Nanoparticles Based on Linear and Star-Shaped Poly(Ethylene Glycol)-Poly(ε-Caprolactone) Copolymers for the Delivery of Antitubulin Drug.

PURPOSE: Biodegradable polymeric nanoparticles of different architectures based on polyethylene glycol-co-poly(ε-caprolactone) block copolymers have been loaded with noscapine (NOS) to study their effect on its anticancer activity. It was intended to use solubility of NOS in an acidic environment and ability of the nanoparticles to passively target drugs into cancer tissue to modify the NOS pharmacokinetic properties and reduce the requirement for frequent injections. METHODS: Linear and star-shaped copolymers were synthetized and used to formulate NOS loaded nanoparticles. Cytotoxicity was performed using a sulforhodamine B method on MCF-7 cells, while biocompatibility was determined on rats followed by hematological and histopathological investigations. RESULTS: Formulae with the smallest particle sizes and adequate entrapment efficiency revealed that NOS loaded nanoparticles showed higher extent of release at pH 4.5. Colloidal stability suggested that nanoparticles would be stable in blood when injected into the systemic circulation. Loaded nanoparticles had IC50 values lower than free drug. Hematological and histopathological studies showed no difference between treated and control groups. Pharmacokinetic analysis revealed that formulation P1 had a prolonged half-life and better bioavailability compared to drug solution. CONCLUSIONS: Formulation of NOS into biodegradable polymeric nanoparticles has increased its efficacy and residence on cancer cells while passively avoiding normal body tissues. Graphical Abstract ᅟ.

Mechanism of mucosal permeability enhancement of CriticalSorb® (Solutol® HS15) investigated in vitro in cell cultures.

PURPOSE: CriticalSorb™, with the principal component Solutol® HS15, is a novel mucosal drug delivery system demonstrated to improve the bioavailability of selected biotherapeutics. The intention of this study is to elucidate mechanism(s) responsible for the enhancement of trans-mucosal absorption of biological drugs by Solutol® HS15. METHODS: Micelle size and CMC of Solutol® HS15 were determined in biologically relevant media. Polarised airway Calu-3 cell layers were used to measure the permeability of a panel of biological drugs, and to assess changes in TEER, tight junction and F-actin morphology. The rate of cell endocytosis was measured in vitro in the presence of Solutol® HS15 using a membrane probe, FM 2-10. RESULTS: This work initially confirms surfactant-like behaviour of Solutol® HS15 in aqueous media, while subsequent experiments demonstrate that the effect of Solutol® HS15 on epithelial tight junctions is different from a 'classical' tight junction opening agent and illustrate the effect of Solutol® HS15 on the cell membrane (endocytosis rate) and F-actin cytoskeleton. CONCLUSION: Solutol® HS15 is the principle component of CriticalSorb™ that has shown an enhancement in permeability of medium sized biological drugs across epithelia. This study suggests that its mechanism of action arises primarily from effects on the cell membrane and consequent impacts on the cell cytoskeleton in terms of actin organisation and tight junction opening.

Surface characterisation of bioadhesive PLGA/chitosan microparticles produced by supercritical fluid technology.

PURPOSE: Novel biodegradable and mucoadhesive PLGA/chitosan microparticles with the potential for use as a controlled release gastroretentive system were manufactured using supercritical CO(2) (scCO(2)) by the Particle Gas Saturated System (PGSS) technique (also called CriticalMix(TM)). METHODS: Microparticles were produced from PLGA with the addition of mPEG and chitosan in the absence of organic solvents, surfactants and crosslinkers using the PGSS technique. Microparticle formulations were morphologically characterized by scanning electron microscope; particle size distribution was measured using laser diffraction. Microparticle surface was analyzed using X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS) to evaluate the presence of chitosan on the surface. Mucoadhesiveness of the microparticles was evaluated in vitro using a mucin assay employing two different kinds of mucin (Mucin type III and I-S) with different degrees of sialic acid contents, 0.5-1.5% and 9-17%, respectively. RESULTS: The two analytical surface techniques (XPS and ToF-SIMS) demonstrated the presence of the chitosan on the surface of the particles (<100 µm), dependent on the polymer composition of the microparticles. The interaction between the mucin solutions and the PLGA/chitosan microparticles increased significantly with an increasing concentration of mucin and chitosan. CONCLUSIONS: The strong interaction of mucin with the chitosan present on the surface of the particles suggests a potential use of the mucoadhesive carriers for gastroretentive and oral controlled drug release.

