Synthesis and in vitro antitumor effect of diclofenac and fenoprofen thiolated and nonthiolated polyaspartamide-drug conjugates.

This paper reports the synthesis and antiproliferative effects of new thiomer-diclofenac and fenoprofen conjugates, hydrophilic, bioadhesive, polymeric prodrugs, as well as antiproliferative effects of diclofenac, fenoprofen and a series of previously described polymer-fenoprofen conjugates on five tumor cell lines. Thiolated and nonthiolated polyaspartamides were the chosen polymeric components. Drug-loading ranged from 5.6 to 22.4%, and the amount of SH groups ranged from 6.9 to 45.6micromol g(-1). Tensile studies demonstrated a clear correlation between the amount of thiol and the mucoadhesive properties of the conjugates. The growth-inhibitory activity of the tested polymer-drug conjugates demonstrates that polyaspartamide-type polymers, especially thiolated polymers, enable inhibition of tumor cell growth with significantly lower doses of the active substance.

Development and bioadhesive properties of chitosan-ethylcellulose microspheres for nasal delivery.

Loratadine-loaded microspheres were prepared by spray-drying of dispersions, emulsions and suspensions differing in polymeric composition and solvents used. Conventional microspheres were obtained by spray-drying of dispersions composed of chitosan (CM) as only polymer, while composed microspheres were obtained by spray-drying of two-phase systems composed of chitosan and ethylcellulose (EC). Microspheres differed in EC/CM weight ratio (0:1, 1:2 and 1:3) and in loratadine/polymers weight ratio (1:6 and 1:8). The entrapment efficiencies were between 67.9 and 86.1%; less loratadine was entrapped as polymer/drug ratio decreased. In comparison to one-phase systems composed of CM as only polymer, spray-drying of two-phase systems composed of both, CM and EC resulted in improved loratadine entrapment (80.1-86.1%). All microspheres were positively charged, indicating the presence of chitosan at the surface, regardless of the drug content and the type of spray-dried system. The highest zeta-potential was measured for loratadine-free conventional microspheres, consisting of chitosan only (32.7+/-1.3 mV). Tensile studies showed that both, EC/CM ratio and the type of spray-dried system influenced the bioadhesive properties of the microspheres in a way that the microspheres with higher chitosan content were more bioadhesive and microspheres prepared from suspensions were more bioadhesive than those prepared from emulsions, regardless of the same polymeric composition. The results suggested that the spray-drying method is useful to produce bioadhesive loratadine-loaded microspheres.

Synthesis and characterization of thiomers of polyaspartamide type.

Synthesis of poly[alpha,beta-(N-2-hydroxyethyl-DL-aspartamide)]-thioglycolic acid (PHEA-TGA) conjugate as a new polyaspartamide thiomer is described. The parent polymer PHEA is chemically modified by introducing sulphydryl-bearing compound thioglycolic acid. By varying the reaction conditions several batches of PHEA-TGA conjugates were prepared and analyzed. Tensile studies revealed that total work of adhesion of PHEA-TGA increased more than twice compared to the unmodified polymer. Microparticles prepared from the thiolated polymer preserved its bioadhesive properties.

Microencapsulation of genetically engineered fibroblasts secreting nerve growth factor.

We demonstrated that genetically modified fibroblasts can be encapsulated into biocompatible, biodegradable spheres retaining their viability and capacity to continuously secrete nerve growth factor (NGF) for at least two months. Genetically engineered rat fibroblasts producing NGF were encapsulated in an alginate-polylysine gel with the ultimate objective of improving transplantation methodologies. Cultures were suspended in a sodium alginate solution and the suspension was extruded drop-wise into a solution of calcium chloride. Morphological properties of the spheres were assessed by light and electron microscopy. The spheres had a homogenous external membrane, without fibroblasts, protruding from the surface of the capsular membrane. The NGF determinations in culture media showed that encapsulated fibroblasts continued to synthesize NGF for at least 60 days. We also confirmed that secreted NGF was biologically active, by assessing the induction of choline acetyltransferase (ChAT) activity in dissociated embryonic rat septal cultures. These results encourage further studies using in vivo models to determine the value of applying microencapsulated genetically modified cells secreting trophic factors as a therapeutic strategy for central nervous system (CNS) injuries.

