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In the present paper we describe the production and characterization of specialized delivery systems for camptothecin, namely liposomes, micellar solutions and microemulsion. For instance, liposomes were prepared by reverse phase evaporation technique followed by extrusion through polycarbonate filters. Liposomes were characterized in term of dimensions, morphology and encapsulation efficacy. All the formulations were designed firstly to increase the solubility of camptothecin in aqueous environment and secondly to reduce the toxicity problems related to the administration of this drug. The analysis of their in vitro antiproliferative activity on cultured human leukemic K562 cells demonstrated that liposomes, micellar solutions and microemulsion containing camptothecin exert similar or slightly enhanced effect as compared to that shown by the free drug. Based on these results, the specialized delivery systems here proposed typify an interesting starting point for a future use in experimental therapy.

Solid lipid nanoparticles (SLNs) consisting of tristearin or tribehenin, and monoolein aqueous dispersions (MADs) consisting of glyceryl-monoolein have been studied as potential nanocarriers for nucleic acids. The cationic character of nanocarriers was obtained by adding cationic surfactants, such as diisobutylphenoxyethyl-dimethylbenzyl ammonium chloride (DEBDA) or PEG-15 Cocopolyamine (PCPA), to the lipid composition. The products were characterised in terms of size and morphology by Cryo-TEM and PCS. The charge properties were determined by measuring the zeta potential. Our experimental protocol enabled us to obtain homogeneous and stable cationic nanosystems within 3-6 months of production. Assessment of cytotoxicity on HepG2 cells by MTT assays indicated that MAD preparations were less toxic than SLN, and in general PCPA-containing formulations are less cytotoxic than DEBDA-containing ones. The formation of electrostatic complexes with salmon sperm or plasmid DNA, used as model nucleic acids, was evaluated by electrophoresis on agarose gel. The results confirmed that all the formulations studied are able to form the complex. Finally, we investigated the ability of SLN and MAD to deliver DNA into HepG2 cells, and to this purpose we exploited expression plasmids for green fluorescent protein or firefly luciferase. Although with reduced efficiency, the results showed that the produced nanocarriers are able to convey plasmids into cells. The data obtained encourage further study aimed at improving these new formulations and proposing them as novel in vitro transfection reagents with potential application to in vivo delivery of nucleic acids.

Curcumin (CUR) is a well-known natural compound showing antioxidant, anti-inflammatory, and antitumor abilities but characterized by poor bioavailability and chemical instability, which drastically reduce its application in the treatment of chronic diseases such as osteoarthritis. The aim of the present study is the design and evaluation of monooleine aqueous dispersion (MAD) as novel carriers for the topical administration of CUR. CUR-loaded MAD was formulated using two different emulsifier systems, namely poloxamer 407 (MAD-A) and sodium cholate-sodium caseinate (MAD-B). These vehicles were characterized, and their influence on in vitro percutaneous absorption of CUR was also evaluated. Furthermore, an oxygen radical absorbance capacity assay was used to determine their antioxidant activity, and a Western blot analysis was performed to evaluate the inhibitory effect of the formulations on inducible nitric oxide synthase and cyclooxygenase 2 expressions. From the obtained results, CUR encapsulation efficiency was higher than 98% for MAD-A and 82% for MAD-B. Shelf-life studies showed that MAD-A maintains CUR stability better than MAD-B, and both vehicles demonstrated, in vitro, control of drug diffusion through the skin. Finally, MAD-A and MAD-B were able to extend the antioxidant/anti-inflammatory effects of CUR, also confirming the protective effect toward CUR chemical stability. © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 102:2349–2361, 2013

The present study describes the production and characterization of monoolein aqueous dispersions (MAD) as drug delivery systems for curcumin (CR). MAD based on monoolein and different emulsifiers have been produced and characterized. Morphology and dimensional distribution have been investigated by Cryogenic Transmission Electron Microscopy (cryo-TEM), X-ray and Photon Correlation Spectroscopy (PCS). Monoolein in different mixtures with sodium cholate, sodium caseinate, bentonite and poloxamer resulted in heterogeneous dispersions constituted of unilamellar vesicles, cubosomes and sponge type phases, depending on the employed components, as found by cryo-TEM and X-ray studies. CR was encapsulated with entrapment efficiencies depending on the MAD composition, particularly the highest was reached in the case of monoolein/poloxamer/sodium cholate mixture. The same mixture was able to maintain CR stability also after 6 months. CR release modalities were in vitro investigated in order to mimic a possible subcutaneous administration of MAD. It was found that MAD constituted of monoolein/poloxamer and monoolein/poloxamer/sodium cholate mixtures were able to sustain CR release. MAD viscous vehicles were produced by xanthan gum. CR percutaneous absorption has been studied in vitro using excised human skin membranes [stratum corneum epidermis (SCE)] mounted into Franz cells. It was found that fluxes (Fn) of CR incorporated in MAD are influenced by the presence of monoolein based nanosystems. In particular xanthan gum based MAD better control CR diffusion from MAD.

