Effects of the type of release medium on drug release from PLGA-based microparticles: experiment and theory.

The major objectives of the present study were: (i) to prepare 5-fluorouracil (5-FU)-loaded, poly(lactic-co-glycolic acid) (PLGA)-based microparticles, which can be used for the treatment of brain tumors, (ii) to study the effects of the type of release medium on the resulting drug release kinetics, and (iii) to get further insight into the underlying drug release mechanisms. Spherical microparticles were prepared by a solvent extraction method and characterized using different techniques, including size exclusion chromatography (SEC), differential scanning calorimetry (DSC), scanning electron microscopy (SEM) and particle size analysis before and upon exposure to various release media. Interestingly, very different drug release patterns (including mono-, bi- and tri-phasic ones) were observed, depending on the pH, osmolarity and temperature of the release medium. An adequate mathematical theory was used to quantitatively describe the experimentally measured 5-FU release patterns. The model considers the limited solubility of the drug, polymer degradation as well as drug diffusion and allowed to determine system and release medium specific parameters, such as the diffusion coefficient of the drug. In particular, the pH and temperature of the release medium were found to be of major importance for the resulting release patterns. Based on the obtained knowledge the selection of an appropriate release medium for in vitro tests simulating in vivo conditions can be facilitated, and "stress tests" can be developed allowing to get rapid feedback on the release characteristics of a specific batch.

Development and characterization of interleukin-18-loaded biodegradable microspheres.

Immunostimulation represents a promising approach designed to specifically eradicate malignant cells. Since glioma tumour cells hole up in the central nervous system (CNS) in a particularly inauspicious milieu to antitumour immune reactions we here propose a new strategy to revert the properties of this microenvironment by administering an antitumour cytokine into the CNS tumour itself. Thus, biodegradable poly(D,L-lactide-co-glycolide) (PLGA) sustained-release microspheres for stereotaxic implantation loaded with interleukin-18 (IL-18), that is known to exert antitumour activity and trigger immune cell-mediated cytotoxicity, were developed. Different tests for assessing IL-18 bioactivity were set-up and evaluated. A specific bioassay was considered as the most reliable test. The stability and integrity of IL-18 was then verified during the encapsulation process. Consequently, two procedures of IL-18 encapsulation in PLGA microparticles (W/O/W and S/O/W) were investigated. As determined by radiolabelling studies using 125I-IL-18 and a continuous flow system, the in vitro release profile of IL-18 was optimum with S/O/W method with a moderate burst effect and a subsequent progressive discharge of 16.5+/-8.4 ng/day during the next 21 days against 6.1+/-4.2 ng/day with the W/O/W method. Considering analytical testing of IL-18 together with its preserved biological activity after release from microspheres, amounts of the active cytokine obtained with S/O/W method were relevant to plan in vivo evaluation to validate the therapeutic strategy.

In vitro investigation of lipid implants as a controlled release system for interleukin-18.

Operating on the inductive and effective phases of an anti-tumor immune response and uncovering pivotal functions that may reduce cancer cell growth, interleukin-18 (IL-18) appears to be an attractive candidate for the sustained local adjuvant immunotherapeutic treatment of brain gliomas. The objective of this work was to develop IL-18 loaded lipid implants as a controlled delivery system. For the preparation of protein loaded triglyceride matrix material, a solid-in-oil (s/o) dispersion technique was chosen for which protein particles in the micrometer range were first prepared by co-lyophilization with polyethylene glycol (PEG). Implants of 1 mm diameter, 1.8 mm height and 1.8 mg weight were manufactured by compression of the powder mixture in a specially designed powder compacting tool. The in vitro release behavior of 125I-Bolton-Hunter-radiolabeled IL-18 was assessed in a continuous-flow system. A cell culture assay was established for the determination of bioactivity of released IL-18. Implants showed a continuous release of 10-100 ng IL-18 per day for 12 days. A progressive integrity loss was observed with ongoing release, which would be related to protein degradation during incubation. The initially released fraction proved complete retention of bioactivity, indicating that the manufacturing procedure had no detrimental effects on protein stability.

Paclitaxel-loaded microparticles and implants for the treatment of brain cancer: preparation and physicochemical characterization.

