Injectable Supramolecular Hydrogel from Insulin-Loaded Triblock PCL-PEG-PCL Copolymer and γ-Cyclodextrin with Sustained-Release Property

Supramolecular hydrogels formed by cyclodextrins and polymers have been widely investigated as a biocompatible, biodegradable and controllable drug delivery system. In this study, a supramolecular hydrogel based on biodegradable poly(caprolactone)–poly(ethylene glycol)–poly(caprolactone) (PCL-PEG-PCL) triblock copolymers and γ-cyclodextrin (γ-CD) was prepared through inclusion complexation as an injectable, sustained-release vehicle for insulin. The triblock copolymer PCL-PEG-PCL was synthesised by the ring-opening polymerisation method, using microwave irradiation. The polymerisation reaction and the copolymer structures were evaluated by nuclear magnetic resonance (NMR) and gel permeation chromatography (GPC). The supramolecular hydrogel was prepared in aqueous solution by blending an aqueous γ-CD solution with an aqueous solution of PCL-PEG-PCL triblock copolymer at room temperature. In vitro insulin release through the hydrogel system was studied. The relative surface hydrophobicity of standard and released insulin from the SMGel was estimated using 8-anilino-1-naphthalene sulfonic acid (ANS). Results of ¹HNMR and gel permeation chromatography revealed that microwave irradiation is a simple and reliable method for synthesis of PCL-PEG-PCL copolymer. Gelation occurred within a minute. The supramolecular hydrogel obtained by mixing 10.54% (w/v) γ-CD and 2.5% (w/v) copolymer had an excellent syringeability. Insulin was released up to 80% over a period of 20 days. Insulin kept its initial folding after formulating and releasing from SMGel. A supramolecular hydrogel based on complexation of triblock PCL-PEG-PCL copolymer with γ-cyclodextrin is a suitable system for providing sustained release of therapeutic proteins, with desirable flow behaviour.Key words: insulin, PCL-PEG-PCL, supramolecular hydrogel, triblock copolymer, γ-CD     

Inflammatory ocular adverse events with the use of oral bisphosphonates: a retrospective cohort study

Background:

There have been several published reports of inflammatory ocular adverse events, mainly uveitis and scleritis, among patients taking oral bisphosphonates. We examined the risk of these adverse events in a pharmacoepidemiologic cohort study.

Methods:

We conducted a retrospective cohort study involving residents of British Columbia who had visited an ophthalmologist from 2000 to 2007. Within the cohort, we identified all people who were first-time users of oral bisphosphonates and who were followed to the first inflammatory ocular adverse event, death, termination of insurance or the end of the study period. We defined an inflammatory ocular adverse event as scleritis or uveitis. We used a Cox proportional hazard model to determine the adjusted rate ratios. As a sensitivity analysis, we performed a propensity-score–adjusted analysis.

Results:

The cohort comprised 934 147 people, including 10 827 first-time users of bisphosphonates and 923 320 nonusers. The incidence rate among first-time users was 29/10 000 person-years for uveitis and 63/10 000 person-years for scleritis. In contrast, the incidence among people who did not use oral bisphosphonates was 20/10 000 person-years for uveitis and 36/10 000 for scleritis (number needed to harm: 1100 and 370, respectively). First-time users had an elevated risk of uveitis (adjusted relative risk [RR] 1.45, 95% confidence interval [CI] 1.25–1.68) and scleritis (adjusted RR 1.51, 95% CI 1.34–1.68). The rate ratio for the propensity-score–adjusted analysis did not change the results (uveitis: RR 1.50, 95% CI 1.29–1.73; scleritis: RR 1.53, 95% CI 1.39–1.70).

Interpretation:

People using oral bisphosphonates for the first time may be at a higher risk of scleritis and uveitis compared to people with no bisphosphonate use. Patients taking bisphosphonates must be familiar with the signs and symptoms of these conditions, so that they can immediately seek assessment by an ophthalmologist.Oral bisphosphonates are the most frequently prescribed class of medications for the prevention of osteoporosis. Most literature about the safety of bisphosphonates has mainly focused on long-term adverse events, including atypical fractures,¹ atrial fibrillation,² and esophageal and colon cancer.³Uveitis and scleritis are ocular inflammatory diseases that are associated with major morbidity. Anterior uveitis is the most common type of uveitis with an estimated 11.4–100.0 cases/100 000 person-years.^4,5 Both diseases require immediate treatment to prevent further complications, which may include cataracts, glaucoma, macular edema and scleral perforation. Numerous case reports and case series have described an association between the use of oral bisphosphonates and anterior uveitis^6–8 and scleritis.^8,9 In most reported cases, severe eye pain was reported within days of taking an oral bisphosphonates, and the symptom resolved after stopping the agent.^6,9 Only one large epidemiologic study has examined the association between the use of bisphosphonates and ocular inflammatory diseases.¹⁰ This study did not find an association, but it was limited by a small number of events and a lack of power. Thus, the association between uveitis or scleritis and the use of oral bisphosphonates is not fully known. Given that early intervention may prevent complications, we performed a pharmacoepidemiologic study to assess the true risk of these potentially serious conditions.     

