A Safe-by-Design Approach for the Synthesis of a Novel Cross-Linked Hyaluronic Acid with Improved Biological and Physical Properties.

Hyaluronic acid (HA) is a polymer with unique biological properties that has gained in interest over the years, with applications in pharmaceutical, cosmetic, and biomedical fields; however, its widespread use has been limited by its short half-life. Therefore, a new cross-linked hyaluronic acid was designed and characterized using a natural and safe cross-linking agent, such as arginine methyl ester, which provided improved resistance to enzymatic action, as compared to the corresponding linear polymer. The antibacterial profile of the new derivative was shown to be effective against S. aureus and P. acnes, making it a promising candidate for use in cosmetic formulations and skin applications. Its effect on S. pneumoniae, combined with its excellent tolerability profile on lung cells, also makes this new product suitable for applications involving the respiratory tract.

Activation of Vegetable Oils by Reaction with Maleic Anhydride as a Renewable Source in Chemical Processes: New Experimental and Computational NMR Evidence.

Vegetable oils are bio-based and sustainable starting materials that can be used to develop chemicals for industrial processes. In this study, the functionalization of three vegetable oils (grape, hemp, and linseed) with maleic anhydride was carried out either by conventional heating or microwave activation to obtain products that, after further reactions, can enhance the water dispersion of oils for industrial applications. To identify the most abundant derivatives formed, trans-3-octene, methyl oleate, and ethyl linoleate were reacted as reference systems. A detailed NMR study, supported by computational evidence, allowed for the identification of the species formed in the reaction of trans-3-octene with maleic anhydride. The signals in the 1H NMR spectra of the alkenyl succinic anhydride (ASA) moieties bound to the organic chains were clearly identified. The reactions achieved by conventional heating were carried out for 5 h at 200 °C, resulting in similar or lower amounts of ASA units/g of oil with respect to the reactions performed by microwave activation, which, however, induced a higher viscosity of the samples.

A rhabdomyosarcoma hydrogel model to unveil cell-extracellular matrix interactions.

Three-dimensional (3D) culture systems have progressively attracted attention given their potential to overcome limitations of classical 2D in vitro systems. Among different supports for 3D cell culture, hydrogels (HGs) offer important advantages such as tunable mechanical and biological properties. Here, a biocompatible hyaluronic acid-polyethylene glycol HG was developed to explore the pro-migratory behavior of alveolar rhabdomyosarcoma (ARMS) cells. Proteomic analysis of ARMS xenografts unveiled the composition of the extracellular matrix (ECM) elucidating the most representative proteins. In parallel, HGs were obtained by the combination of a thiol-containing hyaluronic acid derivative and different polyethylene glycol (PEG) dimaleimide polymers. The selection of the optimal HG for ARMS cell growth was made based on degradation time, swelling, and cell distribution. Rheology measures and mechanical properties were assessed in the presence or absence of ECM proteins (collagen type I and fibronectin), as well as viability tests and cell distribution analysis. The role of ITGA5, the receptor of fibronectin, in determining ARMS cell migration was validated in vitro upon ITGA5 silencing. In vivo, cell dissemination and the capacity for engrafting were validated after injecting ARMS cell populations enriched for the level of ITGA5 in zebrafish embryos. To study the interactions with ARMS-specific ECM proteins (HG + P), the key players from the Rho and heat-shock pathways were investigated by reverse phase protein array (RPPA). Our data suggest that the developed 3D ARMS model is useful for identifying potential physical hallmarks that allow cancer cells to resist therapy, escape from the immune-system and increase dissemination.

Design, Synthesis, Characterization, and In Vitro Evaluation of a New Cross-Linked Hyaluronic Acid for Pharmaceutical and Cosmetic Applications.