Effects of coencapsulated NGF and GM1 in rats with cortical lesions.

Nerve growth factor (NGF) and monosialoganglioside GM1 were microencapsulated in biodegradable co-polymer poly(L-lactide):co-glycolide and administered locally to rats with unilateral cortical devascularizing lesions. Microcapsules were placed directly onto the lesioned cortical surface and rats were sacrificed 30 days post-operatively. Biochemical and quantitative immunocytochemical analyses revealed effective protection from degenerative changes of nucleus basalis magnocellularis (NBM) cholinergic neurones on the lesioned side as well as a significant increase in choline acetyltransferase activity in the frontoparietal cortex surrounding the lesion. Results of this study strongly suggest that the topical application of microcapsules of a biodegradable polymer containing a mixture of two neuroprotective factors is a viable alternative to the use of osmotic minipumps for delivery of these agents into the CNS. Indeed, this approach might find clinical applications.

Preparation, characterization and release of microencapsulated bromodeoxyuridine.

Biodegradable and biocompatible microspheres with bromodeoxyuridine (BrUrd) have been prepared, characterized and tested in vitro and in vivo. Scanning electron microscopy and image analysis revealed regular spherical shapes and an average size +/- SD of 2.47 +/- 0.59 microns. Total content of BrUrd as determined by HPLC was within the range of 0.2-1.5%. Kinetic analyses of two different preparations showed similar release half-times (approx. 12 hrs), kinetic constants -0.0556, -0.0564, and -0.0557, -0.0597 [h-1], and correlation coefficients of 0.998, and 0.999 when fitted to the first order or biphasic first order kinetics, respectively. Preliminary data from immunocytochemical studies revealed efficient incorporation of BrUrd delivered from these microcapsules into nuclei of proliferating cells surrounding brain lesions in rats.

Chitosan microspheres with hydrocortisone and hydrocortisone-hydroxypropyl-beta-cyclodextrin inclusion complex.

In the present study, an inclusion complex composed of hydrocortisone acetate (HC) and hydroxypropyl-beta-cyclodextrin (HPbetaCD) was prepared by the spray-drying method. HC alone, HC inclusion complex or HC with HPbetaCD as a physical mixture were incorporated into chitosan microspheres by spray-drying. The inclusion complex and microspheres were characterized by X-ray powder diffractometry and differential scanning calorimetry (DSC). Microspheres were studied with respect to particle size distribution, drug content and in vitro drug release. The results indicate that the HCHPbetaCD inclusion complex is more water soluble than HC alone. The HC release rates from chitosan microspheres were influenced by the drug/polymer ratio in the manner that an increase in the release rate was observed when the drug loading was decreased. However, release data from all samples showed significant improvement of the dissolution rate for HC, with 25-40% of the drug being released in the first hour compared with about 5% for pure HC. The complexation method and microsphere preparation method (spray-drying) is simple with great potential for industrial production.

Processing of carbamazepine-PEG 4000 solid dispersions with supercritical carbon dioxide: preparation, characterisation, and in vitro dissolution.

The purpose of this study was to apply the attractive technique of the supercritical fluid to the preparation of solvent-free solid dispersions. In particular, the gas antisolvent crystallisation technique (GAS), using supercritical carbon dioxide as processing medium, has been considered to prepare an enhanced release dosage form for of the poorly soluble carbamazepine, employing PEG 4000 as a hydrophilic carrier. The physical characterisation of the systems using laser granulometer, powder X-ray diffraction, thermal analyses, and scanning electron microscopy was carried out in order to understand the influence of this technological process on the physical status of the drug. The results of the physical characterisation attested a substantial correspondence of the solid state of the drug before and after treatment with GAS technique, whereas a pronounced change in size and morphology of the drug crystals was noticed. The dramatic reduction of the dimensions and the better crystal shape, together with the presence of the hydrophilic polymer determined a remarkable enhancement of the in vitro drug dissolution rate.

Preparation in high-shear mixer of sustained-release pellets by melt pelletisation.