Synthesis, properties and crystal structures of new binuclear lead(II) complexes based on phenyl, naphthyl-containing fluorine β-diketones and substituted 2,2′-bipyridines

Four lead(II) complexes with substituted 2,2′-bipyridine adducts and β-diketonates ligands, [Pb(5,5′-dm-2,2′-bpy)(tfpb)₂]₂1, [Pb(4,4′-dmo-2,2′-bpy)(tfpb)₂]₂2, [Pb(4,4′-dm-2,2′-bpy)(tfnb)₂]₂3 and [Pb(5,5′-dm-2,2′-bpy)(tfnb)₂]₂4, (“4,4′-dm-2,2′-bpy”, “5,5′-dm-2,2′-bpy”, “4,4′-dmo-2,2′-bpy”, “Htfpb” and “Htfnb” are the abbreviations of 4,4′-dimethyl-2,2′-bipyridine, 5,5′-dimethyl-2,2′-bipyridine, 4,4′-dimethoxy-2,2′-bipyridine, 4,4,4-trifluoro-1-phenyl-1,3-butanedione and 4,4,4-trifluoro-1-(2-naphthyl)-1,3-butanedione, respectively) have been synthesized and characterized by elemental analysis, IR, ¹H NMR and ¹³C NMR spectroscopy and also studied by thermal and electrochemical as well as X-ray crystallography. The supramolecular features in these complexes are guided/controlled by weak directional intramolecular interactions.     

Proteomic signature of human embryonic stem cells

Human embryonic stem cells (hESC) represent a population of undifferentiated pluripotent cells with both self-renewal and multilineage differentiation characteristics. Proteomics provides a powerful approach for studying the characteristics of hESC and discovering molecular markers. We have analyzed proteome profiles of three hESC lines using 2-DE and MALDI TOF-TOF. Out of 844 spots analyzed with MALDI TOF-TOF, 685 proteins were identified of which 60 proteins were classified as the most abundant proteins on 2-D gels. A large number of proteins particularly high abundant ones were identified as chaperones, heat shock proteins, ubiquitin/proteasome, and oxidative stress responsive proteins underscoring the ability of these cells to resist oxidative stress and increase the life span. Several proteins involved in cell proliferation and differentiation were also among the highly expressed proteins. Although overall expression pattern of three hESC were similar, 54 spots changed quantitatively and 14 spots changed qualitatively among the hESC cell lines. Most of these proteins were identified as proteins involved in cell growth, metabolism and signal transduction, which may affect the self-renewal and pluripotency. To our knowledge, this study represents the first proteomic dataset for hESC and provides a better insight into the biology of hESC. Proteome maps of hESC are accessible at http://www.RoyanProteomics.ir.     

Fcgamma receptor-like activity of hepatitis C virus core protein

We have previously demonstrated that viral particles with the properties of nonenveloped hepatitis C virus (HCV) nucleocapsids occur in the serum of HCV-infected individuals (1). We show here that nucleocapsids purified directly from serum or isolated from HCV virions have FcgammaR-like activity and bind "nonimmune" IgG via its Fcgamma domain. HCV core proteins produced in Escherichia coli and in the baculovirus expression system also bound "nonimmune" IgG and their Fcgamma fragments. Folded conformation was required for IgG binding because the FcgammaR-like site of the core protein was inactive in denaturing conditions. Studies with synthetic core peptides showed that the region spanning amino acids 3-75 was essential for formation of the IgG-binding site. The interaction between the HCV core and human IgG is more efficient in acidic (pH 6.0) than in neutral conditions. The core protein-binding site on the IgG molecule differs from those for C1q, FcgammaRII (CD32), and FcgammaRIII (CD16) but overlaps with that for soluble protein A from Staphylococcus aureus (SpA), which is located in the CH2-CH3 interface of IgG. These characteristics of the core-IgG interaction are very similar to those of the neonatal FcRn. Surface plasmon resonance studies suggested that the binding of an anti-core antibody to HCV core protein might be "bipolar" through its paratope to the corresponding epitope and by its Fcgamma region to the FcgammaR-like motif on this protein. These features of HCV nucleocapsids and HCV core protein may confer an advantage for HCV in terms of survival by interfering with host defense mechanisms mediated by the Fcgamma part of IgG.     

