Enhanced taxane uptake into bladder tissues following co-administration with either mitomycin C, doxorubicin or gemcitabine: association to exfoliation processes.

OBJECTIVE: To investigate the effect of three anticancer drugs (mitomycin c (MMC), doxorubicin or gemcitabine) on bladder wall morphology and the uptake of paclitaxel or docetaxel following coadministration. The primary objective of this study was to measure the uptake of MMC, doxorubicin or gemcitabine with or without exposure of the tissue to amine terminated cationic nanoparticles (CNPs) and to investigate any possible exfoliation effects of the three drugs on intact bladder tissue. The secondary objective was to investigate the uptake of taxane drugs (docetaxel, DTX) and paclitaxel, (PTX) from surfactant micelle formulations in the presence of MMC, doxorubicin or gemcitabine. MATERIALS AND METHODS: Sections of fresh pig bladder tissue were incubated in Franz diffusion cells with the urothelial side exposed to solutions of doxorubicin, MMC and gemcitabine containing radioactive drug for 90 min. Some tissue samples were simultaneously exposed to each of the three drugs in combination with the surfactant micelle formulations of PTX (Taxol) or DTX (Taxotere). Tissue sections were then cryostat sectioned for drug quantitation by liquid scintillation counting or fixed for scanning electron microscopy and haematoxylin and eosin staining. RESULTS: All three drugs caused exfoliation of the urothelial layer of bladder tissues. Drug uptake studies showed that all three drugs effectively penetrated the lamina propria through to the muscular layer over a 2-h incubation and these levels were unaffected by pre-treatment with CNPs. The uptake levels of the taxane drugs PTX and DTX were significantly enhanced following simultaneous treatment of bladders with MMC, doxorubicin or gemcitabine. CONCLUSION: The exfoliation effects of MMC, doxorubicin and gemcitabine allow for good tissue penetration of these drugs with no additional effect from CNP treatment of bladders. The observed exfoliation effect of these amine-containing drugs probably arises from a cationic interaction with the mucus and urothelium cell layer in a manner similar to that previously reported for CNPs. These studies suggest that the lack of long-term clinical efficacy of these drugs may not arise from poor intravesical drug penetration but may result from a rapid diffusion of the drugs into the deeper vascularised muscular region with rapid drug clearance. The enhanced uptake of PTX or DTX following co-administration with MMC, doxorubicin or gemcitabine probably arises from the removal of the urothelial barrier by exfoliation allowing for improved taxane partitioning into superficial layers. These effects may allow for dual drug intravesical strategies offering greatly improved taxane uptake and potential additive drug effects for improved efficacy.

Cardiovascular Manifestations and Complications of Loeys-Dietz Syndrome: CT and MR Imaging Findings.

Loeys-Dietz syndrome (LDS) is a recently described genetic connective tissue disorder with a wide spectrum of multisystem involvement. LDS is characterized by rapidly progressive aortic and peripheral arterial aneurysmal disease. LDS and the other inherited aortopathies such as Marfan syndrome have overlapping phenotypic features. However, LDS is characterized by a more aggressive vascular course; patient morbidity and mortality occur at an early age, with complications developing at relatively smaller aortic dimensions. In addition, there is more diffuse arterial involvement in LDS, with a large proportion of patients developing aneurysms of the iliac, mesenteric, and intracranial arteries. Early diagnosis and careful follow-up are essential for ensuring timely intervention in patients with arterial disease. Cross-sectional angiography has an important role in the baseline assessment, follow-up, and evaluation of acute complications of LDS, the thresholds and considerations of which differ from those of other inherited aortopathies. In this article, LDS is compared with other genetic vascular connective tissue disorders. In addition, the genetic, histopathologic, and cardiovascular manifestations of this disease process are reviewed, with a focus on computed tomographic and magnetic resonance imaging findings. Online DICOM image stacks and supplemental material are available for this article. ©RSNA, 2018.

Tissue Permeability Effects Associated with the Use of Mucoadhesive Cationic Nanoformulations of Docetaxel in the Bladder.

