Tracking immune-related cell responses to drug delivery microparticles in 3D dense collagen matrix.

Beyond the therapeutic purpose, the impact of drug delivery microparticles on the local tissue and inflammatory responses remains to be further elucidated specifically for reactions mediated by the host immune cells. Such immediate and prolonged reactions may adversely influence the release efficacy and intended therapeutic pathway. The lack of suitable in vitro platforms limits our ability to gain insight into the nature of immune responses at a single cell level. In order to establish an in vitro 3D system mimicking the connective host tissue counterpart, we utilized reproducible, compressed, rat-tail collagen polymerized matrices. THP1 cells (human acute monocytic leukaemia cells) differentiated into macrophage-like cells were chosen as cell model and their functionality was retained in the dense rat-tail collagen matrix. Placebo microparticles were later combined in the immune cell seeded system during collagen polymerization and secreted pro-inflammatory factors: TNFα and IL-8 were used as immune response readout (ELISA). Our data showed an elevated TNFα and IL-8 secretion by macrophage THP1 cells indicating that Placebo microparticles trigger certain immune cell responses under 3D in vivo like conditions. Furthermore, we have shown that the system is sensitive to measure the differences in THP1 macrophage pro-inflammatory responses to Active Pharmaceutical Ingredient (API) microparticles with different API release kinetics. We have successfully developed a tissue-like, advanced, in vitro system enabling selective "readouts" of specific responses of immune-related cells. Such system may provide the basis of an advanced toolbox enabling systemic evaluation and prediction of in vivo microparticle reactions on human immune-related cells.

Long-term distribution of biodegradable microparticles in rat muscle quantified noninvasively by MRI.

PURPOSE: To demonstrate the feasibility of MRI to monitor longitudinally the fate of PLGA microparticles in muscle tissue after intramuscular injection in rats using standard equipment. METHODS: MRI was performed at different time points and until day 28 after intramuscular administration of microparticles. Image segmentation was used to quantify the MRI signals. Histology was performed at selected time points to validate the in vivo observations. The SOM230-long acting release formulation was used as test compound. RESULTS: Microparticles were detected in vivo until 28 days following their administration. Imaging and histology data indicated that the MRI signals followed three phases: in an early phase (≤ 48 h after injection), vehicle, edema and hydration of microparticles contributed to the signals. In the second (days 3-17) and third phases (day 17 onward), microparticle hydration was the main contributor. SOM230 in blood displayed peaks at days 2 and 17. CONCLUSION: MRI was suitable to follow longitudinally the presence of PLGA microparticles in the rat muscle without labeling them. This is advantageous, because labeling could potentially alter the properties and pharmacokinetics of the microparticles. Data were consistent with an initial compound release followed by diffusion and microparticle erosion as main mechanisms of SOM230 release.

Chitosan/glucose 1-phosphate as new stable in situ forming depot system for controlled drug delivery.

Chitosan (CS)-based thermosensitive solutions that turn into semi-solid hydrogels upon injection at body temperature have increasingly drawn attention over the last decades as an attractive new type of in situ forming depot (ISFD) drug delivery system. Despite the great potential of the standard CS/ß-glycerophosphate (ß-GP) thermogelling solutions, their lack of stability over time at room temperature as well as at refrigerated conditions renders them unsuitable as ready-to-use drug product. In the present study, we investigated Glucose-1-Phosphate (G1-P) as an alternative gelling agent for improving the stability of CS-based ISFD solutions. The in vitro release performance of CS/G1-P formulations was assessed using several model compounds. Furthermore, the local tolerance of subcutaneously implanted CS/G1-P hydrogels was investigated by histological examination over three weeks. The thermogelling potential of CS/G1-P solutions, determined by rheology, is dependent on the polymer molecular weight (Mw) and concentration as well as on the G1-P concentration. Differential scanning calorimetry (DSC) measurements confirmed that sol/gel transition takes place at around body temperature and is not fully thermo-reversible. The long term storage stability was evaluated through the appearance, pH, viscosity and gelation time at 37°C of the solution. The results emphasized an enhanced stability of the CS/G1-P system compared to the standard CS/ß-GP. CS solution with 0.40 mmol/g G1-P is stable for at least 9 months at 2-8°C, versus less than 1 month when using ß-GP as gelling agent. Furthermore, the solution is easy to inject, as evidenced from injectability evaluation using 23-30 G needles. In vitro release experiments showed a sustained release over days to weeks for hydrophilic model compounds, demonstrating thereby that CS/G1-P may be suitable for the prolonged delivery of drugs. The inflammatory reaction observed in the tissue surrounding the hydrogel in rats was a typical foreign body reaction, similar to the one observed for CS/ß-GP hydrogels. These features confirm the potential of CS/G1-P solutions as an injectable ready-to-use in situ forming hydrogel.

