients, and new treatment options are needed. We used B-cell-enriched peripheral blood lymphocytes from a volunteer immunized with a P. aeruginosa O-polysaccharide-toxin A conjugate vaccine to generate human hybridoma cell lines producing monoclonal antibodies specific for individual P. aeruginosa lipopolysaccharide serotypes. The fully human monoclonal antibody secreted by one of these lines, KBPA101, is an IgM/kappa antibody that binds P. aeruginosa of International Antigenic Typing System (IATS) serotype O11 with high avidity (5.81 x 10(7) M(-1) +/- 2.8 x 10(7) M(-1)) without cross-reacting with other serotypes. KBPA101 specifically opsonized the P. aeruginosa of IATS O11 serotype and mediated complement-dependent phagocytosis in vitro by the human monocyte-like cell line HL-60 at a very low concentration (half-maximal phagocytosis at 0.16 ng/ml). In vivo evaluation of KBPA101 demonstrated a dose-response relationship for protection against systemic infections in a murine burn wound sepsis model, where 70 to 100% of animals were protected against lethal challenges with P. aeruginosa at doses as low as 5 microg/animal. Furthermore, a high efficacy of KBPA101 in protection from local respiratory infections in an acute lung infection model in mice was demonstrated. Preclinical toxicology evaluation on human tissue, in rabbits, and in mice did not indicate any toxicity of KBPA101. Based on these preclinical findings, the first human clinical trials have been initiated.

In vitro and in vivo characterization of novel 18F-labeled bombesin analogues for targeting GRPR-positive tumors

The gastrin-releasing peptide receptor (GRPR) is overexpressed on a number of human tumors and has been targeted with radiolabeled bombesin analogues for the diagnosis and therapy of these cancers. Seven bombesin analogues containing various linkers and peptide sequences were designed, synthesized, radiolabeled with (18)F, and characterized in vitro and in vivo as potential PET imaging agents. Binding studies displayed nanomolar binding affinities toward human GRPR for all synthesized bombesin analogues. Two high-affinity peptide candidates 6b (K(i) = 0.7 nM) and 7b (K(i) = 0.1 nM) were chosen for further in vivo evaluation. Both tracers revealed specific uptake in GRPR-expressing PC-3 tumors and the pancreas. Compared to [(18)F]6b, compound [(18)F]7b was characterized by superior tumor uptake, higher specificity of tracer uptake, and more favorable tumor-to-nontarget ratios. In vivo PET imaging allowed for the visualization of PC-3 tumor in nude mice suggesting that [(18)F]7b is a promising PET tracer candidate for the diagnosis of GRPR-positive tumors in humans.

N-myc downstream regulated gene-1 expression correlates with reduced pancreatic cancer growth and increased apoptosis in vitro and in vivo

The role of N-myc downstream regulated gene-1 (NDRG1) in cancer has recently gained interest, as potential regulator of cell death and tumor suppressor. Although its normal function in the pancreas is largely unknown, loss of NDRG1 expression is associated with a more aggressive tumor phenotype and poor outcome in pancreatic cancer patients.

One-Step (18)F-Labeling of Carbohydrate-Conjugated Octreotate-Derivatives Containing a Silicon-Fluoride-Acceptor (SiFA): In Vitro and in Vivo Evaluation as Tumor Imaging Agents for Positron Emission Tomography (PET)

The synthesis, radiolabeling, and initial evaluation of new silicon-fluoride acceptor (SiFA) derivatized octreotate derivatives is reported. So far, the main drawback of the SiFA technology for the synthesis of PET-radiotracers is the high lipophilicity of the resulting radiopharmaceutical. Consequently, we synthesized new SiFA-octreotate analogues derivatized with Fmoc-NH-PEG-COOH, Fmoc-Asn(Ac?AcNH-?-Glc)-OH, and SiFA-aldehyde (SIFA-A). The substances could be labeled in high yields (38 ± 4%) and specific activities between 29 and 56 GBq/?mol in short synthesis times of less than 30 min (e.o.b.). The in vitro evaluation of the synthesized conjugates displayed a sst2 receptor affinity (IC?? = 3.3 ± 0.3 nM) comparable to that of somatostatin-28. As a measure of lipophilicity of the conjugates, the log P(ow) was determined and found to be 0.96 for SiFA-Asn(AcNH-?-Glc)-PEG-Tyr³-octreotate and 1.23 for SiFA-Asn(AcNH-?-Glc)-Tyr³-octreotate, which is considerably lower than for SiFA-Tyr³-octreotate (log P(ow) = 1.59). The initial in vivo evaluation of [¹?F]SiFA-Asn(AcNH-?-Glc)-PEG-Tyr³-octreotate revealed a significant uptake of radiotracer in the tumor tissue of AR42J tumor-bearing nude mice of 7.7% ID/g tissue weight. These results show that the high lipophilicity of the SiFA moiety can be compensated by applying hydrophilic moieties. Using this approach, a tumor-affine SiFA-containing peptide could successfully be used for receptor imaging for the first time in this proof of concept study.

