Graphene oxide as a functional excipient in buccal films for delivery of clotrimazole: Effect of molecular interactions on drug release and antifungal activity in vitro.

Graphene oxide (GO) is an amphiphilic, high surface area material with great potential as a functional excipient in drug delivery. The present study aimed at incorporating GO in buccal polyelectrolyte films for delivery of antifungal drugs and investigating the effect of GO on the film properties and drug release profiles, as well as antifungal activities. Mucoadhesive excipients chitosan and alginate were selected to form polyelectrolyte films with the antifungal drug clotrimazole. The buccal formulations were prepared by mixing the excipients and clotrimazole via probe sonication, followed by film casting and drying. Inclusion of GO in the formulations increased clotrimazole release from the films in vitro (pH 6.8), possibly due to GO altering the electrostatic interactions between chitosan and alginate. An increase of in vitro activity against Candida albicans was observed when 0.04 wt% GO was added in the formulation containing clotrimazole. However, when the GO amount increased to 0.09 wt%, the films had similar antifungal ability to the films with 0.04 wt% GO, suggesting that the electrostatic and hydrophobic interactions between GO and clotrimazole also affects the antifungal effect of clotrimazole. In summary, GO has a great potential as a functional excipient for delivery of antifungal drugs.

Pain during topical photodynamic therapy: uncomfortable and unpredictable.

BACKGROUND: The major drawback of the widely used photodynamic therapy (PDT) is treatment-related pain. OBJECTIVE: Gain insight into the intensity of and predictive factors for painful burning sensation associated with PDT. METHODS: A prospective cohort study was performed at the department of Dermatology in the Maastricht University Medical Centre in Maastricht, a reference centre for dermatological oncology in The Netherlands. A total of 141 lesions in 108 patients were included, treated from November 2008 until June 2009 with PDT for superficial basal cell carcinoma, Bowen's disease (BD) or actinic keratosis (AK). Painful burning sensation was scored based on an 11-point pain intensity numeric rating scale (PI-NRS) (0=no pain; 10=worst possible pain). RESULTS: The percentage of patients with a PI-NRS score over six was 32.6% and 37.9% during the primary and follow-up PDT session respectively. A total of 76.6% (95/124) of the patients was consistent in pain intensity score reporting. Factors associated with higher PI-NRS scores were treatment of AK or BD, tumour localization in the head/neck region, patient's age over 70, Fitzpatrick skintype I/II, photosensitizer 5-aminolevulinic acid and use of oral analgesics. After mutual adjustment of these factors, Fitzpatrick skintype remained the only independent predictor of PI-NRS scores during PDT. CONCLUSION: It remains difficult to decide which patients should be considered for pain relieving measures. The solution remains to support all patients treated with PDT with pain relieving techniques or to let the support of pain relieving measures depend on the reported pain score for the primary session.

Probing insulin's secondary structure after entrapment into alginate/chitosan nanoparticles.

The aim of the present study was to probe the structural integrity of insulin after being entrapped into chitosan/alginate nanoparticles produced by ionotropic polyelectrolyte pre-gelation. By manipulating the alginate:chitosan mass ratio and the pH during nanoparticle production, desired nanoparticles with a mean size of 850 (+/-88)nm and insulin association efficiency of 81 (+/-2)% were obtained. Insulin secondary structure was assessed by Fourier transform infrared (FTIR) and circular dichroism (CD) after entrapment into nanoparticles and after release from the particles under gastrointestinal simulated conditions. FTIR second-derivative spectra and area-overlap compared to an insulin standard confirmed that no significant conformational changes of insulin occurred in terms of alpha-helix and beta-sheet content. Far-UV-CD spectra corroborated the preservation of insulin structure during the nanoparticle production procedure. The presented nanoparticulate system is a promising carrier for insulin oral delivery since it preserves insulin structure and therefore also, potentially, its bioactivity.

Characterisation of salmon calcitonin in spray-dried powder for inhalation. Effect of chitosan.

Salmon calcitonin (sCT) powders suitable for inhalation, containing chitosan and mannitol as absorption enhancer and protection agent, respectively, were prepared using a spray-drying process. The effect of chitosan on physicochemical stability of sCT in the dry powder was investigated by different analytical techniques. High-performance liquid chromatography (HPLC) analysis indicated that sCT was chemically stable upon spray-drying. With the proportion of chitosan in spray-drying formulation being increased, dissolution of sCT from the dry powders was decreased both in phosphate buffer and acetate buffer. The thioflavine T fluorescence assay showed that no fibrils were present in the spray-dried powder. However, sCT partly fibrillated in the phosphate buffer, but not in acetate buffer. Fourier transform infrared (FTIR) spectra showed that the secondary structure of sCT was slightly changed in the dry powder, yet no aggregate signal was observed. Circular dichroism analysis indicated that the structure of sCT in an aqueous formulation was slightly altered by addition of chitosan. Nevertheless, recovery of sCT was not influenced by chitosan in the aqueous formulation as indicated by HPLC analysis. This study suggested that sCT, in absence of any additives, was stable during the spray-drying process under certain conditions. Addition of chitosan affects recovery of sCT from spray-dried powders, which may be due to formation of a partially irreversible complex between the protein and chitosan during the spray-drying process.