The aim of this study was to prepare different types of paclitaxel-loaded, PLGA-based microparticles and lipidic implants, which can directly be injected into the brain tissue. Releasing the drug in a time-controlled manner over several weeks, these systems are intended to optimize the treatment of brain tumors. The latter is particularly difficult because of the blood-brain barrier (BBB), hindering most drugs to reach the target tissue upon systemic administration. Especially paclitaxel (being effective for the treatment of ovarian, breast, lung and other cancers) is not able to cross the BBB to a notable extent since it is a substrate of the efflux transporter P-glycoprotein. Both, biodegradable microparticles as well as small, cylindrical, glycerol tripalmitate-based implants (which can be injected using standard needles) were prepared with different paclitaxel loadings. The effects of several formulation and processing parameters on the resulting drug release kinetics were investigated in phosphate buffer pH 7.4 as well as in a diethylnicotinamide (DENA)/phosphate buffer mixture. Using DSC, SEM, SEC and optical microscopy deeper insight into the underlying drug release mechanisms could be gained. The presence of DENA in the release medium significantly increased the solubility of paclitaxel, accelerated PLGA degradation, increased the mobility of the polymer and drug molecules and fundamentally altered the geometry of the systems, resulting in increased paclitaxel release rates.

Effect of the size of biodegradable microparticles on drug release: experiment and theory.

The aim of this study was to investigate the effect of the size of biodegradable microparticles (monolithic dispersions) on the release rate of an incorporated drug in a quantitative way. 5-Fluorouracil-loaded, poly(lactic-co-glycolic acid)-based microparticles were prepared with a solid-in-oil-in-water solvent extraction technique. In vitro drug release from different-sized particle fractions was measured in phosphate buffer pH 7.4. Differential scanning calorimetry (DSC), scanning electron microscopy (SEM) and size exclusion chromatography (SEC) were used to monitor the degradation behavior of the polymer and morphological changes of the microparticles upon exposure to the release medium. Based on these experimental results, an appropriate mathematical theory was identified and used to get further insight into the underlying physical and chemical processes, which are involved in the control of drug release. Interestingly, the relative as well as the absolute release rate of the drug increased with increasing microparticle radius, despite of the increasing diffusion pathways. SEC, DSC and SEM analysis revealed that the degradation behavior of the matrix forming polymer was not significantly affected by the size of the devices and that autocatalytic effects do not seem to play a major role. Importantly, the initial drug loading significantly increased with increasing radius of the drug delivery system. Thus, large microparticles became more porous during drug release than small microparticles, leading to higher apparent diffusivities and drug transport rates. This effect overcompensated the effect of the increasing diffusion pathways with increasing microparticle radius, resulting in increased drug release rates with increasing device dimension. The applied mathematical model, considering drug diffusion with non-constant diffusivities (to account for polymer degradation) was able to quantitatively describe the observed drug release patterns. Importantly, an exponential relationship could be established between the diffusion coefficient and the initial loading of the drug. Based on this dependency, it was possible to predict the resulting drug release kinetics for arbitrary microparticle sizes in a quantitative way.

Mathematical modeling of drug release from bioerodible microparticles: effect of gamma-irradiation.

Bioerodible polymers used in controlled drug delivery systems, such as poly(lactic-co-glycolic acid) (PLGA) undergo radiolytic degradation during gamma-irradiation. In spite of the considerable practical importance, yet only little knowledge is available on the consequences of this sterilization method on the resulting drug release patterns in a quantitative way. The major objectives of the present study were: (i) to monitor the effects of different gamma-irradiation doses on the physicochemical properties of drug-free and drug-loaded, PLGA-based microparticles; (ii) to analyze the obtained experimental results using adequate mathematical models; (iii) to get further insight into the occurring physical and chemical phenomena; and (iv) to relate the applied gamma-irradiation dose in a quantitative way to the resulting drug release rate. 5-Fluorouracil-loaded, PLGA-based microparticles were prepared with an oil-in-water solvent extraction method and exposed to gamma-irradiation doses ranging from 0 to 33 kGy. Size exclusion chromatography, differential scanning calorimetry, scanning electron microscopy, particle size analysis, determination of the actual drug loading and in vitro drug release kinetics were used to study the effects of the gamma-irradiation dose on the physicochemical properties of the microparticles. Two mathematical models-a simplified and a more comprehensive one-were used to analyze the experimental results. The simplified model considers drug diffusion based on Fick's second law for spherical geometry and a Higuchi-like pseudo-steady-state approach. The complex model combines Monte Carlo simulations (describing polymer erosion) with partial differential equations quantifying drug diffusion with time-, position- and direction-dependent diffusivities. Interestingly, exponential relationships between the gamma-irradiation dose and the initial drug diffusivity within the microparticles could be established. Based on this knowledge both models were used to predict the resulting drug release kinetics as a function of the gamma-irradiation dose. Importantly, the theoretical predictions were confirmed by experimental results.