Relating the physicochemical characteristics and dispersion of multiwalled carbon nanotubes in different suspension media to their oxidative reactivity in vitro and inflammation in vivo

Reactive oxygen species (ROS) production is important in the toxicity of pathogenic particles such as fibres. We examined the oxidative potential of straight (50 microm and 10 microm) and tangled carbon nanotubes in a cell free assay, in vitro and in vivo using different dispersants. The cell free oxidative potential of tangled nanotubes was higher than for the straight fibres. In cultured macrophages tangled tubes exhibited significantly more ROS at 30 min, while straight tubes increased ROS at 4 h. ROS was significantly higher in bronchoalveolar lavage cells of animals instilled with tangled and 10 mum straight fibres, whereas the number of neutrophils increased only in animals treated with the long tubes. Addition of dispersants in the suspension media lead to enhanced ROS detection by entangled tubes in the cell-free system. Tangled fibres generated more ROS in a cell-free system and in cultured cells, while straight fibres generated a slower but more prolonged effect in animals.

Effect of beam hardening on arterial enhancement in thoracoabdominal CT angiography with increasing patient size: an in vitro and in vivo study

To assess the effect of beam hardening on arterial enhancement in thoracoabdominal computed tomographic (CT) angiography in various body sizes in a phantom and in a clinical study.

Gonadotropin-releasing hormone type II antagonist induces apoptosis in MCF-7 and triple-negative MDA-MB-231 human breast cancer cells in vitro and in vivo

Triple-negative breast cancer does not express estrogen and progesterone receptors, and no overexpression/amplification of the HER2-neu gene occurs. Therefore, this subtype of breast cancer lacks the benefits of specific therapies that target these receptors. Today chemotherapy is the only systematic therapy for patients with triple-negative breast cancer. About 50% to 64% of human breast cancers express receptors for gonadotropin-releasing hormone (GnRH), which might be used as a target. New targeted therapies are warranted. Recently, we showed that antagonists of gonadotropin-releasing hormone type II (GnRH-II) induce apoptosis in human endometrial and ovarian cancer cells in vitro and in vivo. This was mediated through activation of stress-induced mitogen-activated protein kinases (MAPKs) p38 and c-Jun N-terminal kinase (JNK), followed by activation of proapoptotic protein Bax, loss of mitochondrial membrane potential, and activation of caspase-3. In the present study, we analyzed whether GnRH-II antagonists induce apoptosis in MCF-7 and triple-negative MDA-MB-231 human breast cancer cells that express GnRH receptors. In addition, we ascertained whether knockdown of GnRH-I receptor expression affects GnRH-II antagonist-induced apoptosis and apoptotic signaling.

A prokaryotic S1P lyase degrades extracellular S1P in vitro and in vivo: implication for treating hyperproliferative disorders

Sphingosine-1-phosphate (S1P) regulates a broad spectrum of fundamental cellular processes like proliferation, death, migration and cytokine production. Therefore, elevated levels of S1P may be causal to various pathologic conditions including cancer, fibrosis, inflammation, autoimmune diseases and aberrant angiogenesis. Here we report that S1P lyase from the prokaryote Symbiobacterium thermophilum (StSPL) degrades extracellular S1P in vitro and in blood. Moreover, we investigated its effect on cellular responses typical of fibrosis, cancer and aberrant angiogenesis using renal mesangial cells, endothelial cells, breast (MCF-7) and colon (HCT 116) carcinoma cells as disease models. In all cell types, wild-type StSPL, but not an inactive mutant, disrupted MAPK phosphorylation stimulated by exogenous S1P. Functionally, disruption of S1P receptor signaling by S1P depletion inhibited proliferation and expression of connective tissue growth factor in mesangial cells, proliferation, migration and VEGF expression in carcinoma cells, and proliferation and migration of endothelial cells. Upon intravenous injection of StSPL in mice, plasma S1P levels rapidly declined by 70% within 1 h and then recovered to normal 6 h after injection. Using the chicken chorioallantoic membrane model we further demonstrate that also under in vivo conditions StSPL, but not the inactive mutant, inhibited tumor cell-induced angiogenesis as an S1P-dependent process. Our data demonstrate that recombinant StSPL is active under extracellular conditions and holds promise as a new enzyme therapeutic for diseases associated with increased levels of S1P and S1P receptor signaling.