Hyaluronic acid (HA), an excellent biomaterial with unique bio properties, is currently one of the most interesting polymers for many biomedical and cosmetic applications. However, several of its potential benefits are limited as it is rapidly degraded by hyaluronidase enzymes. To improve the half-life and consequently increase performance, native HA has been modified through cross-linking reactions with a natural and biocompatible amino acid, Ornithine, to overcome the potential toxicity commonly associated with traditional linkers. 2-chloro-dimethoxy-1,3,5-triazine/4-methylmorpholine (CDMT/NMM) was used as an activating agent. The new product (HA-Orn) was extensively characterized to confirm the chemical modification, and rheological analysis showed a gel-like profile. In vitro degradation experiments showed an improved resistance profile against enzymatic digestions. Furthermore, in vitro cytotoxicity studies were performed on lung cell lines (Calu-3 and H441), which showed no cytotoxicity.

The Relevance of Assessing Subjective Experiences of Skin Toxicity During Adjuvant Radiotherapy for Breast Cancer.

PURPOSE: Radiodermatitis is likely to be an inevitable side effect of radiotherapy (RT) but experiencing pain relief during RT might contribute making treatment more acceptable and less impairing. The current study aimed to assess the subjective perceptions and experiences of skin toxicity in a sample of women undergoing adjuvant RT for breast cancer. METHODS: Eighty patients were randomly assigned to one out of two groups: treatment (i.e., a newly developed topical product) and control (i.e., standard-of-care). Patients underwent adjuvant RT for 3 weeks. Clinical assessment of radiodermatitis and self-reported levels of pain, relief, and perceptions of treatment response were collected at the initiation of RT (T1), during RT (T2 and T3), and 2 weeks after treatment completion (T4). To assess changes in skin-related QoL, a subgroup of patients completed the Padua Skin-Related QoL questionnaire at T0 (before the initiation of RT) and at T4. RESULTS: A comparable timing of onset and severity of radiodermatitis during treatment was observed in both groups. The treatment group reported lower levels of pain and higher levels of relief compared to the control group when skin toxicity was at its highest levels (T2 and T3). Independent of the group, levels of perceived improvements in clinical status increased over time, whereas skin-related QoL worsened from T0 to T4. CONCLUSION: Current findings outline the relevance of integrating clinical evaluations of radiodermatitis with patients' subjective experiences of skin toxicity in interventional studies. Moreover, they provide preliminary evidence about the soothing effect of a newly developed topical product, thus supporting its usefulness of as a supportive care.

Rosa chinensis in vitro cell cultures: a phytocomplex rich of medium molecular weight polysaccharides with hydrating properties.

In-vitro cell cultures of selected Rosa chinensis meristematic cells cultivated with an innovative CROP® (Controlled Release of Optimized Plants) platform, allowed obtaining a stable and standardized phytocomplex rich of medium molecular weight polysaccharides. The polysaccharides profile of the rose extract has been analysed with the size exclusion chromatography (HPLC-ELSD-SEC) both in the in vitro extract and in the dried petals of Rosa chinensis. The polysaccharides content in the extract was ≥20%, higher than in the dried petals. The 65-80% of total polysaccharides have a medium molecular weight (1000 Da), known for their moisturizing and anti-age properties. Reconstructed human epidermis in homeostatic conditions was used to evaluate its moisturizing action and the ability to maintain homeostasis. The Rosa chinensis extract increased the Aquaporin-3 expression and cell membrane localization and demonstrated to regulate hydration either in topical and systemic exposure.

Red Carrot Cells Cultured in vitro Are Effective, Stable, and Safe Ingredients for Skin Care, Nutraceutical, and Food Applications.