The preparation of sustained-release pellets by melt pelletisation was investigated in a 10-l high shear mixer and ternary mixtures containing stearic acid as a melting binder, anhydrous lactose as a filler and theophylline as a model drug. A translated Doehlert matrix was applied for the optimisation of process variables and quality control of pellets characteristics. After determination of size distribution, the pellets were characterised with scanning electron microscopy, X-ray photoelectron spectroscopy and porosimetric analysis. Finally, the in vitro release from every single size fraction was evaluated and the release mechanism was analysed. Since the drug release rate decreased when enhancing the pellet size fraction, the 2000-microm fraction, exhibiting a substantially zero-order release, was selected for further in vivo biovailability studies. These data demonstrated that pellets based on the combination of stearic acid and lactose can be used to formulate sustained release pellets for theophylline.

Microspheres of human serum albumin with barbiturates: effect of drug partition coefficient on preparation and drug release.

Human serum albumin microspheres were prepared with a series of barbiturates by the thermal denaturation method. The barbiturates were of similar general physicochemical properties but different partition coefficients. The total drug content of microspheres was dependent on the partition coefficient, and an increase in the partition coefficient caused a decrease in the drug content of finished microspheres due to drug migration to the outer organic phase during preparation. The ensemble drug release from microspheres was followed by the paddle method and by potentiometric titration with a pH-stat. Nonlinear regression analysis showed the best fit for the spherical Higuchi equation, especially first-order kinetics (biphasic). The drug partition coefficient affected the release in such a way that drugs with higher partition coefficients were released faster and to a greater extent than those with lower coefficients.

Effect of hydroxypropyl-beta-cyclodextrin on hydrocortisone dissolution from films intended for ocular drug delivery.

The formation of an inclusion complex between hydrocortisone and hydroxypropyl-beta-cyclodextrin can affect the in vitro transfer rate of hydrocortisone from the aqueous to the organic phase. The observed first order transfer rate constants showed that the complexation of hydrocortisone with hydroxypropyl-beta-cyclodextrin decreased significantly the transport of the drug depending on the partition coefficient of the drug, and the relative magnitude of the stability constant of the inclusion complex. To optimize the ocular drug delivery, high molecular weight cellulosis and PVA polymeric films were prepared. No unified mathematical model can predict the release profile of drug and complex from films. The drug and complex-polymer interactions in each system could be responsible for the solubility of the drug, and different release behaviours of hydrocortisone and cyclodextrin inclusion complex from the films prepared.

Albumin-loaded PLA and PLGA microspheres: in vitro evaluation.

Polylactide (PLA) and poly(lactide-co-glycolide) (PLGA) microspheres containing bovine serum albumin (BSA) as a model protein were prepared by an solvent extraction procedure using water-in oil-in water complex emulsion system. The effects of process parameters on morphology, particle size, protein loading and in vitro release properties of microspheres were studied. The encapsulation efficiency was very high ranging from 75% to 99%. The in vitro release profile was described as bi-phasic process with an initial burst effect and the second sustained release phase and it was dependent on the type of polymer used, and BSA loading of microspheres.

A nonionic surfactant/chitosan micelle system in an innovative eye drop formulation.

Micelle systems composed of the polyoxyethylated nonionic surfactant Pluronic F127 (F127) and cationic polyelectrolyte chitosan (CH) were prepared with dexamethasone (DEX) as a hydrophobic model drug. The F127/CH micelles were characterised by their hydrodynamic diameter and a zeta-potential ranging between 25.4 and 28.9 nm and +9.3 and +17.6 mV, respectively. The DEX loading was between 0.48% and 0.56%, and no significant influence of CH on DEX loading was observed. All micelle systems were characterised by prolonged release profiles. The addition of CH significantly enhanced the in vitro DEX release rate and transport across Caco-2 cell monolayers, as compared to the CH-free F127 micelle system. This colloidal carrier was well tolerated in rabbit eyes, and no clinically abnormal signs in various ocular structures were observed. The increase in intraocular pressure (IOP) in rabbits was used to evaluate DEX ocular bioavailability. The AUC values showed a 1.7- and 2.4-fold increase in bioavailability with F127 and F127/0.015 (w/v) % CH micelle systems, respectively, as compared to a standard DEX suspension. These data indicate improved intraocular DEX absorption from the micelle systems, which can be ascribed to both F127 and CH corneal permeability enhancement.