PURPOSE: Recently, efficacy studies in mice have shown that amine-terminated cationic (CNP) nanoparticulate carriers of DTX offer an improved formulation of the drug for intravesical delivery. It is hypothesized that this improved efficacy may arise from a carrier mediated bladder exfoliation process that removes the urothelial barrier allowing for increased drug uptake into bladder tissue. The objective of this study was to investigate exfoliation processes in fresh pig's bladders (ex vivo) exposed to three cationic polyglycerols with increasing degrees of amination (denoted 350, 580 and 780). The study also compared the tissue depth profile of DTX uptake into these tissues using these different carriers. MATERIALS AND METHODS: Aminated polyglycerols were synthesized and characterized in the laboratory with low (CNP-360), medium (CNP-580) and high (CNP-780) levels of amine content. CNP-based DTX solutions and commercial DTX solutions in polysorbate 80 (Taxotere®) were doped with (3)H-radiolabeled DTX and prepared by solvent evaporation from acetonitrile, followed by drying and reconstitution in pH 6.4 buffer. Sections of fresh pig's bladder tissue were clamped into Franz diffusion cells and the urothelial side was exposed to the DTX solutions for 2 h. Tissue sections were then frozen for sectioning by cryotome sectioning and subsequently processed for drug analysis by liquid scintillation counting. Alternatively tissue sections were fixed in 2% glutaraldehyde and 2% paraformaldehyde in 0.1 M sodium cacodylate buffer for the purposes of scanning electron microscopy (SEM). RESULTS: Exposure of the urothelial surface to the amine-terminated polyglycerol solutions resulted in the exfoliation of bladder tissues in a time- and concentration-dependent manner. Exfoliation was significantly more pronounced when using CNPs with a medium or high levels of amination whereas only minor levels of exfoliation were seen with low levels. Following incubation of tissues in Tween-based commercial formulations (Taxotere) of DTX (0.5 mg/mL) the drug was detectable at low levels (10-40 µg/g tissue) in all depths of tissue. Similar drug uptake was observed using the CNP-360 formulation. However drug uptake levels were increased to 60-100 µg/g tissue when samples were incubated with either the CNP-580 or CNP-780 formulations. CONCLUSION: The use of cationic polyglycerols with higher levels of amine termination allows for an enhanced uptake of DTX into bladder tissues as compared to commercial (Taxotere) formulations. These increased drug levels probably arise from exfoliation processes resulting in a temporary elimination of the urothelial permeability barrier and increased drug penetration into the tissue.

Same-single-cell analysis using the microfluidic biochip to reveal drug accumulation enhancement by an amphiphilic diblock copolymer drug formulation.

Multidrug resistance (MDR) is one of the major obstacles in drug delivery, and it is usually responsible for unsuccessful cancer treatment. MDR may be overcome by using MDR inhibitors. Among different classes of these inhibitors that block drug efflux mediated by permeability-glycoprotein (P-gp), less toxic amphiphilic diblock copolymers composed of methoxypolyethyleneglycol-block-polycaprolactone (MePEG-b-PCL) have been studied extensively. The purpose of this work is to evaluate how these copolymer molecules can reduce the efflux, thereby enhancing the accumulation of P-gp substrates (e.g., daunorubicin or DNR) in MDR cells. Using conventional methods, it was found that the low-molecular-weight diblock copolymer, MePEG17-b-PCL5 (PCL5), enhanced drug accumulation in MDCKII-MDR1 cells, but the high-molecular-weight version, MePEG114-b-PCL200 (PCL200), did not. However, when PCL200 was mixed with PCL5 (and DNR) in order to encapsulate them to facilitate drug delivery, there was no drug enhancement effect attributable to PCL5, and the reason for this negative result was unclear. Since drug accumulation measured on different cell batches originated from single cells, we employed the same-single-cell analysis in the accumulation mode (SASCA-A) to find out the reason. A microfluidic biochip was used to select single MDR cells, and the accumulation of DNR was fluorescently measured in real time on these cells in the absence and presence of PCL5. The SASCA-A method allowed us to obtain drug accumulation information faster in comparison to conventional assays. The SASCA-A results, and subsequent curve-fitting analysis of the data, have confirmed that when PCL5 was encapsulated in PCL200 nanoparticles as soon as they were synthesized, the ability of PCL5 to enhance DNR accumulation was retained, thus suggesting PCL200 as a promising delivery system for encapsulating P-gp inhibitors, such as PCL5.

The use of tissue sealants to deliver antibiotics to an orthopaedic surgical site with a titanium implant.