Plant biomasses growing in bioreactor could be developed as production systems for cosmetic ingredients, nutraceuticals and food additives. We previously reported that the red carrot cell line R4G accumulates high levels of anthocyanins, which are potent antioxidants with multiple health-promoting properties. To investigate the industrial potential of this cell line in detail, we tested extract for antioxidant and anti-inflammatory activity in the mouse monocyte/macrophage cell-line J774A.1 and in reconstructed skin tissue models. We also compared the R4G extract to commercial carrot extracts in terms of stability and metabolomic profiles. We found that the R4G extract have potent antioxidant and anti-inflammatory activities, protecting mammalian cells from the oxidative stress triggered by exposure to bacterial lipopolysaccharides and H2O2. The extract also inhibited the nuclear translocation of NF-κB in an epidermal skin model, and induced the expression of VEGF-A to promote the microcirculation in a dermal microtissue model. The anthocyanins extracted from R4G cells were significantly more stable than those found in natural red carrot extracts. Finally, we showed that R4G extract has similar metabolomic profile of natural extracts by using a combination of targeted and untargeted metabolomics analysis, demonstrating the safety of R4G carrot cells for applications in the nutraceutical and food/feed industries.

Evaluating Natural Alternatives to Synthetic Acrylic Polymers: Rheological and Texture Analyses of Polymeric Water Dispersions.

The natural cosmetics market is growing, in line with the interest of public opinion on environmental safety. The availability of polysaccharides for cosmetic use is very wide; each raw material has its own sensorial specificities and hardly matches the performance of synthetic polymers. We developed an instrumental protocol based on rheology and texture analysis to evaluate alternatives to acrylic polymers. The study has been carried out on a set of water dispersions prepared with different synthetic, semisynthetic, and natural polymers at different concentrations. Using statistical principal component analysis, three different clusters have been identified: group A includes polymers with a stringy viscoelastic behavior, group B includes polymers with low firmness and a weak-gel rheological pattern, and group C includes polymers which formed soft and elastic gels. This work showed that this instrumental approach is a powerful tool to comprehensively characterize new rheological modifiers and to forecast their contribution to the formulation based on their applicative features. Moreover, rheology and texture analysis turned out to be complementary tools useful to compare polymeric raw materials and to identify appropriate alternatives to synthetic ones in order to formulate green cosmetic products.

Action of surfactants on the mammal epidermal skin barrier.

BACKGROUND: Daily skin washing routines can promote undesirable effects on skin barrier function. The stratum corneum (SC) lipid matrix is crucial for skin barrier function. Skin cleansing products are mostly composed of surfactants: surface-active molecules that interact with skin lipids in several ways. The main aim of this work was to investigate the effect produced by surfactants on skin barrier permeability. Porcine skin is a well-accepted and readily available model of the human skin barrier. The effect of two cleansing formulations (based on different surfactant mixtures) on the barrier properties of mammalian skin were evaluated. METHODS: Water sorption/desorption (DVS) experiments were used to measure skin permeability. Attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy and confocal Raman were useful to study SC lipid organization. RESULTS: The results showed that while anionic surfactants (SLS) had a negative impact on the skin barrier, with a clear increase of alkyl chain disorder; cosurfactants present in the shampoo formulation diminished the detrimental effect of their primary ionic surfactant, inducing less modification on lipid intramolecular chain disorder. CONCLUSIONS: The obtained results confirmed that the mild cleansing formulations studied had gentle interaction with skin. The capacity to discriminate between detergent systems was clearly established with both DVS and spectroscopy techniques.

Synthesis of Ferrofluids Made of Iron Oxide Nanoflowers: Interplay between Carrier Fluid and Magnetic Properties.

Ferrofluids are nanomaterials consisting of magnetic nanoparticles that are dispersed in a carrier fluid. Their physical properties, and hence their field of application are determined by intertwined compositional, structural, and magnetic characteristics, including interparticle magnetic interactions. Magnetic nanoparticles were prepared by thermal decomposition of iron(III) chloride hexahydrate (FeCl3·6H2O) in 2-pyrrolidone, and were then dispersed in two different fluids, water and polyethylene glycol 400 (PEG). A number of experimental techniques (especially, transmission electron microscopy, Mössbauer spectroscopy and superconducting quantum interference device (SQUID) magnetometry) were employed to study both the as-prepared nanoparticles and the ferrofluids. We show that, with the adopted synthesis parameters of temperature and FeCl3 relative concentration, nanoparticles are obtained that mainly consist of maghemite and present a high degree of structural disorder and strong spin canting, resulting in a low saturation magnetization (~45 emu/g). A remarkable feature is that the nanoparticles, ultimately due to the presence of 2-pyrrolidone at their surface, are arranged in nanoflower-shape structures, which are substantially stable in water and tend to disaggregate in PEG. The different arrangement of the nanoparticles in the two fluids implies a different strength of dipolar magnetic interactions, as revealed by the analysis of their magnetothermal behavior. The comparison between the magnetic heating capacities of the two ferrofluids demonstrates the possibility of tailoring the performances of the produced nanoparticles by exploiting the interplay with the carrier fluid.