The effect of gamma-irradiation on drug release from bioerodible microparticles: a quantitative treatment.

The two major objectives of this study were: (i) to monitor the effect of different gamma-irradiation doses (4-33 kGy) on the release kinetics from 5-fluorouracil (5-FU)-loaded poly(D,L-lactide-co-glycolide) (PLGA)-based microparticles, and (ii) to analyze the obtained experimental data with a new mathematical model giving insight into the occurring mass transport phenomena. Drug release was found to depend significantly on the applied gamma-irradiation dose. Interestingly, the obtained release profiles were all biphasic: a rapid initial drug release phase ("burst") was followed by a slower, approximately constant drug release phase. Surprisingly, only the initial rapid drug release was accelerated by gamma-irradiation; the subsequent zero-order phase was almost unaffected. Importantly, the new mathematical model which is based on Fick's second law of diffusion and which considers polymer degradation was applicable to all the investigated systems. In addition, the gamma-irradiation dose could be quantitatively related to the resulting drug release rate. In conclusion, diffusion seems to be the dominating release rate controlling mechanism in all cases, with a significant contribution of the polymer degradation process.

PLGA-based microparticles: elucidation of mechanisms and a new, simple mathematical model quantifying drug release.

The two major aims of this study were: (i) to elucidate the underlying release mechanisms from drug-loaded, erodible microparticles based on poly(lactic-co-glycolic acid) (PLGA) showing biphasic drug release behavior: an initial 'burst' effect, followed by a zero order release phase; and (ii) to develop a new, simple mathematical model that allows the quantitative description of the observed in vitro drug release patterns from this type of delivery system. PLGA-based microparticles offer various advantages, such as the possibility to control the resulting drug release rate accurately over prolonged periods of time, easiness of administration (e.g., by stereotaxic injection), good biocompatibility and complete erosion (avoiding the removal of empty remnants). Consequently, the practical importance of these advanced drug delivery systems is remarkably increasing. However, only little knowledge is yet available concerning the processes controlling the release rate of the drug out of these devices. Various chemical and physical phenomena are involved, rendering the identification of the crucial mechanisms and the mathematical description of the resulting drug release kinetics difficult. In the present study, different physicochemical characterization methods (e.g., DSC, SEM, SEC, particle size analysis) were used to monitor the changes occurring within anticancer drug-loaded PLGA microparticles upon exposure to phosphate buffer pH 7.4. Based on these experimental findings, the most important underlying drug release rate controlling mechanisms were identified and a new mathematical model was developed that allows the quantitative description of the resulting release patterns.

PLGA microsphere bioburden evaluation for radiosterilization dose selection.

The aim of this study was to determine the bioburden of PLGA microspheres produced by the solvent emulsion/extraction process as a means of determining an appropriate gamma-irradiation dose for sterilization. Bioburden was evaluated on the basis of ISO specifications. The analysis of initial microbial contamination was performed on blank microspheres, prepared by a non-aseptic laboratory scale process. A mean bioburden of 36.04 CFU (colony forming units)/110 mg microspheres was determined. Most of the detected germs originated from human commensal flora. According to the ISO dose-selection method, a gamma-irradiation dose of 19.6 kGy was found sufficient to ensure a sterility level of 10(-6). The effect of the selected irradiation dose on both the molecular weight of the polymer and the kinetics of 5-fluorouracil drug release from the microspheres was compared to the European Pharmacopeia recommended irradiation dose (25 kGy). This 20% reduced dose showed a lower extent of molecular weight reduction of PLGA and a better control of 5-FU release from microparticles. This can be related to reduce polymer radiation damage.

Development of microspheres for neurological disorders: from basics to clinical applications.

