Non-invasive in vivo characterization of microclimate pH inside in situ forming PLGA implants using multispectral fluorescence imaging.

The pH inside drug delivery systems influences directly the physical and chemical behavior of its ingredients specifically their solubility and stability. These properties significantly affect the release performance of the formulations as well as the pharmacological effect. Therefore, the determination of the microclimate pH (µpH) inside the drug delivery systems is of great importance and interest. Implants are considered to be attractive parenteral drug delivery systems used for the long-term application of drugs and of peptides. Poly(lactide-co-glycolide) (PLGA) is the most frequently used and extensively researched polymer for implant preparation. However it is known that the microclimate pH (µpH) within the PLGA implants can also drop dramatically. This pH drop can cause peptide or protein instabilities as well as drug insolubilities and further decomposition. Although the internal pH behavior of PLGA implants and microparticles has been studied in vitro, no data about the µpH behavior in in situ forming implants has yet been described. This is due to the fact, that there is no reliable non-invasive method available to measure directly and continuously the pH in vivo. Therefore, the question if in vitro measurement results are potentially assignable remains unclear. In this study, the µpH of in situ forming PLGA implants were mapped in vitro, in vivo, and ex vivo. A non-invasive in vivo pH measurement method using the multispectral Maestro fluorescence imaging system was developed. The in vivo experiments performed, not only enabled the authors of this article to make certain assumptions about µpH behavior but also emphasized certain expectations regarding the solvent replacement in the core area of the implant as well as the release profile of hydrophilic substances. The experiments emphasized the broad application range of the fluorescence imaging technique for non-invasive monitoring of µpH values in drug delivery systems in vivo.

Accumulation of nanocarriers in the ovary: a neglected toxicity risk?

Several nanocarrier systems are frequently used in modern pharmaceutical therapies. Within this study a potential toxicity risk of all nanoscaled drug delivery systems was found. An accumulation of several structurally different nanocarriers but not of soluble polymers was detected in rodent ovaries after intravenous (i.v.) administration. Studies in different mouse species and Wistar rats were conducted and a high local accumulation of nanoparticles, nanocapsules and nanoemulsions in specific locations of the ovaries was found in all animals. We characterised the enrichment by in vivo and ex vivo multispectral fluorescence imaging and confocal laser scanning microscopy. The findings of this study emphasise the role of early and comprehensive in vivo studies in pharmaceutical research. Nanocarrier accumulation in the ovaries may also comprise an important toxicity issue in humans but the results might as well open a new field of targeted ovarian therapies.

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.

Noninvasive in vivo monitoring of the biofate of 195 kDa poly(vinyl alcohol) by multispectral fluorescence imaging.

A comprehensive knowledge of the in vivo fate of polymers is essential for their potential application in humans. In this study, the body distribution, accumulation, and elimination processes of intraperitoneally (ip) administered poly(vinyl alcohol) (PVA) in mice were investigated in detail. Two derivatives of PVA (195 kDa) having covalently bound fluorescent dye labels were synthesized and used to follow PVA in vivo by noninvasive multispectral fluorescence imaging over several months. Detailed ex vivo fluorescence imaging was performed additionally and combined with tissue accumulation studies using confocal microscopy. Filtration and confocal imaging at appropriate synthetic membranes, used as models for glomerular filtration, confirmed a considerable PVA permeation. This investigation yields new scientific findings about the fate of PVA in vivo. PVA accumulated in fat tissue at high levels, which suggests that PVA is suitable not only for abdominal surgeries but also for controlled release applications after ip or subcutaneous injection.

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.

Monitoring of internal pH gradients within multi-layer tablets by optical methods and EPR imaging.

The high variability of gastrointestinal pH is a general challenge regarding constant release from oral drug delivery systems, especially for ionisable drugs. These drugs often show a pH-dependent solubility and therewith associated intra- and inter-individual variability of emerging drug plasma levels. Several strategies have been investigated with the intention to influence the microenvironmental pH (pH(M)) within solid formulations and therefore achieve pH-independent release profiles. Because of the heterogeneity of solid systems, a precise prediction of the occurring pH(M) is rather difficult. It is therefore important to monitor the pH(M) within the formulations to achieve requested release as well as to minimise pH-dependent degradation processes of the active compound. The purpose of the current study was the analysis of pH(M) gradients within 2- and 3-layer tablets during hydration using 3 different techniques for comparison intensions, in particular a pH indicator dye, fluorescence imaging and EPR imaging. The influence of the presence or absence of pH modifying substances and of an additional lipophilic inter layer on the pH(M) was investigated as well as the variation of matrix forming excipient and buffer pH. The influence of the pH(M) on drug release was analysed as well. In addition, benchtop MRI was accomplished to gain a deeper insight on the hydration and erosion behaviour of 2- and 3-layer tablets.

In-vivo studies on intraperitoneally administrated poly(vinyl alcohol).

The fate of poly(vinyl alcohol) (PVA, 195,000 g/mol) was studied in rabbits and nude mice after intraperitoneal (i.p.) administration. In-vivo fluorescence imaging using nude mice allowed for studies of tetramethylrhodamine labeled PVA distribution in the body and tracking the urinary excretion. The excreted PVA was studied in detail after collecting the urine of rabbits over a time period of 28 days. The PVA was separated from the urine by dialysis and analyzed by FTIR spectroscopy, (1)H-NMR spectroscopy, and size exclusion chromatography (SEC). Even after extensive dialysis, it was found that the excreted PVA showed a characteristic brownish color. The spectroscopic techniques revealed that this color was caused by the urine pigment (a metabolite of bilirubin) that could not be separated completely from the PVA. SEC showed unambiguously that the PVA with the very high molar mass had a glomerular permeability in the kidneys. Simultaneously, histological studies of the kidneys and the liver demonstrated that the tissues did not show any obvious damage.