Star-like oligo-arginyl-maltotriosyl derivatives as novel cell-penetrating enhancers for the intracellular delivery of colloidal therapeutic systems.

A novel nonpeptide, multiarmed oligo-arginyl derivative was engineered as a cell-penetration enhancer for the delivery of bioactive macromolecules and colloidal drug systems. Hepta-arginyl-maltotriosylamido-N-acetyl-dodecanoyl acid (Arg(7)-Malt-NAcC(12) acid) was synthesized through a carefully designed multistep chemical protocol, as follows: (1) maltotriose derivatization with 12-amino-dodecanoic acid and acetylation of the free amino group; (2) esterification of the maltotriosyl hydroxyl groups with 2-bromo-isobutyryl bromide; and (3) synthesis of star-like oligomer bearing multiple copies of arginine moieties under atom transfer radical polymerization (ATRP) conditions. The intermediates and final product were characterized by (1)H NMR, IR, mass spectrometry, colorimetric assays, and elemental analysis. Cytotoxicity studies on the final polymeric material showed that this novel cell-penetrating enhancer does not have significant toxic effects on MCF-7 and MC3T3-E1 cell lines. The IC(50) was greater than 100 µM with both cell lines, while the polyethylenimine with similar average molecular mass (M(n)) that was used as a reference showed an IC(50) of 30 and 40 µM, for MCF-7 and MC3T3-E1, respectively. The biological properties of the novel bioconjugate were investigated using a fluorescein-labeled bovine serum albumin (FITC-BSA) as a hydrophilic cargo model. MCF-7 and MC3T3-E1 cells were incubated for 60 min with the Arg(7)-Malt-NAcC(12)-conjugated FITC-BSA [(Arg(7)-Malt-NAcC(12))(2)-FITC-BSA] or FITC-BSA, and the intracellular fluorescence level was analyzed by spectrofluorimetric analysis of cell lysate, cytofluorimetry, and confocal microscopy. The fluorescence of the lysate of MCF-7 and MC3T3-E1 cells that were incubated with (Arg(7)-Malt-NAcC(12))(2)-FITC-BSA at 37 °C was approximately 4.5 times higher than the fluorescence obtained with cells incubated with FITC-BSA. At 4 °C, the cell uptake of (Arg(7)-Malt-NAcC(12))(2)-FITC-BSA was only 2 times higher than that of FITC-BSA. Cytofluorimetric studies showed that, after (Arg(7)-Malt-NAcC(12))(2)-FITC-BSA treatment, over 80% of MCF-7 cells and over 95% of MC3T3-E1 cells displayed enhanced fluorescence. Confocal investigations showed punctuated fluorescence within the cytosol in both cell lines, indicating that (Arg(7)-Malt-NAcC(12))(2)-FITC-BSA was confined to endosomes, with no fluorescence observed in the nucleus.

New association of surfactants for the production of food and cosmetic nanoemulsions: preliminary development and characterization.