The present study describes the production and characterization of monoolein aqueous dispersions (MAD) and lecithin organogels (ORG) as percutaneous delivery systems for curcumin (CUR). In particular, MAD stabilized by sodium cholate/poloxamer and w0 3 ORG lipid carriers, both in the presence and absence of CUR, have been considered: MAD morphology and dimensional distribution have been investigated by Cryogenic Transmission Electron Microscopy (cryo-TEM) and Photon Correlation Spectroscopy (PCS), while the inner structure of MAD and ORG has been studied by X-ray scattering techniques. As a general result, CUR chemical stability has been found to be better controlled by MAD, probably because CUR is more protected in the case of CUR-MAD with respect to CUR-ORG. To investigate the performance of differently composed lipid formulations as CUR delivery system, in vitro studies, based on Franz cell and stratum corneum–epidermis (SCE) membranes, and in vivo studies, based on skin reflectance spectrophotometry and tape stripping, were then performed. The results indicated that ORG induces a rapid and intense initial penetration of CUR probably due to a strong interaction between the peculiar supramolecular aggregation structure of phospholipids in the vehicle and the lipids present in the stratum corneum. Conversely, CUR incorporated into MAD can be released in a controlled fashion possibly because of the formation of a CUR depot in the stratum corneum. In this respect ORG could be employed in pathologies requiring rapid CUR action, while MAD could be proposed for assuring a prolonged CUR activity.

The effects of glucosinolates (GLs) (sinigrin, gluconapin, progoitrin, epi-progoitrin, sinalbin, glucotropaeolin, glucoerucin, glucocheirolin, and glucoraphenin) and their enzymatic hydrolysis-derived products (EHDPs) have been studied in controlling the proliferation of cancer cell lines. The results of this study indicate the following: (i) neither myrosinase nor intact GLs have any effect on tumor cell growth when used up to 36 U/mL and 500 μM, respectively; (ii) all EHDPs show a clear inhibition of human erythroleukemic K562 cell proliferative growth, which is particularly evident for EHDPs from sinigrin, glucotropaeolin, glucoerucin, and glucocheirolin (IC50 < 20 μM); (iii) the EHDP production by in situ or pre-mix procedures gives rather similar antiproliferative effects; and finally, (iv) the EHDPs from glucoraphenin are active toward several other tumor cells, viz. FL (murine erythroleukemic cells), Jurkat (human T-lymphoid cells), HeLa (human cervix carcinoma cells), H9 (human T-lymphoid cells),...

The purpose of this study was to investigate the potential of intranasal immunization with non-ionic surfactant vesicles (NISV) containing either the secretory recombinant form of glycoprotein B (gBs) of herpes simplex virus type 1 or a related polylysine reach peptides (DTK) for induction of protective immunity against genital herpes infection in mice. NISV were prepared by lipid film hydration method. The mean diameter of vesicles was around 390 nm for DTK-containing NISV (DTK-NISV) and 320 nm for gB1s-containing NISV (gB1s-NISV). The encapsulation efficiency of the molecules was comprised between 57% and 70%. After 7-14 day NISV maintained stable dimensions and a drug encapsulation higher than 48%. We showed that intranasal immunization with gB1s-NISV induces gB-specific IgG antibody and lymphoproliferative responses, whereas vaccination with DTK-NISV was not able to generate a gB-specific immune response. Our results indicate that vaccination of BALB/c mice with gB1s-NISV induced Th1 responses, as characterized by increased titre of IG2a in plasma and IFN-production in CD4+ splenic cells. Intranasal immunization with gB1s-NISV could elicit 90% (almost complete) protection against a heterologous lethal vaginal challenge with herpes simplex virus type 2. These data may have implications for the development of a mucosal vaccine against genital herpes.

The purpose of this study was to investigate the potential of new positively charged solid lipid nanoparticles (SLN) to convey nucleic acids. The cationic character of SLN was obtained by adding as cationic molecules two different long-chain cationic phosphines (CP), namely hexadecyl-PTA iodide (CP16) and octadecyl-PTA iodide (CP18). The obtained CP-SLN are characterized by a positive charge on the surface and reproducible dimensions around 220 nm. These nanosystems are able to efficiently bind nucleic acid molecules and to protect DNA from the activity of serum nucleases up to 120 min. Lastly, in vitro experiments demonstrated that CP-SLN exhibit a quite pronounced antiproliferative effect on cultured human K562 erythroleukemic cells and a limited effect as transfecting adjuvant. These data, and particularly the ability of CP-SLN to protect DNA from degradation, encourages further studies aimed at proposing these nanosystems as a potential approach to deliver nucleic acid to cells in living organisms.

In many cell types extracellular ATP (ATPe) has been shown to cause reversible plasma membrane permeabilization to low molecular weight (900 Da) water-soluble compounds. In the present report we have exploited this technique to incorporate the anticancer drug doxorubicin (DXR), molecular mass 543 Da, into the cytoplasm of two mouse cell lines that had previously been shown to express the ATPe-gated pore, J774 macrophages and tumor necrosis factor (TNF)-resistant L929 fibroblasts. Compared to passively loaded cells, ATPe-mediated reversible permeabilization allowed an at least 4-fold increase in DXR intracellular trapping (0.5 pg/cell versus 2 pg/cell). Analysis of the release kinetics at 37 degrees C showed that about 40% of total intracellular DXR was discharged during the first hour from both ATPe-permeabilized and passively loaded cells; about 15% further release was observed upon incubation up to 4 h. DXR release profiles were similar in ATPe-permeabilized and passively loaded cells. ATPe-permeabilized, DXR-loaded (ATPe-DXR) cells strongly inhibited the proliferation of K562 tumor cells. Taken together these results indicate that ATPe-mediated reversible plasma membrane permeabilization can be effectively used to load cells of different histotypes with high concentrations of DXR. This approach could permit to vehicle high doses of anticancer agents by using living cells while reducing systemic toxic effects.