Effect of PEGylation on the toxicity and permeability enhancement of chitosan.

The aim of the present work is to investigate if conditions can be devised where PEGylation of chitosan would reduce its toxicity toward the nasal mucosa while maintaining its ability to open the cellular tight junctions and, consequently, produce an enhancement of macromolecular permeability. A series of mPEG-g-chitosan copolymers with varying levels of mPEG substitution, mPEG molecular weight, and chitosan molecular weight were synthesized by grafting carboxylic acid-terminated mPEGs (Mw 1.9 and 5.0 × 10(3) g mol(-1)) to chitosans (Mw 28.9 and 82.0 × 10(3) g mol(-1)) using a NHS/EDC coupling system. The synthesized mPEG-g-chitosans were fully characterized using a number of techniques, including FT-IR, (1)H NMR, and SEC-MALLS and their physicochemical properties were analyzed by TGA and DSC. Thereafter, the conjugates were tested for their cytotoxicity and tight junction modulating property in a relevant cell model, a mucus producing Calu-3 monolayer. mPEG-g-chitosan conjugates exhibited reduced toxicity toward cells, as compared to unmodified chitosan counterparts. Furthermore, the conjugates demonstrated a dramatic effect on cell monolayer transepithelial electrical resistance (TEER) and enhancement of permeability of model macromolecules. TEER and permeability-enhancing effects, as measurable indicators of tight junction modulation, were found to be pH-dependent and were notably more pronounced than those exhibited by unmodified chitosans. This work therefore demonstrates that conditions can be contrived where PEGylation improves the toxicity profile of chitosan, while preserving its effect on epithelial tight junctions in the nose.

In vitro and preclinical assessment of an intranasal spray formulation of parathyroid hormone PTH 1-34 for the treatment of osteoporosis.

Osteoporosis treatment with PTH 1-34 injections significantly reduces the incidence of bone fracture. Potential further reductions in fracture rate should be observed through nasal spray delivery to address the poor compliance associated with patient dislike of repeated PTH 1-34 subcutaneous injections. In vitro human osteoblast-like Saos-2 cell intracellular cAMP levels were used to define PTH 1-34 nasal spray formulation bioactivity. The chemically synthesised PTH 1-34 had an EC50 of 0.76nM. Absorption enhancers polyethylene glycol (15)-hydroxystearate (Solutol® HS15), poloxamer 407, chitosan or sodium hyaluronate did not diminish the bioactivity of PTH 1-34 within an in vitro cell culture model (p >0.05). We also demonstrated the effectiveness of the transmucosal absorption enhancer Solutol® HS15 in a nasal spray formulation using a preclinical pharmacokinetic model. In Sprague-Dawley rats without the absorption enhancer the uptake of PTH 1-34 into the blood via intranasal delivery produced a Cmax of 2.1±0.5ng/ml compared to 13.7±1.6ng/ml with Solutol® HS15 enhancer (p=0.016) and a Cmax14.8±8ng/ml in subcutaneous injections. Together these data illustrate that the nasal spray formulation bioactivity in vitro is not affected by the nasal spray absorption enhancers investigated, and the Solutol® HS15 nasal spray formulation had an equivalent pharmacokinetic profile to subcutaneous injection in the rat model. The Solutol® HS15 formulation therefore demonstrated potential as a PTH 1-34 nasal spray formulation for the treatment of osteoporosis.

Nanoparticulate systems for nasal delivery of drugs: a real improvement over simple systems?