Feasibility of macrophage mediated on-demand drug release from surface eroding poly(ethylene carbonate).

Macrophage induced surface degradation of poly(ethylene carbonate) (PEC) was investigated under in vitro conditions. Degradation of PEC with the MW of 41 kDa (PEC41) was slower than that of PEC with the MW of 200 kDa (PEC200). In terms of macrophage mediated drug release from PEC matrix, in cell-free medium, less than 1% of levofloxacin was released from both PEC samples in 10 days, while more than 60 and 20% of the drug, levofloxacin, can be detected in medium with macrophages from PEC200 and PEC41 films, respectively. This work indicated that on-demand drug delivery induced by macrophages can be achieved with PEC polymer.

Thermosensitive chitosan/glycerophosphate-based hydrogel and its derivatives in pharmaceutical and biomedical applications.

INTRODUCTION: Thermogelling chitosan (CS)/glycerophosphate (GP) solutions have been reported as a new type of parenteral in situ forming depot system. These free-flowing solutions at ambient temperature turn into semi-solid hydrogels after parenteral administration. AREAS COVERED: Formulation parameters such as CS physico-chemical characteristics, CS/gelling agent ratio or pH of the system, were acknowledged as key parameters affecting the solution stability, the sol/gel transition behavior and/or the final hydrogel structure. We discuss also the use of the standard CS/GP thermogels for various biomedical applications, including drug delivery and tissue engineering. Furthermore, this manuscript reviews the different strategies implemented to improve the hydrogel characteristics such as combination with carrier particles, replacement of GP, addition of a second polymer and chemical modification of CS. EXPERT OPINION: The recent advances in the formulation of CS-based thermogelling systems already overcame several challenges faced by the standard CS/GP system. Dispersion of drug-loaded carrier particles into the thermogels allowed achieving prolonged release profiles for low molecular weight drugs; incorporation of an additional polymer enabled to strengthen the network, while the use of chemically modified CS led to enhanced pH sensitivity or biodegradability of the matrix.

In situ forming parenteral depot systems based on poly(ethylene carbonate): effect of polymer molecular weight on model protein release.

The objective of this study was to investigate the effect of molecular weight (MW) on the drug release from poly(ethylene carbonate) (PEC) based surface-eroding in situ forming depots (ISFD). In phosphate buffered saline (PBS) pH 7.4, 63.7% of bovine serum albumin BSA was released from high MW PEC of 200 kDa (PEC200) in DMSO (15%, w/w) in 2 days, while during the same time period, the release of BSA from PEC41 samples was only 22.5%. At higher concentrations of PEC41 (25%, w/w), the initial burst was further reduced, and even after 6 days, only 16.3% was released. Compared to depots based on PEC200, there was lower rate of solvent release, slower phase inversion, and a denser surface in PEC41 samples. An expansion in size of PEC41 depots suggested that the polymer barrier of PEC41 impeded the diffusion of solvent out of the samples effectively. In conclusion, the initial burst of protein from ISFD of PEC41 was significantly reduced, which would be a promising candidate as polymeric carrier.

Enzyme-responsive surface erosion of poly(ethylene carbonate) for controlled drug release.