Spray-dried chitosan/ethylcellulose microspheres for nasal drug delivery: swelling study and evaluation of in vitro drug release properties.

The aim of this study was to develop spray-dried chitosan-based microspheres, suitable for nasal delivery of loratadine, and to evaluate their potential of modifying loratadine release. The microspheres were composed with ethylcellulose (EC) and chitosan (CM) in two different weight ratios, 1:2 and 1:3. One-phase systems (dispersions) and two-phase systems (emulsions and suspensions) were subjected to spray-drying, resulting in conventional and composed microspheres, respectively. The microspheres were evaluated with respect to the yield, particle size, encapsulation efficiency, physical state of the drug in the polymer matrix, swelling properties and in vitro drug release profile. It was shown that particle size, swelling ability and loratadine release from spray-dried microspheres were significantly affected by the polymeric composition and feed concentration in spray-drying process. Emulsifying method to produce composed EC/CM microspheres resulted in improved loratadine entrapment and moderate swelling, when compared to conventional chitosan microspheres. It seems like better formation of EC cores and chitosan coating were obtained when higher feed concentration and ultrasonic homogenization were employed in the preparation of emulsion systems and when EC to CM weight ratio was 1:3.

Mucoadhesive chitosan-coated liposomes: characteristics and stability.

Lecithin liposomes, empty or containing FITC-dextran, were prepared by the ethanol injection method. Three different types of chitosans with different molecular weight and degrees of deacetylation were used (Seacure 113, 210 and 311). Chitosan coating was carried out by mixing the liposomal suspension with the chitosan solution followed by incubation. The size of liposomes was measured before and after polymer coating by an image analysis technique. The mean diameter of liposomes containing FITC-dextran was in the size range 250-280 nm, whereas the size after coating was 300-330 nm, regardless of chitosan type. All chitosan-coated liposomes were of spherical shape and no morphological differences between uncoated and coated liposomes were observed. Liposomes with FITC-dextran, originally entrapping 50% of the marker substance taken in the preparation and coated in the presence of unentrapped marker substance, contained 60-65% of the marker substance. The highest entrapment was found for liposomes coated with medium molecular weight chitosan. The stability of chitosan-coated liposomes in simulated gastric fluid was significantly higher as compared to uncoated liposomes. One can conclude that chitosan is stabilizing the original liposomal structure and protecting liposomally entrapped drug.

Microparticles with neuroactive agents.

An overview of biodegradable and biocompatible microcapsules and microspheres loaded with neuroactive substances, or cells producing neuroactive substances, and their role as drug delivery systems (DDS) for drug administration to the central nervous system (CNS) is given. In addition, closely related systems are also summarized.

Microencapsulated ciliary neurotrophic factor: physical properties and biological activities.

Controlled drug release in the CNS and PNS is still an obstacle to the treatment of neurodegenerative disorders. We have prepared a variety of microspheres containing either ciliary neurotrophic factor (CNTF) or genetically engineered cells able to synthesize and release this cytokine. CNTF is a multifunctional cytokine that can regulate the survival and differentiation of many types of developing and adult neurons. However, when given in therapeutically effective doses by systemic injections, it produces numerous adverse side effects. In order to minimize these effects we have microencapsulated it in biopolymers (chitosans, alginates, and copolymers in various proportions to achieve different kinetic properties). Size distribution profiles were determined by an image analysis system and surface characteristics were assessed by electron microscopy. The total content of CNTF as well as the amounts released per day were determined by ELISA and in vitro bioassays. The results from the release kinetics demonstrate that long-term secretion (up to 24 days) of CNTF is achieved by combining chitosan with copolymerized lactic and glycolic acid, whereas microspheres made of alginate provided only relatively short-term release (2-12 days). Neuron survival and neurite outgrowth in cultures of ciliary ganglia were supported by microencapsulated CNTF, indicating biological stability of CNTF. Genetically engineered human kidney cells 293 continued synthesizing CNTF within spheres and the released amounts of CNTF in the culture medium were comparable to the amounts secreted from monolayers (1 ng/ml of supernatant from confluent cultures) or even higher. These studies provide a basis for future testing of CNTF in encapsulated preparations using animal models of neurodegenerative disorders.