No abstract

The rate and extent of drug dissolution and absorption from solid oral dosage forms is highly dependent upon the volumes and distribution of gastric and small intestinal water. However, little is known about the time courses and distribution of water volumes in vivo in an undisturbed gut. Previous imaging studies offered a snapshot of water distribution in fasted humans and showed that water in the small intestine is distributed in small pockets. This study aimed to quantify the volume and number of water pockets in the upper gut of fasted healthy humans following ingestion of a glass of water (240 mL, as recommended for bioavailability/bioequivalence (BA/BE) studies), using recently validated noninvasive magnetic resonance imaging (MRI) methods. Twelve healthy volunteers underwent upper and lower abdominal MRI scans before drinking 240 mL (8 fluid ounces) of water. After ingesting the water, they were scanned at intervals for 2 h. The drink volume, inclusion criteria, and fasting conditions matched the international standards for BA/BE testing in healthy volunteers. The images were processed for gastric and intestinal total water volumes and for the number and volume of separate intestinal water pockets larger than 0.5 mL. The fasted stomach contained 35 ± 7 mL (mean ± SEM) of resting water. Upon drinking, the gastric fluid rose to 242 ± 9 mL. The gastric water volume declined rapidly after that with a half emptying time (T50%) of 13 ± 1 min. The mean gastric volume returned back to baseline 45 min after the drink. The fasted small bowel contained a total volume of 43 ± 14 mL of resting water. Twelve minutes after ingestion of water, small bowel water content rose to a maximum value of 94 ± 24 mL contained within 15 ± 2 pockets of 6 ± 2 mL each. At 45 min, when the glass of water had emptied completely from the stomach, total intestinal water volume was 77 ± 15 mL distributed into 16 ± 3 pockets of 5 ± 1 mL each. MRI provided unprecedented insights into the time course, number, volume, and location of water pockets in the stomach and small intestine under conditions that represent standard BA/BE studies using validated techniques. These data add to our current understanding of gastrointestinal physiology and will help improve physiological relevance of in vitro testing methods and in silico transport analyses for prediction of bioperformance of oral solid dosage forms, particularly for low solubility Biopharmaceutics Classification System (BCS) Class 2 and Class 4 compounds.

Nanosized materials have been investigated as potential medicines for several decades. Consequently, a great deal of work has been conducted on how to exploit constructs of this size range in a beneficial way. Similarly, a number of the consequences from the use of these materials have already been considered. Nanosized materials do behave differently to low-molecular-weight drugs, the biological properties of nanomaterials being mainly dependent on relevant physiology and anatomy, which are reviewed in this article. Biodistribution, movement of materials through tissues, phagocytosis, opsonization and endocytosis of nanosized materials are all likely to have an impact on potential toxicity. In turn these processes are most likely to depend on the nanoparticle surface. Evidence from the literature is considered which suggests that our understanding of these areas is incomplete, and that biodistribution to specific sites can occur for nanoparticles with particular characteristics. However, our current knowledge does indicate which areas are of concern and deserve further investigation to understand how individual nanoparticles behave and what toxicity may be expected from them.

Nanoparticles assembled from poly(D,L-lactic acid)-poly(ethylene glycol) (PLA-PEG) block copolymers may have a therapeutic application in site-specific drug delivery. A series of AB block copolymers based on a fixed PEG block (5 kDa) and a varying PLA segment (2-110 kDa) have been synthesized by the ring-opening polymerization of D,L-lactide using stannous octoate as a catalyst. These copolymers assembled to form spherical nanoparticles in aqueous media following precipitation from a water-miscible organic solvent. 1 H NMR studies of the PLA-PEG nanoparticles in D2O confirmed their core-shell structure, with negligible penetration of the hydrated PEG chains into the PLA core. The influence of the PLA block molecular weight on the hydrodynamic size and micellar aggregation number of the assemblies was determined by dynamic and static light scattering techniques. The hydrodynamic radius of the PLA-PEG 2:5-30:5 nanoparticles was solely dependent on the copolymer architecture and scaled linearly as NPLA 1/3 , where NPLA is the number of monomeric units in the PLA block. The PEG chains of the small PLA-PEG 2:5 and 3:5 assemblies appeared to be fairly splayed as a consequence of their relatively low aggregation number and high surface coverage. However, as NPLA was increased to 6 kDa the area available per PEG chain at the periphery of the shell decreased significantly and then remained fairly constant with further increases in the molecular weight of the PLA block. The aggregation number and hence particle size of nanoparticles produced from copolymers with a PLA block of 45 kDa or more was found to also depend on the concentration of copolymer dissolved in the organic phase during preparation. This suggested that that the PEG chains had little influence on the assembly of the higher molecular weight copolymers.

Three-dimensional cell culture has many advantages over monolayer cultures, and spheroids have been hailed as the best current representation of small avascular tumours in vitro. However their adoption in regular screening programs has been hindered by uneven culture growth, poor reproducibility and lack of high-throughput analysis methods for 3D. The objective of this study was to develop a method for a quick and reliable anticancer drug screen in 3D for tumour and human foetal brain tissue in order to investigate drug effectiveness and selective cytotoxic effects. Commercially available ultra-low attachment 96-well round-bottom plates were employed to culture spheroids in a rapid, reproducible manner amenable to automation. A set of three mechanistically different methods for spheroid health assessment (Spheroid volume, metabolic activity and acid phosphatase enzyme activity) were validated against cell numbers in healthy and drug-treated spheroids. An automated open-source ImageJ macro was developed to enable high-throughput volume measurements. Although spheroid volume determination was superior to the other assays, multiplexing it with resazurin reduction and phosphatase activity produced a richer picture of spheroid condition. The ability to distinguish between effects on malignant and the proliferating component of normal brain was tested using etoposide on UW228-3 medulloblastoma cell line and human neural stem cells. At levels below 10 µM etoposide exhibited higher toxicity towards proliferating stem cells, whereas at concentrations above 10 µM the tumour spheroids were affected to a greater extent. The high-throughput assay procedures use ready-made plates, open-source software and are compatible with standard plate readers, therefore offering high predictive power with substantial savings in time and money.