This review discusses the possible benefits of using nanoparticles for nasal delivery of drugs and vaccines. It considers the various factors affecting particle transport across the nasal tissue. The evidence for the improved transport of drugs, such as peptides and proteins, across the nasal epithelium when formulated in a nanoparticulate system, as compared to an optimal solution formulation, is not convincing. For instance it has been shown that a chitosan solution and especially a chitosan powder formulation was superior in enhancing the nasal absorption of insulin as compared to chitosan nanoparticles. On the other hand, the use of nanoparticles for vaccine delivery seems beneficial in that good immune responses are achieved. This could be due to the fact that small particles can be transported preferentially by the lymphoid tissue of the nasal cavity (NALT). However, apparently no studies have been published comparing directly other adjuvant nasal systems with nanoparticulate systems.

Evaluation of thermosensitive poloxamer 407 gel systems for the sustained release of estradiol in a fish model.

The purpose of this study was to develop and evaluate a delivery system comprising a thermosensitive gel for the sustained release of steroidal hormones in fish, over an extended period of time after a single intramuscular (i.m.) injection and for the improved reproductive performance in fish. Controlled delivery systems based on thermosensitive gels are easy to prepare, low cost and high versatility dosage forms, which have been shown to be effective in several animal species for sustained release of hormones. In this work, a thermosensitive gel system based on poloxamer 407 in water:ethanol medium, able to work as a prolonged release carrier for 17ß-estradiol (E2), has been developed. Such a system was able to solubilize the lipophilic E2 and to gel at the required water temperature for fish rearing (20°C). Moreover, the system exhibited the best injection condition at temperatures below 15°C when the system behaved as a low viscosity Newtonian liquid. The thermosensitive gel system was tested in vivo in the fish model, Carassius auratus, and the results compared with a single i.m. injection of E2 dissolved in corn-oil and other relevant control systems not containing E2. The results were particularly interesting, since fish injected with the E2 thermosensitive gel formulation, showed significantly higher levels of the circulating hormone than corn oil-E2 treated animals at 72 and 96h after injection. In addition, the thermogel system was able to sustain the plasma level of E2 for about 11days. The increased plasma levels of E2 were also accompanied by maintained higher values of plasma vitellogenin (VTG), thus suggesting that the thermosensitive polymer based delivery system could prevent rapid hepatic clearance of E2, resulting in prolonged stimulation of estrogen receptor-mediated pathways in goldfish.

Suitability of polymer materials for production of pulmonary microparticles using a PGSS supercritical fluid technique: preparation of microparticles using PEG, fatty acids and physical or chemicals blends of PEG and fatty acids.

The production of microparticles using a supercritical carbon dioxide based PGSS technique (CriticalMix™) has been exploited to develop blended systems targeted at pulmonary delivery. Hence, PEG based polymers of different molecular weights (1000-6000 Da) were blended in situ with fatty acids (stearic, palmitic or myristic acid) or with commercially available PEG-stearates. The effect of the different thermodynamic properties of the polymers was evaluated by characterising the microparticles produced in terms of their melting temperature by conventional DSC and in the presence of high pressure CO(2) using a high pressure variable volume view cell. The microparticles produced were also assessed by SEM and particle size distribution. It is well known that as the molecular weight of the PEG chains increases, so does the viscosity of the melt and this leads to an increase in the particle size. In the paper we show that blending with myristic acid provides optimal control of particle size when the blend is sprayed from scCO(2) leading to high yields in the optimal aerodynamic size range of 2-5 µm for the deep lung delivery. The highest yield and smallest particles (~5 µm) were produced with a blend of PEG 3000 and myristic acid (1:1) whereas the batches containing palmitic acid and stearic acid showed lower yields and larger particle sizes.

CriticalSorb: a novel efficient nasal delivery system for human growth hormone based on Solutol HS15.

The absorption enhancing efficiency of CriticalSorb for human growth hormone (MW 22 kDa) was investigated in the conscious rat model. The principle absorption enhancing component of CriticalSorb, Solutol HS15, comprises polyglycol mono- and di-esters of 12-hydroxystearic acid combined with free polyethylene glycol. When administering hGH nasally in rats with increasing concentrations of Solutol HS15, it was found that for a 10%w/v solution formulation a bioavailability of 49% was obtained in the first 2h after administration. Furthermore it was shown that the most effective ratio of Solutol HS15 to hGH was 4:1 on a mg to mg basis. Histopathology studies in rats after 5 days repeated nasal administration showed that Solutol HS15 had no toxic effect on the nasal mucosa. These results have been confirmed in a 6 month repeat nasal toxicity study in rats. It can be concluded that the principle absorption enhancing component of CriticalSorb - Solutol HS15 - is a potent and non- toxic nasal absorption enhancer that warrants further development.