This paper describes a comparative study on the performances of ethosomes and solid lipid nanoparticle as delivery systems for acyclovir. Ethosomes were spontaneously produced by dissolution of phosphatidylcholine and acyclovir in ethanol followed by addition of an aqueous buffer while solid lipid nanoparticle were produced by homogenization and ultrasonication. Both colloidal systems were morphologically characterized by cryo-transmission electron microscopy. The encapsulation efficiency was 94.2±2.8% for ethosomes and 53.2±0.2% for solid lipid nanoparticle. Concerning Z potential, both formulations are close to neutrality. The diffusion coefficients of the drug from ethosomes and solid lipid nanoparticle, determined by a Franz cell method, were 9.4 and 1.2-fold lower as compared to the free acyclovir in solution, thus evidencing the ability of both colloidal systems in enhancing the diffusion of the drug. The antiviral activity against HSV-1 of both systems was tested by plaque reduction assay in monolayer cultures of Vero cells. Data showed that no significant differences in the antiviral activity were observed by acyclovir in the free or loaded forms. Taken together these results, colloidal systems could be interesting to mediate the penetration of acyclovir within Vero cells.

The present article describes a comparative study of the performances of liposomes and ethosomes as specialized delivery systems for distamycin A (DA) and two of its derivatives. Liposomes and ethosomes were prepared by classical methods, extruded through polycarbonate filters, and characterized in terms of dimensions, morphology, and encapsulation efficiency. It was found that DA was associated with vesicles (either liposomes or ethosomes) by around 16.0%, while both derivatives of DA showed a percentage of association around 80% in the case of liposomes and around 50% in the case of ethosomes. In vitro antiproliferative activity experiments performed on cultured human and mouse leukemic cells demonstrated that vesicles were able to increase the activity of both derivatives of DA. In addition, it was demonstrated that the aging of both liposomes- and ethosomes-associated distamycin suspensions did not heavily influence the vesicle size, while all samples showed a relevant drug leakage with time. Moreover, according to the different physicochemical characteristics of DA and its derivatives (i.e., log P), vesicle-associated DA showed the highest loss of drug with respect to both its derivatives. In conclusion, the enhancement of drug activity expressed by these specialized delivery systems-associated DD could be interesting to obtain an efficient therapeutic effect aimed at reducing or minimizing toxic effects occurring with distamycins administration.

In the present study are reported the differential DNA binding activity of the anti-tumor polyamidine tetra-p-amidinophenoxyneopentane (TAPP-H) and its 2'-halo derivative (TAPP-Br), and their effects on the binding of the recombinant Epstein-Barr virus (EBV) nuclear antigen to a synthetic oligonucleotide mimicking the target DNA sequence present in the EBV genome. In addition, the proliferation kinetics and cell cycle analysis of human leukemia K562 cells treated with TAPP-H and TAPP-Br are reported. The possible in vivo relationship between DNA binding affinity and cytotoxicity is also discussed.

The present study concerns the percutaneous absorption of naproxen (NPX), as model anti-inflammatory drug, included in liposome formulations constituted of different lipids: stratum corneum lipids (SCL) and phosphatidylcholine/cholesterol (PC/CHOL). Liposome dispersions were produced using two different methods: reverse-phase evaporation (REV) and thin layer evaporation (TLE). Morphology and dimensions of the disperse phase were characterized by cryo-transmission electron microscopy (cryo-TEM) and photon correlation spectroscopy, respectively. X-ray diffraction was employed to determine the structural organization of the vesicles. In vitro diffusion was studied by Franz cell on liposome dispersions viscosized by carbomer. Tape stripping was performed to investigate in vivo the performance of differently composed liposomes as NPX delivery system. Cryo-TEM showed spherical vesicles and bigger irregular elongated nanoparticles for TLE SCL liposomes. REV resulted in spherical and elongated multilamellar vesicles. Also X-ray diffraction evidenced La or Lp multilamellar vesicles for PC/CHOL and SCL liposome respectively. The in vitro study showed a lower NPX flux for SCL with respect to PC/CHOL liposome. Tape stripping corroborate the in vitro findings regarding SCL, suggesting that liposomes create a drug reservoir mixing with SC lipids, whilst PC/CHOL liposome promoted NPX permeation through the skin. Liposome lipid composition seems to affect NPX permeation through the skin. © 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 99:2819-2829, 2010

This paper describes the production and characterization of polyacrylic polymer (Eudragit RL, RS and NE) microparticles by spray drying method. Microparticles were designed for ophthalmic administration of acyclovir. Microparticle morphology was characterized by optical and electron microscopy. The release kinetics of the drug from microspheres were determined by a dialysis method. The spray drying method described allows the production of microparticles with acceptable encapsulation efficiency and appropriate dimensional characteristics for ophthalmic administration. Release profile data indicate that acyclovir is released from microparticles in a controlled manner. In addition the release pattern of the drug is influenced by the type of Eudragit used for microparticle production. Moreover the plaque reduction efficiency of acyclovir containing microparticles (except for RS/NE microspheres) is comparable to that displayed by the free drug. Finally our results suggest that acyclovir containing microparticles could represent an interesting system for the release of this antiviral drug at the eye site.