Preparing and evaluating delivery systems for proteins.

From a formulation perspective proteins are complex and therefore challenging molecules to develop drug delivery systems for. The success of a formulation depends on the ability of the protein to maintain the native structure and activity during preparation and delivery as well as during shipping and long-term storage of the formulation. Therefore, the development and evaluation of successful and promising drug delivery systems is essential. In the present review, some of the particulate drug delivery systems for parenteral delivery of protein are presented and discussed. The challenge for incorporation of protein in particulate delivery systems is exemplified by water-in-oil emulsions.

Stability aspects of salmon calcitonin entrapped in poly(ether-ester) sustained release systems.

Poly(ether-ester)s composed of hydrophilic poly(ethylene glycol)-terephthalate (PEGT) blocks and hydrophobic poly(butylene terephthalate) (PBT) blocks were studied as matrix for the controlled release of calcitonin. Salmon calcitonin loaded PEGT/PBT films were prepared from water-in-oil emulsions. The initial calcitonin release rate could be tailored by the copolymer composition, but incomplete release of calcitonin was observed. FTIR measurements indicated aggregation of calcitonin in the matrix, which was not due to the preparation method of the matrices, but due to the instability of calcitonin in an aqueous environment. Release experiments showed the susceptibility of calcitonin towards the composition of the release medium, in particular to the presence of metal ions. With increasing amount of sodium ions, a decrease in the total amount of released calcitonin was observed due to enhanced aggregation. The calcitonin had to be stabilized in the matrix to prevent aggregation. Incorporation of sodium dodecyl sulphate (SDS) as a stabilizer in PEGT/PBT matrices increased the percentage of calcitonin released, but could not avoid aggregation on a longer term.

Influence of neutron irradiation on holmium acetylacetonate loaded poly(L-lactic acid) microspheres.

Holmium-loaded microspheres are useful systems in radio-embolization therapy of liver metastases. For administration to a patient, the holmium-loaded microspheres have to be irradiated in a nuclear reactor to become radioactive. In this paper. the influence of neutron irradiation on poly(L-lactic acid) (PLLA) microspheres and films, with or without holmium acetylacetonate (HoAcAc), is investigated, in particular using differential scanning calorimetry (MDSC), scanning electron microscopy, gel permeation chromatography (GPC), infrared spectroscopy, and X-ray diffraction. After irradiation of the microspheres, only minor surface changes were seen using scanning electron microscopy, and the holmium complex remained immobilized in the polymer matrix as reflected by a relatively small release of this complex. GPC and MDSC measurements showed a decrease in molecular weight and crystallinity of the PLLA, respectively, which can be ascribed to radiation induced chain scission. Irradiation of the HoAcAc loaded PLLA matrices resulted in evaporation of the non-coordinated and one coordinated water molecule of the HoAcAc complex, as evidenced by MDSC and X-ray diffraction analysis. Infrared spectroscopy indicated that some degradation of the acetylacetonate anion occurred after irradiation. Although some radiation induced damage of both the PLLA matrix and the embedded HoAcAc-complex occurs, the microspheres retain their favourable properties (no marginal release of Ho, preservation of the microsphere size), which make these systems interesting candidates for the treatment of tumours by radio-embolization.

Fourier transform infrared spectrometric analysis of protein conformation: effect of sampling method and stress factors.

Changes in the amide bands in Fourier transform infrared spectra of proteins are generally attributed to alterations in protein secondary structure. In this study spectra of five different globular proteins were compared in the solid and solution states recorded with several sampling techniques. Spectral differences for each protein were observed between the various sampling techniques and physical states, which could not all be explained by a change in protein secondary structure. For example, lyophilization in the absence of lyoprotectants caused spectral changes that could (partially) have been caused by the removal of hydrating water molecules rather than secondary structural changes. Moreover, attenuated total reflectance spectra of proteins in H2O were not directly comparable to transmission spectra due to the anomalous dispersion effect. Our study also revealed that the amide I, II, and III bands differ in their sensitivities to changes in protein conformation: For example, strong bands in the region 1620-1630 and 1685-1695 cm(-1) were seen in the amide I region of aggregated protein spectra. Surprisingly, absorbance of such magnitudes was not observed in the amide II and III region. It appears, therefore, that only the amide I can be used to distinguish between intra- and intermolecular beta-sheet formation. Considering the differing sensitivity of the different amide modes to structural changes, it is advisable to utilize not only the amide I band, but also the amide II and III bands, to determine changes in protein secondary structure. Finally, it is important to realize that changes in these bands may not always correspond to secondary structural changes of the proteins.

Characterization of poly(L-lactic acid) microspheres loaded with holmium acetylacetonate.