BACKGROUND: Orthopaedic surgery is associated with unacceptable infection rates that respond poorly to systemic antibiotics. The objective of this study was to use an animal model for orthopaedic implant infection to examine the ability of a new-generation fibrin tissue sealant to effectively deliver antibiotics to the surgical site. METHODS: The antibiotics cefazolin, fusidic acid or 5-fluorouracil were blended into Vitagel tissue sealant. The release rate of the drugs was measured using HPLC methods and bioactivity was measured by the zone of inhibition method with pathogenic Staphylococcus aureus. The antibiotic activity of the drug-loaded sealant was then tested in rats using infected orthopaedic surgical sites (titanium clip on spine). Efficacy was evaluated by residual bacterial counts on clips, clinical observations of infection, and histological findings. RESULTS: The drugs were released in a controlled manner over 2-4 days. All three antibiotics demonstrated strong antibacterial activity when released from the sealants. None of the treated animals demonstrated systemic illness. Post mortem dissection revealed a well-encapsulated abscess surrounding the titanium clip with erosion of the bony process. Using an inoculum of 1-5 × 10(3) CFU, treatment with antibiotic-loaded fibrin sealant demonstrated reduced infective swelling and reduced bacterial counts on surgical clip swabs compared to control rats or rats treated with antibiotic only. This model allowed for almost 100 % infectivity with a 0 % mortality rate due to infection, mimicking the clinical features of human implant infection. CONCLUSION: The results support the use of antibiotic-loaded commercially available fibrin sealants to prevent infection after implant surgery.

Copolymer micelles and nanospheres with different in vitro stability demonstrate similar paclitaxel pharmacokinetics.

Paclitaxel loaded amphiphilic block copolymer nanoparticles have been demonstrated to enhance the aqueous solubility and improve the toxicity profile as compared to the commercially available product Taxol; however, in many cases long circulation of the drug is not achieved due to rapid partitioning of the drug from the carrier and/or carrier instability upon injection. In this work we investigated the effect of increasing the hydrophobic block length of methoxy poly(ethylene glycol)-block-poly(ε-caprolactone) (MePEG-b-PCL) copolymers on the physicochemical properties and in vitro stability of the formed nanoparticles as well as the pharmacokinetics and biodistribution of both the copolymer and solubilized drug. We hypothesized that copolymers composed of high molecular weight hydrophobic blocks (MePEG114-b-PCL104) that form nanoparticles with a kinetically "frozen core" (which we term nanospheres) would better retain their PTX payload as compared to micelles composed of shorter hydrophobic blocks (MePEG114-b-PCL19), thus leading to prolonged drug circulation. Nanospheres solubilized PTX more efficiently, released the drug in a more sustained fashion and were characterized by enhanced stability and drug retention in the presence of plasma proteins as compared to micelles. Using radiolabeled copolymers and PTX, it was found that, upon injection, MePEG114-b-PCL104 circulated for longer than MePEG114-b-PCL19; however, the drug was rapidly eliminated from the blood regardless of the formulation. These results suggest that, despite formulation in more stable nanospheres, PTX was still rapidly extracted from these nanoparticles.

Phase separation behavior of fusidic acid and rifampicin in PLGA microspheres.

The purpose of this study was to characterize the phase separation behavior of fusidic acid (FA) and rifampicin (RIF) in poly(d,l-lactic acid-co-glycolic acid) (PLGA) using a model microsphere formulation. To accomplish this, microspheres containing 20% FA with 0%, 5%, 10%, 20%, and 30% RIF and 20% RIF with 30%, 20% 10%, 5%, and 0% FA were prepared by solvent evaporation. Drug-polymer and drug-drug compatibility and miscibility were characterized using laser confocal microscopy, Raman spectroscopy, XRPD, DSC, and real-time video recordings of single-microsphere formation. The encapsulation of FA and RIF alone, or in combination, results in a liquid-liquid phase separation of solvent-and-drug-rich microdomains that are excluded from the polymer bulk during microsphere hardening, resulting in amorphous spherical drug-rich domains within the polymer bulk and on the microsphere surface. FA and RIF phase separate from PLGA at relative droplet volumes of 0.311 ± 0.014 and 0.194 ± 0.000, respectively, predictive of the incompatibility of each drug and PLGA. When coloaded, FA and RIF phase separate in a single event at the relative droplet volume 0.251 ± 0.002, intermediate between each of the monoloaded formulations and dependent on the relative contribution of FA or RIF. The release of FA and RIF from phase-separated microspheres was characterized exclusively by a burst release and was dependent on the phase exclusion of surface drug-rich domains. Phase separation results in coalescence of drug-rich microdroplets and polymer phase exclusion, and it is dependent on the compatibility between FA and RIF and PLGA. FA and RIF are mutually miscible in all proportions as an amorphous glass, and they phase separate from the polymer as such. These drug-rich domains were excluded to the surface of the microspheres, and subsequent release of both drugs from the microspheres was rapid and reflected this surface location.