The present investigation describes a formulative study for the development of innovative drug delivery systems for bromocriptine. Solid lipid nanoparticles (SLN) based on different lipidic components have been produced and characterized. Morphology and dimensional distribution have been investigated by electron microscopy and Photon Correlation Spectroscopy. The antiparkinsonian activities of free bromocriptine and bromocriptine encapsulated in nanostructured lipid carriers were evaluated in 6-hydroxydopamine hemilesioned rats, a model of Parkinson’s disease. Tristearin/tricaprin mixture resulted in nanostructured lipid carriers with stable mean diameter up to 6 months from production. Bromocriptine was encapsulated with high entrapment efficiency in all of the SLN samples, particularly in the case of tristearin/tricaprin mixture. Bromocriptine encapsulation did not change nanoparticle dimensions. In vitro release kinetics based on a dialysis method demonstrated that bromocriptine was released in a prolonged fashion for 48 h. Tristearin/tricaprin nanoparticles better controlled bromocriptine release. Both free and encapsulated bromocriptine reduced the time spent on the blocks (i.e. attenuated akinesia) in the bar test, although the action of encapsulated bromocriptine was more rapid in onset and prolonged. It can be concluded that nanostructured lipid carriers encapsulation may represent an effective strategy to prolong the half-life of bromocriptine.

Poly[(N-isopropylacrylamide-co-acrylamide-co-(hydroxyethylmethacrylate))] [poly(NIPAAm-co-AAm-co-HEMA)] copolymer was synthesized as a new thermoresponsive material possessing a lower critical solution temperature (LCST) around 37 °C in phosphate buffer, pH 7.4, at a solution concentration of 1%, w/v. The influence of polymer concentration on LCST was determined by cloud point measurements and by microcalorimetric analysis. The copolymer was transformed in hydrogel microspheres by suspension reticulation of OH groups with glutaraldehyde. The volume phase transition temperature (VPTT) of microspheres was determined by a new approach, which involves measurement of the increase in concentration of a blue dextran (BD) solution at different temperatures in the presence of dry microspheres. The minimum BD concentration that gives reliable and reproducible results was determined to be 1 mg/ml. However, the higher is the concentration of BD in solution the smaller is the error. Contrary to solution of the linear polymer which displays a sharp phase transition temperature, the dependence of water regain of the hydrogel with temperature lasts from 4 °C to 50 °C.

The aim of this work is to safely transport bioadhesive microspheres loaded with DNA to intestine and to test their bioadhesive properties. Poly(vinyl alcohol) (PVA) microspheres were prepared by dispersion reticulation with glutaraldehyde and further aminated. These microspheres were firstly loaded with plasmid DNA by electrostatic interactions and then entrapped in cellulose acetate butyrate (CAB) microcapsules for gastric protection. The entrapped PVA microspheres do not have enough force by swelling to produce the rupture of CAB shell, therefore the resistance of microcapsules was weakened by incorporating different amount of the pH/thermosensitive polymer (SP) based on poly(N-isopropylacrylamide-co-methyl methacrylate-co-methacrylic acid) (NIPAAm-co-MM-co-MA). This polymer is insoluble in gastric juice at pH 1.2 and 37 degrees C, but quickly solubilized in intestinal fluids (pH 6.8 and pH 7.4). Therefore, DNA loaded PVA microspheres were not expelled in acidic media but were almost entirely discharged in small intestine or colon. The integrity of DNA after entrapment was tested by agarose gel electrophoresis indicating that no DNA degradation occurs during encapsulation. The percentage of adhered microspheres on the mucus surface of everted intestinal tissue was 65+/-18% for aminated PVA microspheres without DNA and almost 50+/-15% for those loaded with DNA. Non-aminated PVA microspheres display the lowest adhesive properties (33+/-12%). In conclusion DNA loaded microspheres were progressively discharged in intestine. The integrity of DNA was not modified after entrapment and release, as proved by agarose gel electrophoresis. Both loaded and un-loaded aminated microspheres display good bioadhesive properties.

Poly(vinyl alcohol) (PVA) microspheres were prepared by dispersion reticulation with glutaraldehyde and further aminated. These microspheres were firstly loaded with diclofenac (DF) and then entrapped in cellulose acetate butyrate (CAB) microcapsules by an o/w solvent evaporation technique for intestinal delivery of drug. The encapsulated PVA microspheres due to their low swelling degree in intestinal fluids, do not have enough force to produce the disruption of CAB shell, therefore different amounts of succinoylated pullulan microspheres (SP-Ms) (exchange capacity up to 5.2 meq/g) were co-encapsulated. The SP-Ms do not swell in acidic pH, but swell up to 20-times in intestinal fluids causing the rupture of CAB shell and facilitating the escape of loaded PVA microspheres.

In the present study the preparation, characterization and activity of cationic liposomes containing the secretory form of herpes simplex virus type 1 (HSV-1) glycoprotein B (gB1s) or two related polylysine rich peptides, namely DTK1 and DTK2, were described. The immunotherapeutic potential of these HSV antigens containing liposomes was examined with a rabbit ocular model of HSV-1 infection. Our study indicates that the liposomes (i) are able to encapsulate quantitatively gB1s and around 30% the DTK peptides, (ii) are characterized by dimensions compatible with ocular applications and (iii) can release the peptide comparably to the free solution. In addition, neutralization studies demonstrated that an anti-DTK specific polyclonal antiserum can inhibit HSV-1 infection, indicating that such peptides could be a good immunogen/antigen in an anti-HSV vaccine formulation. Although the vaccination protocol did not induce protection against the eye disease, a significative protection against a lethal ocular challenge was detectable together with the absence of reactivation episodes from latency on the survived animals. In this respect, the use of cationic liposomes coupled to gB1s and DTK peptides, as a local ocular vaccine, could represent an interesting approach in order to obtain a possible efficacy in protecting animals against a subsequent HSV-1 ocular challenge.