Fragile-X-associated tremor/ataxia syndrome (FXTAS) in females with the FMR1 premutation

We describe five female carriers of the FMR1 premutation who presented with symptoms of tremor and ataxia and who received a diagnosis of definite or probable fragile-X-associated tremor/ataxia syndrome (FXTAS). Unlike their male counterparts with FXTAS, none of the women had dementia. Females had not been reported in previous studies of FXTAS, suggesting that they may be relatively protected from this disorder. Brain tissue was available from one of the five subjects, a women who died at age 85 years; microscopic examination revealed intranuclear neuronal and astrocytic inclusions, in accord with the findings previously reported in males with FXTAS. The work-up of families with the FMR1 mutation should include questions regarding neurological symptoms in both older male and female carriers, with the expectation that females may also manifest the symptoms of FXTAS, although more subtly and less often than their male counterparts.     

Affinity recovery of Moloney Murine Leukaemia Virus

Lipid enveloped retroviruses such as Moloney Murine Leukaemia Virus (MoMuLV) are commonly used gene therapy vectors. Downstream processing protocols used for their purification are time consuming and a potentially generic, single step capture method for the recovery of retroviral particles is proposed that exploits streptavidin-biotin affinity chromatography. The ability of four conventional adsorbent solid phases, Fractogel, Sepharose, Magnespheres and STREAMLINE immobilised with streptavidin, to capture and recover biotinylated Moloney Murine Leukaemia Virus was studied. MoMuLV can be biotinylated whilst retaining infectivity and the biotinylated virus can be adsorbed to Streptavidin Magnespheres yielding a 2298-fold increase in titre. For optimal virus biotinylation purification using Fractogel streptavidin can yield a 1896-fold increase in cfu/mg of protein and a 1191-fold decrease in DNA/cfu. Infectious virus can be recovered from Fractogel streptavidin with a maximum recovery of 16.7%.     

Caloric restriction reduces cell loss and maintains estrogen receptor-alpha immunoreactivity in the pre-optic hypothalamus of female B6D2F1 mice

Life-long calorie restriction (CR) remains the most robust and reliable means of extending life span in mammals. Among the several theories to explain CR actions, one variant of the neuroendocrine theories of aging postulates that changing hypothalamic sensitivity to endocrine feedback is the clock that times phenotypic change over the life span. If the feedback sensitivity hypothesis is correct, CR animals should display a significantly different pattern of hormone-sensitive cell density and distribution in the hypothalamus. Of the many endocrine signal receptors that may be involved in maintaining hypothalamic feedback sensitivity, our study has selected to begin mapping those for estrogen (E). Altered hypothalamic sensitivity to E is known to schedule reproductive maturation and influence reproductive senescence. Taking estrogen receptor-alpha (ERalpha) immunoreactivity as an index of sensitivity to E, we counted ERalpha immunoreactive and non-immunoreactive cells in the pre-optic hypothalamus of young (6 weeks), ad-libitum (Old-AL) fed old (22 months), and calorie restricted (Old-CR) old (22 months) female B6D2F1 mice. An automated imaging microscopy system (AIMS) was used to generate cell counts for each sampled section of pre-optic hypothalamus. Results show a 38% reduction in ERalpha immunoreactive cells and an 18% reduction in total cell numbers in AL-old mice in comparison to young mice. However, CR mice only show a 19% reduction in ERalpha immunoreactive cells and a 13% reduction in total cell numbers in comparison to young mice. This indicates CR prevents age-related cell loss and maintains estrogen sensitivity in the pre-optic hypothalamus of old female B6D2F1 mice.     

Conjugation of lentivirus to paramagnetic particles via nonviral proteins allows efficient concentration and infection of primary acute myeloid leukemia cells

Nonviral producer cell proteins incorporated into retroviral vector surfaces profoundly influence infectivity and in vivo half-life. We report the purification and concentration of lentiviral vectors using these surface proteins as an efficient gene transduction strategy. Biotinylation of these proteins and streptavidin paramagnetic particle concentration enhances titer 400- to 2,500-fold (to 10(9) CFU/ml for vesicular stomatitis virus G protein and 5 x 10(8) for amphotropic murine leukemia virus envelope). This method also uses newly introduced membrane proteins (B7.1 and DeltaLNGFR) directed to lentiviral surfaces, allowing up to 17,000-fold concentrations. Particle conjugation of lentivirus allows facile manipulation in vitro, resulting in the transduction of 48 to 94% of human acute myeloid leukemia blasts.