A new nanoemulsifing system has been developed. This study refers to an innovative association of polysorbate 80 and palmitic ester of l-ascorbic acid for the production of good stability and very thin nanostructured emulsions with average micellar diameter size ranging from 100 to 300 nm. This system has showed to be very performing to create nanoemulsions with moderate stirring rate and warming regimen thanks to the high efficiency of the association between ascorbyl palmitate and polysorbate-80 (PS-80). This nanoemulsified system is very easy to achieve and shows a very good capability to encapsulate several substrates of both nutritional and cosmetic usage such as melatonin, resveratrol, essential oils and steroidic terpenes such as boswellic acids and others. This system has been optimally applied to nutraceutical and cosmetic formulations and particularly to develop sprayable sublingual delivery systems for melatonin and the aforesaid molecules. This study describes developing and analytical characterization of this system containing melatonin.

New cyclodextrin bioconjugates for active tumour targeting.

A new cyclodextrin-based carrier for active targeting of low soluble and degradable drugs has been synthesized and characterized. Beta-cyclodextrins were first reacted with excess hexamethylene diisocyanate and the resulting CD-(C6-NCO)5 derivative was reacted with 700 Da diamino-PEG to yield CD-(C6-PEG-NH2)5. About one out of five free amino groups of PEG were functionalised with folic acid (FA) as a tumour targeting moiety. The chemical structures of the intermediates as well as the final product, CD-(C6-PEG)5-FA, were characterized by 1H and 13C NMR, reverse phase and gel permeation chromatography, and UV-Vis spectroscopy. After modification, the haemolytic activity of beta-cyclodextrins decreased by about 70%. In the presence of the new carrier, the beta-estradiol solubility increased by more than 300 fold and the chlorambucil degradation rate decreased by 50-60%. CD-(C6-PEG)5-FA formed an inclusion complex with curcumin displaying an association constant of 954,732 M(-1). The new carrier increased the curcumin solubility by about 3200 fold as compared to native beta-cyclodextrins and reduced its degradation rate at pH 6.5 and 7.2 by 10 and 45 fold, respectively. FA receptor-overexpressing human nasopharyngeal tumour KB cell lines and non-folic acid receptor-expressing human breast cancer MCF7 cells were used to evaluate the targeting properties of the new drug delivery system. The in vitro studies demonstrate that the new carrier possesses potential selectivity for the folate receptor-overexpressing tumour cells as ED50 values of 52 microM, 58 microM and 21 microM were obtained with curcumin-loaded CD-(C6-PEG-NH2)5, curcumin in foetal serum medium and CD-(C6-PEG)5-FA, respectively.

Cyclodextrin/PEG based hydrogels for multi-drug delivery.

Cyclodextrin-PEG hydrogels were prepared by reaction of hexamethlyene isocyanate-activated beta-cyclodextrins with 1.9kDa NH(2)PEGNH(2). The reaction was carried out in anhydrous dimethylsulfoxide by using 0.25:1, 0.33:1, 0.5:1, 0.67:1, 1:1, and 2:1 CD/PEG molar ratios. The addition of acetic acid to the reaction mixture was found to slow the cross-linking reaction, yielding homogeneous matrices. The mechanical characterization indicated that the elasticity of the matrices increased as the CD content in the hydrogel increased while the elongation was irrespective of the hydrogel composition. By incubation in water and ethanol, the hydrogels underwent complete swelling in 5-10min. The water up-take increased logarithmically as the CD/PEG ratio decreased to reach a swelling degree of 800% (swollen hydrogel/dry hydrogel, w/w%). The ethanol uptake increased with a power correlation as the CD/PEG ratio decreased to reach a swelling degree of about 1000% with 0.25:1 CD/PEG hydrogel. Lysozyme, beta-estradiol, and quinine were loaded by swell embedding. The lysozyme loading increased as the CD/PEG ratio decreased while the incorporation of beta-estradiol and quinine displayed inverse correlation with respect to the CD/PEG ratio. The maximal incorporation (loaded drug/dry hydrogel, w/w%) for lysozyme, beta-estradiol and quinine was 2, 0.6, and 2.4%, respectively. Lysozyme was quickly released from the matrices, and the release was faster as the CD/PEG ratio decreased. Also, beta-estradiol and quinine release rates were inversely proportional to the CD/PEG ratio, but in these cases, the release profiles were strongly affected by the drug interaction with the hexamethylated beta-cyclodextrins in the matrices.