The application of layered double hydroxide clay (LDH)-poly(lactide-co-glycolic acid) (PLGA) film composites for the controlled release of antibiotics.

Many sites of bacterial infection such as in-dwelling catheters and orthopedic surgical sites require local rather than systemic antibiotic administration. However, currently used controlled release vehicles, such as polymeric films, release water-soluble antibiotics too quickly, whereas nonporous bone cement, used in orthopedics, release very little drug. The purpose of this study was to investigate the use of nanoparticulates composed of layered double hydroxide clays to bind various antibiotics and release them in a controlled manner. Mg-Al (carbonate) layered double hydroxides were synthesized and characterized using established methods. These clay particles were suspended in solutions of the antibiotics tetracycline, doxorubicin (DOX), 5-fluorouracil, vancomycin (VAN), sodium fusidate (SF) and antisense oligonucleotides and binding was determined following centrifugation and quantitation of the unbound fraction by UV/Vis absorbance or HPLC analysis. Drug release from layered double hydroxide clay/drug complexes dispersed in polymeric films was measured by incubation in phosphate-buffered saline (pH 7.4) at 37 °C using absorbance or HPLC analysis. Antimicrobial activity of drug released from film composites was determined using zonal inhibition studies against S. epidermidis. All drugs bound to the clay particles to various degrees. Generally, drugs released with a large burst phase of release (except DOX) with little further drug release after 4 days. Dispersion of drug/clay complexes in poly(lactic-co-glycolic acid) films resulted in a reduced burst phase of release and a slow continuous release for many weeks with effective antimicrobial amounts of VAN and SF released at later time points. Layered double hydroxide clays may be useful for controlled release applications at sites requiring long-term antibiotic exposure as they maintain the drug in a non-degraded state and release effective amounts of drug over long time periods. LDH clay/drug complexes are amenable to homogenous dispersion in polymeric films where implant coating may be optimal or required.

Synthesis and characterization of carboxylic acid conjugated, hydrophobically derivatized, hyperbranched polyglycerols as nanoparticulate drug carriers for cisplatin.

Hyperbranched polyglycerols (HPGs) with hydrophobic cores and derivatized with methoxy poly(ethylene glycol) were synthesized and further functionalized with carboxylate groups to bind and deliver cisplatin. Low and high levels of carboxylate were conjugated to HPGs (HPG-C(8/10)-MePEG(6.5)-COOH(113) and HPG-C(8/10)-MePEG(6.5)-COOH(348)) and their structures were confirmed through NMR and FTIR spectroscopy and potentiometric titration. The hydrodynamic diameter of the HPGs ranged from 5-10 nm and the addition of COOH groups decreased the zeta potential of the polymers. HPG-C(8/10)-MePEG(6.5)-COOH(113) bound up to 10% w/w cisplatin, whereas HPG-C(8/10)-MePEG(6.5)-COOH(348) bound up to 20% w/w drug with 100% efficiency. Drug was released from HPG-C(8/10)-MePEG(6.5)-COOH(113) over 7 days at the same rate, regardless of the pH. Cisplatin release from HPG-C(8/10)-MePEG(6.5)-COOH(348) was significantly slower than HPG-C(8/10)-MePEG(6.5)-COOH(113) at pH 6 and 7.4, but similar at pH 4.5. Release of cisplatin into artificial urine was considerably faster than into buffer. Carboxylated HPGs demonstrated good biocompatibility, and drug-loaded HPGs effectively inhibited proliferation of KU-7-luc bladder cancer cells.

In vitro and in vivo evaluation of intravesical docetaxel loaded hydrophobically derivatized hyperbranched polyglycerols in an orthotopic model of bladder cancer.