Holmium-loaded PLLA microspheres are useful systems in radioembolization therapy of liver metastases because of their low density, biodegradability and favourable radiation characteristics. Neutron activated Ho-loaded microspheres showed a surprisingly low release of the relatively small holmium complex. In this paper factors responsible for this behaviour are investigated, in particular by the use of differential scanning calorimetry, scanning electron microscopy, infrared spectroscopy and X-ray diffraction. The holmium complex is soluble in PLLA up to 8% in films and 17% in microspheres. Interactions between carbonyl groups of PLLA, and the Ho-ion in the HoAcAc complex, explain very satisfactorily the high stability of holmium-loaded microspheres.

The effect of a water/organic solvent interface on the structural stability of lysozyme.

The effect of emulsification of lysozyme solutions with methylene chloride on protein recovery and structural integrity was investigated. Total lysozyme recovery in the aqueous phase was found to be concentration dependent, and ranged between 65 and 80%. The unrecovered lysozyme was observed at the interface as a white precipitate. No structural changes of the soluble lysozyme were observed by enzymatic activity assay, size-exclusion chromatography (SEC), gel electrophoresis (SDS-PAGE), and circular dichroism (CD). The lyophilized precipitated protein was analyzed by FTIR, and evidence of intermolecular beta-sheet formation was found. In addition, the precipitate was analyzed after redissolution in 1 M guanidine hydrochloride by enzymatic activity assay, CD, SDS-PAGE, and SEC. No differences with control lysozyme samples or samples in aqueous buffer solutions were observed. This indicates that lysozyme precipitates as non-covalent aggregates upon emulsification, and these precipitates can refold into their native state in 1 M guanidine hydrochloride. Protein recovery could not be improved by the addition of sucrose, Tween 20, or Tween 80. Excipients competing for the water/organic solvent interface, such as BSA and partially hydrolyzed polyvinylalcohol (PVA) significantly improved lysozyme recovery to >95%. Emulsions which contained poly(lactic-co-glycolic acid) (PLGA) in the organic phase gave irreproducible protein recovery. Here also, partially hydrolyzed PVA significantly increased lysozyme recovery. Thus, we found that emulsification of lysozyme-containing aqueous solutions with methylene chloride causes incomplete protein recovery and non-covalent aggregation of lysozyme. These aggregates are also encapsulated in controlled drug delivery systems which are prepared using a water-in-oil emulsification procedure. The use of surface-active additives, such as partially hydrolyzed PVA significantly reduces lysozyme aggregation, and can be used to prevent encapsulation of inactive and potentially immunogenic protein species.

Lysozyme distribution and conformation in a biodegradable polymer matrix as determined by FTIR techniques.

Lysozyme distribution and conformation in poly(lactic-co-glycolic acid)(PLGA) microspheres was determined using various infrared spectroscopic techniques. Infrared microscopy and confocal laser scanning microscopy indicated that the protein was homogeneously distributed inside the microspheres in small cavities resulting from the water-in-oil emulsification step. Part of the protein was observed at or near the cavity walls, while the rest was located within these cavities. Attenuated total reflectance (ATR) and photoacoustic spectroscopy (PAS) also showed that there is hardly any protein at the surface of the microspheres. Since this microsphere formulation gave a large burst release (ca. 50%), this burst release can not be caused by protein at the surface of the particles. Probably, the protein is rapidly released through pores in the PLGA matrix. Conformational analysis of lysozyme in the PLGA microspheres by KBr pellet transmission suffered from band shape distortion and baseline slope. Despite incomplete subtraction of the PLGA background, a characteristic band of non-covalent aggregates at 1625 cm(-1) was observed in the second derivative spectrum of the protein Amide I region. The other Fourier-transform infrared (FTIR) methods yielded similar results, indicating that the sample preparation procedure did not introduce artifacts. The observed aggregation signal may correspond to the protein adsorbed to the cavity walls inside the microspheres.

Protein instability in poly(lactic-co-glycolic acid) microparticles.

In this review the current knowledge of protein degradation during preparation, storage and release from poly(lactic-co-glycolic acid) (PLGA) microparticles is described, as well as stabilization approaches. Although we have focussed on PLGA microparticles, the degradation processes and mechanisms described here are valid for many other polymeric release systems. Optimized process conditions as well as stabilizing excipients need to be used to counteract several stress factors that compromise the integrity of protein structure during preparation, storage, and release. The use of various stabilization approaches has rendered some success in increasing protein stability, but, still, full preservation of the native protein structure remains a major challenge in the formulation of protein-loaded PLGA microparticles.

Identification of oxidized methionine in peptides.

The positive- and negative-ion collision-induced dissociation spectra of peptides containing methionine, methionine sulphoxide and methionine sulphone have been studied. Characteristic fragmentations were identified and evaluated as possible indicators for the presence of oxidized methionine residues in peptides. It was found that the elimination of CH3SOH (-64 u) from [M + H]+ is unique for peptides that contain methionine sulphoxide. Sequence ions containing the oxidized methionine undergo the same elimination, allowing unambiguous sequence determination. Methionine sulphone exhibits an analogous elimination of CH3SO2H (-80 u) from the protonated molecule, but not from sequence ions.