Nanotechnologies in protein delivery.

The growth rate for biotech drugs, namely proteins, peptides, and oligonucleotides, is dictated by the parallel progresses in biotechnology and nanotechnology. Actually, biotechnology techniques have expanded enormously the arsenal of therapeutically useful peptides and proteins making these products of primary interest for future pharmaceutical market. Nevertheless, the exploitation of protein and peptide drugs is strictly related to the development of innovative delivery systems which should provide for controlled, prolonged, or targeted delivery, improved stability during storage and delivery, reduced adverse effects, increased bioavailability, improved patient compliance and allow for administration through the desired route and cope with cost-containment therapeutic protocols. Colloidal formulations ideally possess the physicochemical and biopharmaceutical requisites for protein delivery. Pharmaceutical nanotechnology is a tool of techniques applied to design, develop and produce these systems. It involves the investigation of innovative materials and production procedures for preparation of a variety of nanosized dosage forms, which range from solid nanoparticles to soluble bioconjugates. The research and development of innovative tailor made protein delivery systems, which must be designed according to the drug candidate pharmacological and physicochemical properties, is one of the primary aim of modern pharmaceutical technology. Therefore, as an unmet need exists for technologies that combine innovative drug delivery solutions, a close un-prejudicial interaction between academic and industrial researchers as well as business thought leaders is required.

Preparation and characterization of active site protected poly(ethylene glycol)-avidin bioconjugates.

Avidin was modified with poly(ethylene glycol) in the presence of a biotin binding site protective agent synthesised by imminobiotin conjugation to branched 20 kDa PEG. Avidin was incubated with imminobiotin-PEG and reacted with high amounts of 5, 10 or 20 kDa PEG to modify the protein amino groups. Circular dichroism demonstrated that the extensive PEGylation does not alter the protein conformational structure. The affinity of avidin-PEG conjugates for biotin and biotinylated antibodies depended on the PEG size or the use of a protective agent. Avidin-PEG 10 and 20 kDa prepared in the presence of imminobiotin-PEG maintained 100% of the native affinity for biotin. The 5 kDa PEG derivative and the ones obtained without biotin site protection maintained 79-85% of the native affinity. The affinity for biotinylated antibodies decreased to 35% when the conjugation was performed without imminobiotin-PEG, while the conjugates obtained with high-molecular-weight PEGs in the presence of protective agent displayed high residual affinity. All conjugates possessed negligible antigenicity and immunogenicity. PEGylation greatly prolonged the avidin permanence in the circulation, reduced its disposition in the liver and kidneys and promoted accumulation into solid tumors. PEGylation was found to prevent the protein cell uptake, either by phagocytosis or pinocytosis.

Glycosylated macromolecular conjugates of antiviral drugs with a polyaspartamide.

Two new polymeric conjugates for specific liver targeting were prepared by conjugation of sugar moieties and antiviral drugs to alpha, beta-poly[N-2-(hydroxyethyl)-DL-aspartamide] (PHEA). PHEA-galactopyranosylphenylthiocarbamide-mono-O-succinylganciclovir (conjugate 7) and PHEA-mannopyranosylphenylthiocarbamide-O-succinylacyclovir (conjugate 8) were synthesized according to a multi-step procedure which allowed for obtaining high product yield and process standardization. Conjugate 7 contained 7.5 and 8.5% of galactose and ganciclovir (substituent/repeating unit, mol/mol), respectively, and conjugate 8 contained 14.2 and 10.8% of mannose and acyclovir, respectively. In vitro studies demonstrated that both acyclovir and ganciclovir are released from the polymeric adducts at a release rate, which depended on the incubation medium. Though a detailed study evidenced that the two bioconjugates undergo different hydrolysis pathways, in both cases high drug release rate was found in plasma, while the glycosidic moiety was not released. Pharmacokinetic studies carried out by intravenous administration of the bioconjugates to Balb/c mice demonstrated that the conjugation of glycosidic moieties promotes the disappearance of the polymer from the bloodstream. The two derivatives displayed a different pharmacokinetic profile. In particular, the mannosyl conjugation promoted the rapid disposition of the macromolecule in the kidneys and in the liver, while prevented the accumulation in the spleen. On the contrary, the galactosyl derivative was found to dispose in the liver at the same extent of the naked polymer. Few considerations on the different behavior of the conjugates were reported.