The objective of this study was to evaluate the tolerability, to establish a dosing regimen, and to evaluate the efficacy of intravesical docetaxel (DTX) formulations in a mouse model of bladder cancer. DTX in commercial formulation (Taxotere, DTX in Tween 80) or loaded in hyperbranched polyglycerols (HPGs) was evaluated. The synthesis and characterization of HPGs with hydrophobic cores and derivatized with methoxy poly(ethylene glycol) in the shell and further functionalized with amine groups (HPG-C(8/10)-MePEG and HPG-C(8/10)-MePEG-NH(2)) is described. Intravesical DTX in either commercial or HPGs formulations (up to 1.0 mg/mL) was instilled in mice with orthotopic bladder cancer xenografts and was well tolerated with no apparent signs of local or systemic toxicities. Furthermore, a single dose of intravesical DTX (0.5 mg/mL) loaded in HPGs was significantly more effective in reducing the tumor growth in an orthotopic model of bladder cancer than the commercial formulation of Taxotere. In addition, DTX-loaded HPG-C(8/10)-MePEG-NH(2) was found to be more effective at lower instillation dose than DTX (0.2 mg/mL)-loaded HPG-C(8/10)-MePEG. Overall, our data show promising antitumor efficacy and safety in a recently validated orthotopic model of bladder cancer. Further research is warranted to evaluate its safety and efficacy in early phase clinical trials in patients refractory to standard intravesical therapy.

The Design, Characterization and Antibacterial Activity of Heat and Silver Crosslinked Poly(Vinyl Alcohol) Hydrogel Forming Dressings Containing Silver Nanoparticles.

The prompt treatment of burn wounds is essential but can be challenging in remote parts of Africa, where burns from open fires are a constant hazard for children and suitable medical care may be far away. Consequently, there is an unmet need for an economical burn wound dressing with a sustained antimicrobial activity that might be manufactured locally at low cost. This study describes and characterizes the novel preparation of a silver nitrate-loaded/poly(vinyl alcohol) (PVA) film. Using controlled heating cycles, films may be crosslinked with in situ silver nanoparticle production using only a low heat oven and little technical expertise. Our research demonstrated that heat-curing of PVA/silver nitrate films converted the silver to nanoparticles. These films swelled in water to form a robust, wound-compatible hydrogel which exhibited controlled release of the antibacterial silver nanoparticles. An optimal formulation was obtained using 5% (w/w) silver nitrate in PVA membrane films that had been heated at 140 °C for 90 min. Physical and chemical characterization of such films was complemented by in vitro studies that confirmed the effective antibacterial activity of the released silver nanoparticles against both gram positive and negative bacteria. Overall, these findings provide economical and simple methods to manufacture stable, hydrogel forming wound dressings that release antibiotic silver over prolonged periods suitable for emergency use in remote locations.

The Development of Solvent Cast Films or Electrospun Nanofiber Membranes Made from Blended Poly Vinyl Alcohol Materials with Different Degrees of Hydrolyzation for Optimal Hydrogel Dissolution and Sustained Release of Anti-Infective Silver Salts.

INTRODUCTION: We previously described the manufacture and characterization of hydrogel forming, thin film, anti-infective wound dressings made from Poly Vinyl Alcohol (PVA) and silver nanoparticles, crosslinked by heat. However, these films were designed to be inexpensive for simple manufacture locally in Africa. In this new study, we have further developed PVA dressings by manufacturing films or electrospun membranes, made from blends of PVA with different degrees of hydrolyzation, that contain silver salts and degrade in a controlled manner to release silver in a sustained manner over 12 days. METHODS: Films were solvent cast as films or electrospun into nanofibre membranes using blends of 99 and 88% hydrolyzed PVA, containing 1% w/w silver sulphadiazine, carbonate, sulphate, or acetate salts. Dissolution was measured as weight loss in water and silver release was measured using inductively coupled plasma (ICP) analysis. RESULTS: Cast films generally stayed intact at PVA 99: PVA 88% ratios greater than 40:60 whereas electrospun membranes needed ratios greater than 10:90. Films (40:60 blend ratio) and membranes (10:90) all released silver salts in a sustained fashion but incompletely and to different extents. Electrospun membranes gave more linear release patterns in the 2-12 day period and all salts released well. CONCLUSION: Blended PVA cast films offer improved control over hydrogel dissolution and silver release without the need for high temperature crosslinking. Blended PVA electrospun membranes further improve membrane dissolution control and silver release profiles. These blended PVA films and membranes offer improved inexpensive systems for the manufacture of long lasting anti-infective hydrogel wound dressings.