The present study concerns the production and characterization of monooleine (MO) dispersions as drug delivery systems for indomethacin, taken as model anti-inflammatory drug. Dispersions were produced by emulsification and homogenization of MO and poloxamer in water. Morphology and dimensional distribution of the disperse phase have been characterized by cryo-transmission electron microscopy and photon correlation spectroscopy, respectively. X-ray diffraction has been performed to determine the structural organization of the disperse phase. Sedimentation field flow fractionation (SdFFF) has been performed to investigate drug distribution in the dispersion. An in vitro diffusion study was conducted by Franz cell associated to stratum corneum epidermis membrane on cubosome dispersions viscosized by carbomer. In vivo studies based on skin reflectance spectrophotometry and tape stripping were performed to better investigate the performance of cubosome as indomethacin delivery system. Microscopy studies showed the coexistence of vesicles and cubosomes. X-ray diffraction revealed the presence of a bicontinuous cubic phase of spatial symmetry Im3m (Q229). SdFFF demonstrated that no free drug was present in the dispersion. Indomethacin incorporated in viscosized MO dispersions exhibited a lower flux with respect to the analogous formulation containing the free drug in the aqueous phase and to the control formulation based on carbomer gel. Reflectance spectroscopy demonstrated that indomethacin incorporated into MO dispersions can be released in a prolonged fashion. Tape-stripping experiments corroborated this finding. MO dispersions can be proposed as nanoparticulate systems able to control the percutaneous absorption of indomethacin.

Despite the large number of publications and patents concerning pH/thermoresponsive polymers, few data are available concerning the preparation of thermoresponsive cross-linked microspheres from preformed polymers. Therefore, N-isopropylacrylamide-co-acrylamide-co-(2-hydroxyethyl acrylate) copolymers were obtained as a new thermoresponsive material with a lower critical solution temperature (LCST) around 36 degrees C, in phosphate buffer at pH 7.4, and with a cross-linkable OH group in their structure. The LCST value was determined both by UV spectroscopy and microcalorimetric analysis. These copolymers were solubilised in acidified aqueous solution below their LCST, dispersed in mineral oil, and transformed into stable microspheres by cross-linking with glutaraldehyde. The thermoresponsive microspheres were characterised by optical and scanning electron microscopy, degree of swelling, and water retention. The pore dimensions of the microspheres and the retention volumes of some drugs and typical compounds were evaluated at different temperatures by liquid chromatography. Indomethacin, as a model drug, was included in the microspheres by the solvent evaporation method. Finally, the influence of temperature and of temperature cycling on drug release was investigated.

The basic properties and the in vitro release rate kinetics of azelaic acid (AA), alternatively vehiculated in different phospholipid-based vesicles such as ethosomes or liposomes, were investigated. Ethosomes were produced by a simple method based on addition of an aqueous phase to an ethanol solution (comprised between 20\% and 45%, v/v) of soy phosphatidyl choline (5%, w/w) and AA (0.2%, w/w) under mechanical stirring. Liposomes were obtained by the same composition in the absence of ethanol with the reverse-phase evaporation method. Vesicle size was measured by photon correlation spectroscopy (PCS), evidencing smaller mean diameters and narrower dimensional distributions in the case of ethosomes with respect to liposomes. In order to obtain homogeneously sized vesicles, both ethosomal and liposomal dispersions were extruded through polycarbonate membranes with pores of calibrated diameter (400 nm and 200 nm). Vesicle morphology was characterized by freeze-fracture scanning electron microscopy (SEM) showing the presence of unilamellar vesicles both in liposome- and in ethosome-based dispersions. Free energy measurements of the vesicle bilayers were conducted by differential scanning calorimetry (DSC). AA diffusion from ethosomal or liposomal dispersions and from ethosomes and liposomes incorporated in a viscous gel was investigated by a Franz cell assembled with synthetic membranes. The release rate was more rapid from ethosomal systems than from liposomal systems. In particular, ethosomes produced by the highest ethanol concentration released AA more rapidly, and the same trend was found using viscous forms.

Poly(vinyl alcohol) (PVA) microspheres containing cyclodextrin (CD) were obtained by chemical cross-linking with glutaraldehyde of an acidified mixture solution of PVA and alpha-, beta- or gamma-CD. The amount of linked CD in microspheres, estimated by tetrazolium blue method, decreases in the order beta-gamma-alpha-CD. The dimensions of PVA/gamma-CD microspheres are much higher than those of PVA/alpha- and beta-CD. The cross-linking density of microspheres was estimated by the amount of iodine retained by the polymer matrix. The pore size as well as the porous volume of PVA/CD microspheres decrease significantly on increasing the amount of glutaraldehyde, but are enough large to permit the access of drugs to the CD cavity. In order to test the PVA/CD microsphere inclusion ability, the microspheres were packed in a glass column and the liquid chromatographic behaviour by isocratic elution of different drugs or typical organic compounds, taken as model drugs, was investigated.

Ursodeoxycholic acid (UDCA), a hydrophilic bile acid effective in dissolving cholesterol gallstones, is usually administered by capsules and tablets. However, oral solid dosage forms are generally inadequate for pediatric needs, since, in order to individualize the preparations, hospitals have to prepare, from capsules, doses corresponding to age and weight of the children. Aim of the present paper was to produce and characterize UDCA suspensions for a personalized regimen of therapy. Four different suspensions were prepared by dissolving preservatives, sodium chloride sweeteners and/or rheology modifiers in hot pure water. After cooling 2.5% (w/v) of UDCA powder was added. After production the formulations were subjected to UDCA content analysis by HPLC, rheological measurements and stability test. The physical and chemical stability of UDCA containing suspensions were investigated for 28 days after production. For physical stability the rate of sedimentation, the height of the sediment and the ease of redispersion, were measured. Particularly, the sedimentation volumes of the four suspensions were between 88 and 95%. The results demonstrated that all UDCA suspensions are characterized by good chemical stability (nominal concentration over 96%). The results indicated that suspensions could be proposed as alternative formulations for the pediatric administration of UDCA.