Suitability of polymer materials for production of pulmonary microparticles using a PGSS supercritical fluid technique: thermodynamic behaviour of fatty acids, PEGs and PEG-fatty acids.

The thermodynamic behaviour of selected polymeric components for preparation of controlled release microparticles using supercritical carbon dioxide (scCO(2)) processing was investigated. The polymeric materials selected were egg lecithin (a model for the lung surfactant phospholipid), poly(ethyleneglycol) (PEG) of different molecular weights, fatty acids (C18, C16, and C14), and physical blends of PEGs and fatty acids. In addition a range of PEG-stearates was also assessed. Analysis of thermodynamic behaviour was performed by differential scanning calorimetry (DSC) and by assessment of their interaction with scCO(2) in a high-pressure variable volume view cell. The key criterion was to demonstrate a strong interaction with scCO(2) and to show liquefaction of the polymeric material at acceptable processing temperatures and pressures. Positive results should then indicate the suitability of these materials for processing by the Particle from Gas Saturated Solutions (PGSS) technique using scCO(2) to create microparticles for pulmonary administration. It was found that the materials tested interacted with scCO(2) and showed a sufficient lowering of their melting temperature (T(m)) to make them suitable for use in the PGSS microparticle production rig. Fatty acids of low T(m) were shown to act as a plasticising agent and to lower the T(m) of PEG further during interaction with scCO(2).

Stability of human growth hormone in supercritical carbon dioxide.

The instability of human growth hormone (hGH) to temperature and interfaces makes its formulation into injectable, sustained-release drug delivery systems challenging. A novel method of encapsulating hGH in polymeric microparticles has been developed using supercritical CO(2) (scCO(2)) technology, but there is limited understanding of the stability of hGH within this system. The aim of this study was to evaluate the stability of hGH in scCO(2) processing. The integrity of the protein was assessed following exposure to scCO(2) using a range of different analytical techniques. Mass spectrometry showed that no peptide cleavage occurred as a result of processing or exposure to scCO(2). Size-exclusion chromatography detected formation of aggregates at high temperatures, but not as a result of the encapsulation process. Reverse-phase chromatography demonstrated that the production of deamidation products occurred as a function of temperature, but only at temperatures higher than those used for the encapsulation process. Circular dichroism and infrared spectroscopy demonstrated that the use of scCO(2) was not detrimental to the secondary molecular structure of hGH. Together, these results show that the structural integrity of hGH is unaffected by scCO(2) processing and that hGH can be successfully encapsulated in polymer microparticles using this technique.

Effect of physicochemical properties on intranasal nanoparticle transit into murine olfactory epithelium.

Small molecular weight drugs, peptides, and nanoparticles have previously been shown to localize in the central nervous system after intraneural administration. A basic understanding of direct nose-to-brain drug delivery, particularly for nanoparticles with different physicochemical characteristics, remains unclear. In this study, fluorescence microscopy and stereology were used to track intranasally administered chitosan-coated polystyrene (C-PS) or polysorbate-coated polystyrene (P80-PS) nanoparticles (100 nm or 200 nm in diameter) in olfactory and respiratory nasal epithelia and olfactory bulbs in mice. Chitosan coating caused particles to adhere to the extracellular mucus which could provide useful modality for paracellular drug transport. Nanoparticle transport was exclusively transcellular. None of the nanoparticle formulations showed preference for uptake into olfactory axons over other nasal epithelial cells. Both 100 nm PS and 100 nm P80-PS were observed in olfactory epithelial cells but were absent from the olfactory bulbs; therefore, it is speculated that an optimal nanoparticle diameter for axonal transport is <100 nm in mice.