The Combined Use of Gentamicin and Silver Nitrate in Bone Cement for a Synergistic and Extended Antibiotic Action against Gram-Positive and Gram-Negative Bacteria.

Using bone cement as a carrier, gentamicin was for years the default drug to locally treat orthopedic infections but has lost favor due to increasing bacterial resistance to this drug. The objective of this study was to investigate the effect of combining gentamicin with silver nitrate in bone cement against S. aureus and P. aeruginosa. Antibacterial effects (CFU counts) of gentamicin and silver were initially studied followed by studies using subtherapeutic concentrations of each in combination. The release rates from cement were measured over 10 days and day 7 release samples were saved and analyzed for antibiotic activity. A strong synergistic effect of combining silver with gentamicin was found using both dissolved drugs and using day 7 bone cement release media for both Gram-positive and Gram-negative bacteria. The cement studies were extended to vancomycin and tobramycin, which are also used in bone cement, and similar synergistic effects were found for day 7 release media with P. aeruginosa but not S. aureus. These studies conclude that the combined use of low loadings of gentamicin and silver nitrate in bone cement may offer an economical and much improved synergistic method of providing anti-infective orthopedic treatments in the clinic.

Calcium pyrophosphate dihydrate crystal-induced inhibition of neutrophil apoptosis: involvement of Bcl-2 family members.

INTRODUCTION: The inflammation associated with calcium pyrophosphate dihydrate (CPPD) crystal-induced arthritis arises from the activation of neutrophils with crystals in the synovial joint. Furthermore, constitutive neutrophil apoptosis is inhibited by this interaction with CPPD so that the lifetime of the cells and the duration of the inflammatory response are extended. The objective of this study was to investigate the role of bcl-2 protein family members in the CPPD-induced prosurvival response. METHODS: Apoptosis was measured using DNA fragmentation and Caspase 3 assays. The expression and activation levels of the bcl-2 protein family members A1, Mcl-1, Bcl-xl, Bim, Bad and Bax-alpha were measured using western blot analysis. RESULTS: The prosurvival proteins Mcl-1 and Bcl-xl were both found to be strongly expressed but unaffected by CPPD-induced neutrophil activation over 3 h. The expression of proapoptotic proteins Bim and Bax-alpha was found to decrease over the time course of a 3 h incubation of neutrophils with CPPD crystals (but not the bacterial chemoattractant fMLP). Furthermore, expression of the unphosphorylated (active, proapoptotic) form of Bim was dominant in control cells at 0.5 h, whereas the status of this protein switched to the phosphorylated form following cell activation by both CPPD and fMLP. For CPPD (but not fMLP) this phosphorylation effect reversed over a 3 h incubation. CONCLUSION: Upon stimulation by CPPD crystals, the expression of both Bim and Bax-alpha decreased after 3 h suggesting a reduced proapoptotic effect of these proteins so that the static expression of the prosurvival proteins Bcl-xl and Mcl-1 might allow for a temporary shift in the balance to a prosurvival state of the cells. Because a sudden (but transient) increase in the phosphorylated form of Bim was observed in CPPD-stimulated neutrophils it is possible that this species might act as a signaling intermediate, resulting in the observed downregulation of Bax-alpha.

The inhibition of collagenase induced degradation of collagen by the galloyl-containing polyphenols tannic acid, epigallocatechin gallate and epicatechin gallate.

Collagen based cosmetic fillers require repeat treatments due to collagenase derived degradation of the filler in the intradermal injection site. The objective of this study was to investigate the inhibition of this degradation by the galloyl-containing compounds tannic acid, epigallocatechin gallate (EGCG), epicatechin gallate (ECG) and gallic acid (GA). A gel permeation chromatography assay was developed to quantitate the collagenase induced reductions in collagen molecular weight. The binding of the compounds to collagen was measured using HPLC. The stabilization of collagen was measured using Differential Scanning Calorimetry (DSC). Tannic acid, EGCG and ECG (but not GA) were found to strongly inhibit collagen degradation at concentrations in the low micromolar range. The compounds bound strongly to collagen and stabilized collagen. It is concluded that tannic acid, EGCG and ECG bind to collagen via extensive hydrogen bonding augmented by some hydrophobic interactions and prevent the free access of collagenase to active sites on the collagen chains.