Bioadhesive and biodegradable microspheres were obtained by chemical cross-linking with epichlorohydrin of an alkaline solution of a mixture of starch and alpha-, beta-, or gamma-cyclodextrin (CyD). Microspheres were characterized by scanning electron microscopy, swelling degree, and water retention. The percentage of the effective CyD in microspheres was estimated by measuring the amount of iodine and typical organic compounds (TOCs) retained in the hydrophobic cavity of CyD. Gabexate Mesylate (trade name Foy); GM), an antiallergic drug, was included in microspheres by soaking in an aqueous solution containing the drug, followed by solvent evaporation or lyophilization. UV, IR, and DSC data indicated that despite the fact that GM is a hydrophilic drug, its hydrophobic moiety close to the benzene ring is able to penetrate the CyD cavity and to form stable inclusion complexes. Values of the association equilibrium constant for GM binding to CyD, obtained by UV differential spectroscopy, indicated that the affinity of the drug for alpha- and gamma-CyD is higher than that for beta-CyD. In vitro, GM was gradually released during 1h. Even if the release rate of the drug is relatively fast, the microspheres might actually provide the best platform since the material adheres to the nasal mucosa which was proved by adhesion tests. The GM integrity was checked by comparing its anti-trypsin activity before and after release.

Pullulan microspheres containing cyclodextrin (CyD) were obtained by chemical crosslinking with epichlorohydrin of an alkaline solution of pullulan (Pul) and alpha-, beta- or gamma-CyD. The amount of alpha-, beta- and gamma-CyD in microspheres was 120, 156, and 138 micromol/g, respectively, as determined from the percentage of iodine incorporated in the hydrophobic cavity of CyD's. Microspheres were packed in a glass column and the liquid chromatographic behaviour by isocratic elution of different drugs or typical organic compounds (TOC), taken as model drugs, was investigated. The increase of the retention volume (V(R)) of each compound, depending on the interaction(s) between CyD's cavity and the considered molecule, is characterized by a broadening of the peaks. The interaction coefficient K, corresponding to the ratio between the V(R) value of each tested molecule on Pul-alpha-, Pul-beta- and Pul-gamma-CyD active stationary phase and the V(R) value of benzoic acid on St/maltodextrin neutral stationary phase, was determined. According to K values, the accurate prediction can be done on the potential drugs to be conditioned in suitable CyD cavity. Values of K allow to anticipate the release profiles of drugs considered.

This article describes the production and characterization of monoglyceride-based supramolecular systems by a simple processing technique, avoiding time-consuming procedures, high energy input, and the use of organic solvents. A preformulatory study was performed to study the influence of the experimental parameters on the production of monoglyceride-based disperse systems. In particular the effects of (1) stirring speed, (2) type and concentration of monoglyceride mixture, and (3) type and concentration of surfactant were investigated on the recovery, fraction of larger particles, mean diameter, and shape of smaller particles (so called nanosomes). Dispersions were first characterized by optical microscopy and freeze-fracture electron microscopy. The mean diameter of standard nanosomes, analyzed by photon correlation spectroscopy (PCS) after elimination of larger particles by filtration, was 193.5 nm. Cryotransmission electron microscopy studies, conducted in order to investigate the structure of dispersions, showed the coexistence of vesicles and particles characterized by a cubic organization. X-ray diffraction data revealed the coexistence of 2 different cubic phases, the first being a bicontinuous cubic phase of spatial symmetry Im3m (Q229) and the second belonging to the Pn3m spatial symmetry. A study on the stability of monoglyceride-based dispersions based on macroscopical analysis of organoleptic properties and dimensional analysis by time was performed after elimination of larger particles by filtration. Organoleptic and morphological features do not change by time, appearing free from phase-separation phenomena for almost 1 year from production. PCS studies showed that nanosomes undergo an initial increase in mean diameter within the first month following production; afterwards they generally maintain their dimensions for the next 4 months.

Aim of the present paper was to investigate the influence of preparation parameters on the production of lipospheres (LS) for drug delivery. LS composed of triglycerides and monoglycerides were alternatively produced by melt dispersion technique, solvent evaporation or w/o/w double emulsion method. The influence of preparation parameters, such as (a) type and amount of lipids, (b) presence and concentration of surfactants, (c) stirring speed and (d) type of stirrer was studied. In the case of LS prepared by melt dispersion, the use of a lipid composition of cetyl alcohol/cholesterol (2:1, w/w), a 5% (w/w) gelatin solution (50 bloom grades) and 1000 rpm stirring speed resulted in the production of spherical particles, with high percentage of recovery (82%, w/w) a mean diameter of 80 microm and a narrow size distribution. In the case of LS prepared by solvent evaporation, the best results in terms of LS morphology, recovery and size distribution were obtained by the use of a lipid composition of tristearin/monostearate (66:34, w/w), a 1% (w/w) PVA solution, a 750 rpm stirring speed and a 55 mm three-blade turbine rotor. The solvent evaporation method resulted in the production of LS characterised by a smaller size (20 microm mean diameter) but poor mechanical properties with respect to particles with the same composition obtained by the melt dispersion technique (170 microm mean diameter). The use of a combination of lipids and a methacrylic polymer (Eudragit RS 100) overcame this problem, resulting in the production of spherical particles, with a narrower size distribution and good mechanical properties. Two lipophilic drugs, such as retinyl acetate and progesterone, and one hydrophilic drug, sodium cromoglycate (SCG), were encapsulated in LS as model compounds. Lypophilic drugs displayed satisfactory encapsulation efficiencies (over 70% w/w), while SCG was very scarcely encapsulated (about 2% w/w). To solve this drawback, the use of a w/o/w double emulsion strategy was proposed, enabling to increase the encapsulation of SCG up to 50% w/w. Finally, in vitro drug release studies were performed, showing that all drugs were released in a control manner. In particular. the retinyl acetate release efficacy within the first 8 h was 27% of the total amount of the drug, while in the same period, the amount of progesterone released was 63%. With regard to SCG containing LS, the release of the drug was largely influenced by the type of stabiliser of the primary emulsion, in any case the SCG release reached the 100% of the total amount of drug after 5 h from the beginning of the experiment.