Copolymers of polylactide and poly(ethylene glycol) (PLA−PEG), which self-disperse in water to form spherical nonionic micelles, have been investigated as a novel biodegradable drug delivery system. These copolymers are defined by the molecular weight ratios of their polylactide to poly(ethylene glycol) components (1.5:2 PLA−PEG and 2:5 PLA−PEG) and gave two peaks when purified by gel permeation chromatography (GPC). The first peak consisted of spherical micelles with a diameter of 15.6 nm for 1.5:2 PLA−PEG, and 18.9 nm for 2:5 PLA−PEG micelles after analysis by dynamic light scattering (DLS) and by transmission electron microscopy (TEM). The second peak was a PLA-depleted species resulting from the synthesis and did not form micelles. Testosterone and sudan black B (SBB), which have different hydrophobicities, were used as “model drugs” to evaluate the drug loading ability of the micelles. Ultracentrifugation sedimentation velocity studies confirmed that solubilization of the model drugs had occurred by micellar incorporation. Higher drug loading was obtained for the 1.5:2 PLA−PEG micelles (63.9% (w/w) of SBB, 0.74% (w/w) of testosterone) than for the 2:5 PLA−PEG micelles (59.0% (w/w) of SBB, 0.34% (w/w) of testosterone). The amount of testosterone solubilized was therefore significantly lower than SBB for both copolymers. Stability testing in the presence of salt suggested that the micelles had sterically stabilized surfaces. In vivo studies in the rat, using a radioactive marker, showed that PLA−PEG micelles demonstrated extended circulation times in the blood during the period of study (3 h). The 1.5:2 PLA−PEG showed increased blood levels and lower uptake of the micelles by the liver compared to the 2:5 PLA−PEG micelles. This is thought to be due to differences in the packing density of the copolymer molecules on the micelle surface.

NMR studies on a series of poly(lactic acid)-poly(ethylene oxide) (PLA-PEG) diblock copolymers have been carried out in d6-acetone and in D2O. Nanoparticles of the PLA-PEG copolymers were obtained using a modified interfacial polymer deposition-solvent evaporation technique (Fessi, H.; Devissaguet, J. P.; Puisieux, F.; Thies, C. French Patent 2 608 988, 1986). In D2O, the PLA block forms a central hydrophobic core, while the PEG block forms an hydrophilic corona layer. In D2O, the hydrophobic core of the nanoparticle is generally not seen, while the PEG corona is observed. Only the PLA methyl protons at the interface between the two regions are observed, and these are seen as a double doublet structure. For nanoparticles with a low molecular weight PLA block length, an additional methyl multiplet signal is seen suggesting that PLA methyl groups are in more than one chemical environment. This is not seen for nanoparticles with a high molecular weight PLA block length indicating more uniform structure in the core interfacial region. As temperature is increased, the core of the latter becomes more liquidlike. Quantitative calibration studies of the PEG corona layer show that most of the PEG layer is seen indicating that it is in the liquid phase and on the surface of the nanoparticle. 13 C solid-state NMR spectroscopy studies indicate the presence of a central solidlike core and a more mobile interfacial region at the PLA-PEG interface, while the relaxation rate of the nanoparticle obtained from T1 studies indicates that the PEG corona is a much more mobile environment than the interfacial methyl protons.

Animal models are effective for assessing tumor localization of nanosystems but difficult to use for studying penetration beyond the vasculature. Here, we have used wellcharacterized HCT116 colorectal cancer spheroids to study the effect of nanoparticle (NP) physicochemical properties on penetration and uptake. Incubation of spheroids with Hoechst 33342 resulted in a dye gradient, which facilitated discrimination between the populations of cells in the core and at the periphery of spheroids by flow cytometry. This approach was used to compare doxorubicin and liposomal doxorubicin (Caelyx) and a range of model poly(styrene) nanoparticles of different sizes (30 nm, 50 nm, 100 nm) and with different surface chemistries (50 nm uniform plain, carboxylated, aminated and a range of NPs and polyethylene glycol modified NPs prepared from a promising new functionalized biodegradable polymer (poly(glycerol-adipate), PGA). Unmodified poly(styrene) nanoparticles (30 nm/50 nm) were able to penetrate to the core of HCT116 spheroids more efficiently than larger poly(styrene) nanoparticles (100 nm). Surprisingly, penetration of 30 and 50 nm particles was as good as clinically relevant doxorubicin concentrations. However, penetration was reduced with higher surface charge. PGA NPs of 100 nm showed similar penetration into spheroids as 50 nm poly(styrene) nanoparticles, which may be related to polymer flexibility. PEG surface modification of polymeric particles significantly improved penetration into the spheroid core. The new model combining the use of spheroids Hoechst staining and flow cytometry was a useful model for assessing NP penetration and gives useful insights into the effects of NPs' physical properties when designing nanomedicines.