The use of ultrasound to increase the uptake and cytotoxicity of dual taxane and P-glycoprotein inhibitor loaded, solid core nanoparticles in drug resistant cells.

The objective of this study was to use ultrasound in combination with nanoparticulate formulations of taxane drugs for an additive approach to overcome multidrug resistance (MDR). Polymeric nanoparticulate formulations containing both chemotherapeutic taxane drugs and a polymeric inhibitor (MePEG17-b-PCL5) of drug resistant proteins have been previously developed in an attempt to overcome MDR in cells. High frequency (>1 MHz) ultrasound has been shown to increase the uptake of cytotoxic drugs in MDR proliferating cells and has been suggested as a different way to overcome MDR, resensitize drug resistant cancer cells and allow for chemotherapeutic efficacy. MDCK-MDR cells were incubated with docetaxel (DTX) or paclitaxel (PTX) loaded, solid core, nanoparticles made from a 50:50 ratio of two diblock copolymers, MePEG114-b-PCL200 and MePEG17-b-PCL5 (PCL200/PCL5). The accumulation of drug in MDCK-MDR cells was measured using radiolabeled drug and the viability of cells was determined using an MTS cell proliferation assay. The effect of ultrasound (4 MHz, 32 W/cm2, 10 s, 25% duty cycle) on drug uptake and cell viability was studied. Using free DTX or PTX, MDCK-MDR cells were killed at sublethal doses of drug with the P-gp inhibitor (MePEG17-b-PCL5) present at a concentration of just 0.006% (m/v) and cell death began after just 3 h of incubation. Using sublethal incubation doses of PTX or DTX in PCL200/PCL5 nanoparticles for 90 min, followed by a second exposure to blank PCL200/PCL5 nanoparticles, cell viability dropped by approximately 60% at 24 h. Drug accumulation increased by 1.43-1.9 fold following five bursts of ultrasound applied at 90 min. Both, increased ultrasound exposure and increased concentrations of blank nanoparticles during the second incubation allowed for increased levels of cell death. The combined use of ultrasound with taxane and P-gp inhibitor loaded polymeric nanoparticles may allow for increased accumulation of drug and inhibitor which may then release both agents inside cells in a controlled manner to overcome drug resistance in MDR cells.

Effects of continuous and pulsatile PTH treatments on rat bone marrow stromal cells.

Bone marrow stromal cells (MSCs) differentiation and proliferation are controlled by numerous growth factors and hormones. Continuous parathyroid hormone (PTH) treatment has been shown to decrease osteoblast differentiation, whereas pulsatile PTH increases osteoblast differentiation. However, the effects of PTH treatments on MSCs have not been investigated. This study showed continuous PTH treatment in the presence of dexamethasone (DEX) promoted osteogenic differentiation of rat MSCs in vitro, as demonstrated by increased alkaline phosphatase (ALP) activity, number of ALP expressing cells, and up-regulation of PTH receptor-1, ALP, and osteocalcin mRNA expressions. In contrast, pulsatile PTH treatment was found to suppress osteogenesis of rat MSCs, possibly by promoting the maintenance of undifferentiated cells. Additionally, the observed effects of PTH were strongly dependent on the presence of DEX. MSC proliferation however was not influenced by PTH independent of treatment regimen and presence or absence of DEX. Furthermore, our work raised the possibility that PTH treatment may modulate stem/progenitor cell activity within MSC cultures.

Paclitaxel incorporated in hydrophobically derivatized hyperbranched polyglycerols for intravesical bladder cancer therapy.