This paper reports on (a) the production of pectin microspheres and (b) the influence of different experimental parameters and ionic crosslinking on morphological and dimensional characteristics of pectin microspheres. Morphological and dimensional characteristics of pectin were analyzed as a function of the type of pectin, the dispersing phase, the stirring speed, and the emulsifying agent. Crosslinking by calcium chloride and the encapsulation of antibiotics (i.e., metronidazol and tetracycline) gave particles morphologically similar to empty particles but with slower swelling kinetic.

Peptide nucleic acids (PNAs) have been recently proposed as useful molecules in pharmacogenetic therapy, especially due to the fact that they show a very high stability with respect to DNA and RNA. However, PNAs are not efficient decoy molecules, are characterized by negligible cell internalization and low solubility and are not suitable to be delivered by liposomes. With respect to the biological activity of PNA-based molecules, PDP deserve great consideration, due to the fact that they exhibit high levels of solubility, and are expected to be resistant to proteinases and exonucleases. In this manuscript we determined whether double-stranded molecules based on PNA–DNA chimeras containing NF-κB binding sites, exhibit decoy activity against NF-κB transcription factors. In addition, we determined whether they can be complexed by cationic liposomes. The results obtained demonstrated that hybrids based on PNA–DNA chimeras are powerful decoy molecules against NF-κB p52 transcription factor. In addition, we found that cationic liposomes can be proposed for in vitro delivery to target cells of these decoy molecules. The results presented in this paper are thus of practical importance, since the simplicity and the versatility of the cationic liposome technology have made cationic liposomes useful nonviral gene delivery systems for human gene therapy.

Despite the large number of publications describing the synthesis and physicocharacterization of the binding between drugs and DNA, relatively few examine drug delivery systems (DDSs) for these molecules. The aim to find DDSs for DNA-binding drugs (DBDs) was prompted mainly to reduce the toxicity and/or enhance the tumor specificity of systemically administered drugs. With this in mind, we have reviewed the biological effects of some DBDs that are currently used as antitumor drugs and describe a brief selection of DDSs currently in clinical trials or on the market.

A comparison of the effect of isothiocyanates and nitriles derived from some glucosinolates, namely, epi-progoitrin, sinalbin, glucotropaeolin, glucocheirolin, and glucoraphenin, on human erythroleukemic in vitro cultured cells was studied. Many studies have in fact evidenced that a consumption of vegetable containing glucosinolates could reduce the development of colorectal cancer. In the experimental conditions used, the production of isothiocyanates and nitriles from glucosinolates is almost quantitative as confirmed by HPLC or GC-MS analysis. The obtained results demonstrated that in general nitriles are considerably less potent than the corresponding isothiocyanates in inhibiting cancer cell growth. Particularly, the isothiocyanates inhibitory activity on K562 cells growth is higher in the case of products derived from epi-progoitrin, glucotropaeolin, glucoraphenin, and glucocheirolin; while for nitriles the higher activity in inhibiting K562 cells growth is showed by sinalbin-derived product. Considering the antiproliferative activity found for isothiocyanates and nitriles, further studies will be aimed to the possible application of glucosinolate-derived products as chemopreventive cancer agents for the reduction of colorectal cancer.

The production and characterization of cationic microparticles based on Eudragit RS and cationic agents (i.e. a cationic acrylic polymer and three different cationic surfactants) for the delivery of nucleic acids is here described. It was found that morphological and dimensional characteristics of microparticles were influenced by the type and concentration of cationic agent employed and by some experimental parameters such as stirring speed, emulsifying agent and type of rotor. The desoxiribonucleotide Defibrotide (DFT) was associated with positively charged microparticles and its in vitro release kinetics from microparticles were determined. A study of the in vitro toxicity of cationic microparticles on cultured human cell line K562 was also performed, demonstrating that DDAB 18 microparticles display very low cytotoxicity.

The administration of retinoids has been demonstrated to be of potential utility in the therapy of a wide spectrum of neoplastic pathologies due to the ability to induce differentiation in a large variety of primary tumor cells as well as in vitro cultured cell lines. Moreover, a number of compounds, including hemin, cytosine arabinoside, and 5-azacytidine are able to induce erythroid differentiation of the erythroleukemic cell line K562. In this paper we determined whether a combined treatment of K562 cells with suboptimal concentrations of cytosine arabinoside and retinoids containing liposomes lead to a full expression of differentiated functions. Liposomes were prepared by reverse phase evaporation technique followed by extrusion through polycarbonate filters. Cell growth kinetics studies and intracellular detection of hemoglobin by benzidine staining were performed. The results obtained showed that the combined treatment with liposomes containing retinoids and sub-optimal concentration of ara-C is an effective strategy to induce K562 cell differentiation, minimizing at the same time toxic effects. Control experiments aimed to determine possible selection of subpopulations of K562 cells suggest that the observed results are not related to toxicity and/or potential selection of induced cells. In conclusion, liposomally delivered retinoids could be proposed for differentiation therapy as an effective strategy in the treatment and management of malignancy. In addition, the finding that liposomally delivered retinoids increase the capacity of cytosine arabinoside to induce erythroid differentiation, could be of interest in studies aimed at the development of treatment able to reactivate fetal globin genes in beta-thalassemia patients.