Cholesterol esterase (CE) induced surface erosion of poly(ethylene carbonate) (PEC) and drug release from PEC under mild physiological environment was investigated. The degradation process was monitored by changes of mass and molecular weight (MW) and surface morphology of polymer films. During the whole period of degradation, MW of PEC was unchanged. Water uptake of the polymer was only 2.8% and 0.2% for PEC with the MW of 200 kDa (PEC200) and PEC with the MW of 41 kDa (PEC41), respectively. Degradation of less hydrophilic PEC41 with higher density was slower than that of PEC200. By this mechanism, CE-responsive drug in vitro release from PEC in situ forming depots (ISFD) was conducted successfully. As expected, less bovine serum albumin (BSA) was released from PEC41 compared with that of PEC200 in the same time period. In conclusion, this work enabled the in vitro drug release evaluation of existing PEC devices and implied a new candidate for the development of enzyme-responsive systems.

Poly(ethylene carbonate) nanoparticles as carrier system for chemotherapy showing prolonged in vivo circulation and anti-tumor efficacy.

The aim of this study is to investigate the feasibility and efficacy of PEC nanoparticles as delivery system for cancer chemotherapy. Assembly of paclitaxel-loaded nanoparticles with high loading efficiency and narrow-size distribution is successful. For non-invasive in vivo tracing, nanoparticle blends of chelator bearing poly(lactide) with PEC and PLGA are successfully prepared. Pharmacokinetic studies in mice reveal a twofold higher circulation time of PEC as compared to PLGA. A tumor model shows an accumulation of PEC NPs in cancerous tissue and a higher anti-tumor efficiency compared to the standard Taxol™, which is reflected in a significantly slower tumor growth compared to the NaCl control group.

Drug eluting stents based on Poly(ethylene carbonate): optimization of the stent coating process.

First generation drug eluting stents (DES) show a fivefold higher risk of late stent thrombosis compared to bare metal stents. Therefore, new biodegradable and biocompatible polymers for stent coating are needed to reduce late stent thrombosis. In this study, a reproducible spray-coating process for stents coated with Poly(ethylene carbonate), PEC, and Paclitaxel was investigated. PEC is a biocompatible, thermoelastic polymer of high molecular weight. The surface degradation of PEC is triggered by superoxide anions produced by polymorphonuclear leukocytes and macrophages during inflammatory processes. Stents with different drug loading were reproducibly produced by a spray-coating apparatus. Confocal laser scanning micrographs of fluorescent dye loaded stents were made to investigate the film homogeneity. The abluminal stent site was loaded more than the luminal site, which is superior for DES. The deposition of the layers was confirmed by TOF-SIMS investigations. Referring to the stent surface, the drug loading is 0.32 µg (± 0.05) (once coated), 0.53 µg (± 0.11) (twice coated), or 0.73 µg (± 0.06) (three times coated) Paclitaxel per mm(2) stent surface. The in vitro release mechanism during non-degradation conditions can be explained by diffusion-controlled drug release slightly influenced by swelling of PEC, revealing that 100% of the loaded Paclitaxel will be released via diffusion within 2 months. So, the in vivo release kinetic is a combination of diffusion-controlled drug release and degradation-controlled drug release depending on the presence or absence of superoxide anions and accordingly depending on the presence or absence of macrophages. We conclude that the specific release kinetics of PEC, its biocompatibility, and the favorable mechanical properties will be beneficial for a next generation drug eluting stent meriting further investigations under in vivo conditions.

Biodegradable poly(ethylene carbonate) nanoparticles as a promising drug delivery system with "stealth" potential.

The goal of this study was to investigate the suitability of poly(ethylene carbonate) (PEC) nanoparticles as a novel drug delivery system, fulfilling the requirements for a long circulation time. Particles were obtained with a narrow size distribution and nearly neutral zeta potential. Adsorption studies with human plasma proteins revealed that PEC nanoparticles bind much less proteins in comparison to polystyrene (PS) nanoparticles. Cell experiments with fluorescently labeled PEC showed no uptake of the nanoparticles by macrophages. These novel PEC nanospheres with their unique surface properties are a promising candidate for long circulating drug delivery systems in vivo.

Poly(ethylene carbonate) as a surface-eroding biomaterial for in situ forming parenteral drug delivery systems: a feasibility study.