OBJECTIVE: To develop paclitaxel incorporated into unimolecular micelles based on hydrophobically derivatized hyperbranched polyglycerols (dHPGs) for use as mucoadhesive intravesical agents against non-muscle-invasive bladder cancer. MATERIALS AND METHODS: Two different types of dHPGs (HPG- C10-polyethylene glycol (PEG) and polyethyleneimine (PEI)-C18-HPG) were synthesized and paclitaxel was loaded into these using a solvent evaporation method. After physicochemical characterization of the resulting nanoparticles, four human bladder cancer cell lines were incubated with various concentrations of paclitaxel incorporated in dHPGs and the results were compared with those of paclitaxel formulated in Cremophor-EL (Taxol(R), Bristol-Myers-Squibb). In vivo, nude mice with orthotopic KU7-luc tumours were intravesically instilled with phosphate buffered saline, Taxol, or paclitaxel/HPG-C10-PEG. RESULTS: dHPGs are mucoadhesive nanoparticles with hydrodynamic radii of <10 nm and incorporation of paclitaxel did not affect their size. The release profiles of paclitaxel from dHPGs were characterized by a rapid-release phase followed by a slower sustained-release phase. While the PEI-C18-HPG formulation released only approximately 40% of the initially incorporated paclitaxel, up to 80% was released from HPG-C10-PEG. Moreover, only paclitaxel/HPG-C10-PEG was stable in acidic urine. In vitro, all paclitaxel formulations potently decreased bladder cancer proliferation although paclitaxel/HPG-C10-PEG was slightly less cytotoxic than standard Taxol. By contrast, in vivo, the mucoadhesive HPG-C10-PEG formulation of paclitaxel was significantly more effective in reducing orthotopic tumour growth than Taxol and was well tolerated. CONCLUSION: Intravesical administration of mucoadhesive nanoparticulate formulations of paclitaxel might be a promising approach for instillation therapy of patients with non-muscle-invasive bladder cancer.

Intra-articular treatment of inflammatory arthritis with microsphere formulations of methotrexate: pharmacokinetics and efficacy determination in antigen-induced arthritic rabbits.

INTRODUCTION: Methotrexate (MTX) encapsulated microspheres release MTX in the joint in a slow, controlled manner following intra-articular injection in healthy rabbits. The objective of this study was to determine the pharmacokinetics of MTX and to evaluate the efficacy following intra-articular treatment of MTX-loaded microspheres in an antigen-induced inflammatory arthritis rabbit model. METHODS: Arthritis was induced in both knee joints of rabbits using ovalbumin. Rabbits were intra-articularly treated with MTX solution or MTX microspheres and plasma concentrations of MTX were determined in the first 8 h following intra-articular treatment. Rabbits were killed 14 days following treatment and histological analysis of rabbit joints was conducted to determine efficacy. RESULTS: Arthritis was successfully induced in the joints of rabbits with the observation of histopathological features resembling rheumatoid arthritis. No significant differences were detected between MTX solution and MTX microspheres treated groups compared to phosphate buffered saline (control) animals. CONCLUSIONS: MTX microspheres effectively delivered the drug to the intra-articular space. However, a high degree of inter-animal variability, the severity of the disease induced and insufficient length of the observation period were suggested to be possible causes for the lack of therapeutic responses to MTX-loaded microspheres treatment.

PLGA and PHBV microsphere formulations and solid-state characterization: possible implications for local delivery of fusidic acid for the treatment and prevention of orthopaedic infections.

PURPOSE: To develop and characterize the solid-state properties of poly(DL-lactic-co-glycolic acid) (PLGA) and poly(3-hydroxybutyric acid-co-3-hydroxyvaleric acid) (PHBV) microspheres for the localized and controlled release of fusidic acid (FA). METHODS: The effects of FA loading and polymer composition on the mean diameter, encapsulation efficiency and FA released from the microspheres were determined. The solid-state and phase separation properties of the microspheres were characterized using DSC, XRPD, Raman spectroscopy, SEM, laser confocal and real time recording of single microspheres formation. RESULTS: Above a loading of 1% (w/w) FA phase separated from PLGA polymer and formed distinct spherical FA-rich amorphous microdomains throughout the PLGA microsphere. For FA-loaded PLGA microspheres, encapsulation efficiency and cumulative release increased with initial drug loading. Similarly, cumulative release from FA-loaded PHBV microspheres was increased by FA loading. After the initial burst release, FA was released from PLGA microspheres much slower compared to PHBV microspheres. CONCLUSIONS: A unique phase separation phenomenon of FA in PLGA but not in PHBV polymers was observed, driven by coalescence of liquid microdroplets of a DCM-FA-rich phase in the forming microsphere.