Allergic contact sensitivity in elderly patients.

BACKGROUND AND AIMS: Aging has been shown to be correlated with the rate and type of contact sensitization, but only a few studies have evaluated patch test reactivity in elderly subjects with an adequately large population. METHODS: The response patterns to patch testing in 1444 elderly subjects (>65 years) with suspected allergic contact dermatitis were studied, and the results compared with a control group of individuals with suspected allergic contact dermatitis, aged between 20 and 40 years. RESULTS: The prevalence of the positive patch test to at least one hapten was significantly lower in the group of elderly patients compared with adult patients (40.7 vs 47.8%, p<0.0001). However, some allergens, i.e., primin, diaminodiphenylmethane, neomycin, lanolin alcohols, paraben mix, Euxyl K400 and quinoline mix, showed an increased sensitization rate in elderly patients compared with adult patients. These allergens are now less frequently employed in the workplace, or are substances particularly used in the formulation of topical treatment of age-related diseases, i.e., leg ulcer and xerosis. It was also found that the intensity of positive patch test reactions was significantly lower in elderly patients compared with younger subjects, with higher proportions of weak (+) positive reactions. Moreover, elderly patients showed a dynamic pattern of increasing intensity of patch test reactions at the second reading after 3 days compared with the first reading after 2 days more frequently than younger patients (60 vs 53%, p<0.0001). CONCLUSIONS: These findings suggest an age-dependent decline of overall positive patch test reactions, but a higher sensitization rate to some allergens frequently used in the composition of topical treatments. The development of an allergic response in elderly patients was found to be delayed, and this may require an additional reading after 7 days and the interpretation of even weak reactions as valid positive patch test reactions.

Specific antitumor targetable beta-cyclodextrin-poly(ethylene glycol)-folic acid drug delivery bioconjugate.

The tumor targeting properties of a new drug carrier synthesized by bioconjugation of folic acid (FA) to beta-cyclodextrins through a poly(ethylene glycol) (PEG) spacer (CD-PEG-FA) were investigated. Surface plasmon resonance demonstrated that CD-PEG-FA specifically interacts with immobilized folate binding protein (FBP) while the naked beta-cyclodextrins do not display any specific interaction. In vitro studies demonstrated that CD-PEG-FA was devoid of cell toxicity. [(3)H]-folic acid/CD-PEG-FA competition binding investigations performed with folate receptor overexpressing human epidermal carcinoma KB cells showed that CD-PEG-FA had about 14 times lower tumor cell binding capacity than free folic acid. The carrier cell trafficking properties were investigated using rhodamine-B as fluorescent probe, which possesses 3000 and 4580 M(-)(1) inclusion constants for CD-PEG-FA and beta-cyclodextrins, respectively. Cell-associated fluorescence measurements showed that CD-PEG-FA does not promote the rhodamine-B uptake into non-folate receptor expressing human lung carcinoma MCF7 cells while 19% higher accumulation in KB cells was found with respect to rhodamine-B loaded beta-cyclodextrins. Confocal laser scanning microscopy indicated the presence of cytosolic red fluorescent spots after 2 h of incubation of KB cells with rhodamine-B included CD-PEG-FA. The fluorescent dye resided primarily in small spots, namely, endosomes and multivesicular bodies. At 1 h after pulsed incubation, wider red fluorescent cellular structures appeared as a fusion of previous structures.