To evaluate the technical feasibility of poly(ethylene carbonate), PEC, for injectable in situ forming drug delivery systems, the physical properties of PEC solutions were characterized. The solubility of PEC was investigated in different solvents, and the Hildebrand solubility parameters and Flory-Huggins interaction parameters of PEC were determined. By turbidity titration, the experimental ternary phase diagram of water-NMP/DMSO-PEC was constructed. NMP solution required more water to precipitate PEC compared to DMSO solution. The dynamic viscosity of PEC solution increased at lower temperature, higher polymer concentration and longer aging time. Differential scanning calorimetric (DSC) measurements confirmed only weak physical interactions in the system after aging, and the physical aging effect was thermo-reversible. Release of NMP from PEC formulations was twofold slower than that of DMSO at similar concentrations. The morphology of PEC depots after injection into aqueous solution was studied using scanning electron microscopy (SEM). A DMSO formulation of bovine serum albumin displayed less burst release than a NMP formulation. In summary, our investigations demonstrate that in situ depot forming systems can be obtained from PEC solutions. Moreover, a solution of PEC in DMSO would be preferred over NMP due to the reduced burst release.

Non-invasive assessment of the effect of formulation excipients on stratum corneum barrier function in vivo.

The objective of the present work was to investigate the effect of formulation excipients on human stratum corneum (SC) barrier function in vivo. Two formulations, an ointment and an oil-in-water cream, were applied to the skin of human volunteers under both occlusive and non-occlusive conditions. The effects of each treatment were then evaluated using three non-invasive biophysical techniques: transepidermal water loss (TEWL), impedance spectroscopy (IS) and attenuated-total-reflectance Fourier transform infrared (ATR-FTIR) spectroscopy. These measurements were combined with a simple tape-stripping protocol to allow information to be derived across the entire SC. IS and TEWL provided basic information on the effect of each formulation on skin barrier function, while ATR-FTIR enabled (i) the tracking of formulation excipients and evaluation of their concentration profiles within the SC, and (ii) deduction of mechanistic detail with which to explain the TEWL and IS results. It was found that occlusion of the skin either in the presence or absence of the cream caused TEWL to be increased when the treatment was terminated at 6 h. Uptake of ointment into the SC, on the other hand, inhibited the post-application TEWL rate. In parallel, treatment with the ointment caused an increase in relative low-frequency skin impedance, consistent with the entry of additional lipophilic constituents into the SC. The latter was confirmed by ATR-FTIR spectroscopic measurements. Overall, the combined use of the three biophysical measurements allowed formulation effects on, and uptake into, the SC to be deduced and evaluated, and the approach may prove useful for the future selection and optimization of topical drug delivery vehicles.

Post-iontophoresis recovery of human skin impedance in vivo.

The objective of this study was to better understand the recovery of human skin impedance following iontophoresis in vivo. Volunteers were subjected to a 15-min period of iontophoresis in the presence of aqueous solutions of either NaCl, KCl, CaCl(2) or MgCl(2) at 133 mM. Subsequently, the low-frequency impedance (at 1 Hz) recovery was followed for a further 30 min. Assuming direct proportionality between the reciprocal impedance and the ion concentration in the membrane, the experimental data were fitted to the appropriate solutions of Fick's second law of diffusion to derive characteristic diffusion parameters (D/L(2)), apparent diffusivities (D), diffusion pathlengths (L) and mobilities, and ion concentrations in the skin immediately post-iontophoresis. Ion fluxes out of the membrane after termination of current flow were also deduced. In general, recovery was relatively independent of the background electrolyte as previously reported, and the data were consistent with ion transport in predominantly aqueous pathways. Compared to its mobility in aqueous solution, however, the apparent Cl- mobility in the skin was smaller, presumably due to the fact that, under normal physiological conditions, the human skin barrier supports a net negative charge. In parallel, the initial "release" of Na+ and K+ from the skin post-iontophoresis was faster than that of Ca(2+) and Mg(2+), the latter cations of higher charge density being able to associate more strongly, it seems, with the negatively-charged skin. The simple physicochemical analysis of the data presented serves to emphasize that a decrease in skin impedance is not a manifestation of damage to the barrier--rather, it is a natural response to the relevant electrical potential and ion concentration gradients involved.