The aim of the present paper was to find a 'biocompatible' means to cross-link gelatin-based pharmaceutical devices. In particular, we have studied the ability of native and oxidized mono- and di-saccharides to induce the cross-linking of gelatin. To this end, gelatin discs and gelatin microspheres were produced and their dissolution kinetics at 37 degrees C were examined. In order to find evidence of sugar-mediated cross-linking, DSC and FTIR experiments were performed. The obtained results indicated that both native and oxidized sugars resulted to different extents, in the formation of a cross-linked gelatin network able to reduce the dissolution of gelatin. These results suggest that oxidized mono- and disaccharides could be an interesting method by which to cross-link gelatin thereby reducing the risk of toxic side effects arising from the use of synthetic cross-linkers.

The aim of this paper was to investigate the influence of preparation parameters on gelatin microspheres production, chemico-physical characteristics and drug encapsulation. In particular, we focussed our attention on the manufacturing parameters such as amount of polymer, stirring speed, presence and concentration of surfactants. As model drugs, TAPP-Br, clonidine hydrochloride and bromocriptine mesylate were chosen in order to compare their encapsulation and release characteristics with microspheres. In the second part of this work, a study of the influence of thermal treatment on the microspheres is reported, performed with the aim to possibly modify gelatin dissolution and drug release. In particular, the effect of this treatment was evaluated on microsphere characteristics such as swelling, porosity and dissolution, and finally on the release profiles of the encapsulated drugs.

In this paper we evaluate the influence of phospholipid based formulations (PBFs) on skin absorption. In particular we describe the production and characterization of different PBFs, namely liposomes and w/o microemulsion gels, and their influence on in vitro and in vivo absorption of methyl nicotinate (MN) used as model compound. In order to compare the influence of various vehicles on skin absorption, Franz cell and MN induced erythema were used as in vitro and in vivo experimental models respectively. The formulations tested were: (a) unilamellar liposomes consisting of soybean lecithin/ cholesterol (9:1 w/w) suspended in water or incorporated into hydrophilic gels (Carbomer and carboxymethyl cellulose based gels) and (b) soybean lecithin based gels. The results indicate that vehicles containing phospholipids in liposomal form provided enhanced in vivo MN skin permeation compared to the corresponding vehicles without phospholipids. Lecithin gel showed a different behaviour characterized by a short and intense persistence of MN induced erythema.

The preparation and characterization of gelatin microspheres containing the antitumor drug 1-β-D-arabinofuranosylcytosine (ara-C) and its release kinetics from microspheres are reported. Spherical gelatin microspheres, showing high encapsulation efficiency (58%) and an average diameter of 18 μm, were obtained by a coacervation method. In vitro release experiments demonstrated that ara-C is released from gelatin microspheres in a controlled and reproducible fashion. The biological effect of ara-C released from gelatin microspheres was determined on in vitro cultured tumor cell lines, showing that after encapsulation ara-C still retains differentiating and antiproliferative activities comparable with those exhibited by the free drug. Taken together, the results obtained suggest that gelatin based microspheres offer excellent potential as carriers for the controlled release of ara-C after in vivo administration.

This study compares the behaviour of budesonide-containing microparticles made of Eudragit(®)RS or Eudragit(®)RS/Eudragit(®)RL 70:30 (w/w) prepared either by solvent evaporation or spray-drying technique. The loading efficiency of budesonide within microparticles was about 72% for microparticles prepared by solvent evaporation and around 78% for spray-dried microparticles. Thermal analyses were assessed to collect information about the structural stability of budesonide within the polymeric microspheres. The in vitro release was performed using simulating gastric (fasted state simulated gastric fluid) and intestinal (fasted state simulated intestinal fluid) fluids as the receiving solutions. After 3 h the drug release from Eudragit(®)RS/Eudragit(®)RL microparticles was about 6-fold higher than that obtained in the case of monopolymer microparticles. Using fasted state simulated intestinal fluid the drug was released between 4 and 30% in both types of preparations. Eudragit(®)RS microparticles showed a better protection of the drug from gastric acidity than those of Eudragit(®)RS/Eudragit(®)RL allowing us to propose Eudragit(®)RS microparticles as a hypothetical system of colon specific controlled delivery.

This paper describes how the use of different in vitro experimental systems can influence the determination of (a) the drug release profile from microparticles and (b) the interpretation of the release mechanism(s). We employed, as model dosage form, the Parlodel LA, a recently marketed microsphere system especially designed for bromocriptine-controlled delivery. The release kinetics of bromocriptine from microspheres were determined by using two different experimental approaches: a dialysis method and a flow-through cell method. From the comparison of the obtained data it clearly appears that different in vitro experimental models lead to distinct results in terms of drug availability. On the contrary both series of data can be convincingly fitted with the same mathematical equation, giving almost identical results in terms of postulated release mechanism. Taken together these results indicate that different experimental approaches should always be employed to determine drug release kinetics from microparticles in order to obtain more reliable information on the therapeutic dose (bioavailable drug, for in vivo experiments) and on the uniformity of different batches of microspheres.