Drug delivery to the central nervous system remains a challenging area of investigation for both basic and clinical neuroscientists. Numerous drugs are generally excluded from blood to brain transfer due to the negligible permeability of the brain capillary endothelial wall, which makes up the blood brain barrier in vivo. For several years, we have explored the potential applications of the microencapsulation of therapeutic agents to provide local controlled drug release in the central nervous system. Due to their size, these microparticles can be easily implanted by stereotaxy in discreet, precise and functional areas of the brain without damaging the surrounding tissue. This type of implantation avoids the inconvenient insertion of large implants by open surgery and can be repeated if necessary. We have established the compatibility of poly(lactide-co-glycolide) microspheres with brain tissues. Presently, the most developed applications concern Neurology and Neuro-oncology, with local delivery of neurotrophic factors and antimitotic drugs into neurodegenerative lesions and brain tumours, respectively. The drugs that had been encapsulated by our group included nerve growth factor (NGF), 5-fluorouracil (5-FU), idoxuridine and BCNU. Preclinical studies have been performed with each drug. Studies with NGF are reported as an example. A phase I/II clinical trial has been carried out in patients with newly diagnosed glioblastomas to assess the potentialities of 5-FU-loaded microspheres when intracranially implanted.

Banana starch breakdown in the human small intestine studied by electron microscopy.

OBJECTIVE: To determine the origin of the poor digestibility of banana starch granules in the human small intestine. DESIGN: The subjects received the same experimental meal. SETTING: Nutrition Research Unit, Laënnec Hospital, CHU, Nantes. SUBJECTS: Six healthy young subjects. INTERVENTIONS: The digestion of raw green banana flour in the upper part of the gut was studied by the intubation technique. After ingestion of 30 g banana flour mixed with a complex meal, ileal samples were continuously collected during 14 h. In order to determine the structural nature of this resistant starch, the dried ileal samples were observed with scanning and transmission electron microscopy. Transmission electron microscopy was performed after treatment with periodic acid-thiosemicarbazide-silver nitrate. RESULTS: Banana starch proved very resistant to in vivo amylase hydrolysis since 84% of the starch ingested reached the terminal ileum. The microscopic observations showed that raw banana flour contained irregularly shaped dense starch granules with smooth surfaces. After their passage through the small intestine, starch granules appeared exocorroded, with porous surfaces, and some exhibited several irregular pits, crevices or holes by which the enzymes had penetrated and hydrolysed the inner part. Cell walls closely associated with starch granules could have hindered enzyme access to starch. CONCLUSIONS: Encapsulation could be partly responsible for the low digestibility of starch in banana flour, together with the intrinsic resistance of banana starch granules.

Digestion of raw banana starch in the small intestine of healthy humans: structural features of resistant starch.

The digestion of freeze-dried green banana flour in the upper gut was studied by an intubation technique in six healthy subjects over a 14 h period. Of alpha-glucans ingested, 83.7% reached the terminal ileum but were almost totally fermented in the colon. Structural study of the resistant fraction showed that a small part of the alpha-glucans which escaped digestion in the small intestine was composed of oligosaccharides from starch hydrolysis, whereas the rest was insoluble starch in granule form with physical characteristics similar to those of raw banana starch. Passage through the small intestine altered granule structure by increasing susceptibility to further alpha-amylase hydrolysis. Compared with resistant starch values in vivo, those obtained with the in vitro methods tested were inadequate to estimate the whole fraction of starch reaching the terminal ileum.

Structural features of resistant starch at the end of the human small intestine.

Structural features of in vivo resistant starch were assessed using the ileal contents of four humans. Two of the latter were collected by ileostomy after ingestion of bean flakes or potato flakes and the other two were collected by an intubation technique after ingestion of retrograded high-amylose maize starch or complexed high-amylose maize starch. The degree of polymerizations (DP), solubility and crystallinity were assessed. For all samples, starch fractions which escaped digestion in the small intestine were composed of three populations of alpha-glucans with proportions differing according to the substrate. Small quantities of oligosaccharides made up the first population, illustrating a limitation of absorption in the small intestine. The second population, the main resistant fraction, was comprised of retrograded amylose of mean DPn of about 35 glucose units with a melting temperature at 150 degrees C and exhibiting a B-type pattern. Finally high molecular weight semi-crystalline alpha-glucans were attributed to fragments of starch. This study showed that some potentially digestible starch could reach the colon and crystalline fractions constituted only part of the starch that escaped digestion in the human small intestine.