Step-by-step build-up of covalent poly(ethylene oxide) nanogel films.

Hydrogels based on poly(ethylene glycol) (PEG) are commonly used for studies related to cell fate and tissue engineering. Here we present a new covalent layer-by-layer build-up process leading to PEG coatings of nanometer size called "nanogel films". Compared to macroscopic hydrogels, such nanogels should provide a fine control over the structure and the thickness of the coating. Alternated deposition of bifunctional and tetra functional PEG molecules reacting through thiol/maleimide click chemistry is evaluated by quartz crystal microbalance. We first study parameters influencing the build-up process of such coatings and demonstrate the importance of (i) the nature of the first deposited layer, (ii) the PEG concentrations and (iii) the length of the PEG chains that appears to be the most significant parameter influencing film growth. The build-up process can be extended to a large variety of substrates like SiO2 or polymers by using an appropriate anchoring layer. Covalent functionalization of these nanogel films by proteins or enzymes is suited by modifying the biomolecules with thiol or maleimide groups and immobilizing them during the build-up process. Activity of the embedded enzymes can be maintained. Moreover ligands like biotin can be incorporated into the film and recognition by streptavidin can be modulated by playing with the number of PEG layers covering biotin. Compared to well-known PEG hydrogels, these new coatings are promising as they allow to (i) build thin nanometric coatings, (ii) finely control the amount of deposited PEG and (iii) organize the position of the embedded biomolecules inside the film layers.

Layersome: development and optimization of stable liposomes as drug delivery system.

This paper describes the development of stable drug delivery systems named layersomes. The layersomes are conventional liposomes coated with one or multiple layers of biocompatible polyelectrolytes in order to stabilise their structure. The formulation strategy is based on an alternative coating procedure of positive poly(lysine) (pLL) and negative poly(glutamic acid) (pGA) polypeptides on initially charged small unilamellar liposomes (SUVs). The size distribution and the zeta potential of the final entity depend on the number of polyelectrolyte layers and the charge of the last coating layer. Morphological studies were achieved by flux cytometry and cryo electron microscopy. Release studies of encapsulated hydrophilic 5(6)-carboxyfluorescein (5,6CF) in the presence of Triton or ethanol showed an increased membrane resistance of the layersomes compared to classical SUVs. Finally, encapsulation of piroxicam (PX) was performed with success.

Induction of effective and antigen-specific antitumour immunity by a liposomal ErbB2/HER2 peptide-based vaccination construct.

Efficient delivery of tumour-associated antigens to appropriate cellular compartments of antigen-presenting cells is of prime importance for the induction of potent, cell-mediated antitumour immune responses. We have designed novel multivalent liposomal constructs that co-deliver the p63-71 cytotoxic T Lymphocyte epitope derived from human ErbB2 (HER2), and HA307-319, a T-helper (Th) epitope derived from influenza haemagglutinin. Both peptides were conjugated to the surface of liposomes via a Pam3CSS anchor, a synthetic lipopeptide with potent adjuvant activity. In a murine model system, vaccination with these constructs completely protected BALB/c mice from subsequent s.c. challenge with ErbB2-expressing, but not ErbB2-negative, murine renal carcinoma (Renca) cells, indicating the induction of potent, antigen-specific immune responses. I.v. re-challenge of tumour-free animals 2 months after the first tumour cell inoculation did not result in the formation of lung tumour nodules, suggesting that long-lasting, systemic immunity had been induced. While still protecting the majority of vaccinated mice, a liposomal construct lacking the Th epitope was less effective than the diepitope construct, also correlating with a lower number of CD8+ IFN-gamma+ T-cells identified upon ex vivo peptide restimulation of splenocytes from vaccinated animals. Importantly, in a therapeutic setting treatment with the liposomal vaccines resulted in cures in the majority of tumour-bearing mice and delayed tumour growth in the remaining ones. Our results demonstrate that liposomal constructs which combine Tc and Th peptide antigens and lipopeptide adjuvants can induce efficient, antigen-specific antitumour immunity, and represent promising synthetic delivery systems for the design of specific antitumour vaccines.

Polyelectrolyte multilayer films with pegylated polypeptides as a new type of anti-microbial protection for biomaterials.

Adhesion of bacteria at the surface of implanted materials is the first step in microbial infection, leading to post-surgical complications. In order to reduce this adhesion, we show that poly(L-lysine)/poly(L-glutamic acid) (PLL/PGA) multilayers ending by several PLL/PGA-g-PEG bilayers can be used, PGA-g-PEG corresponding to PGA grafted by poly(ethylene glycol). Streaming potential and quartz crystal microbalance-dissipation measurements were used to characterize the buildup of these films. The multilayer films terminated by PGA and PGA-g-PEG were found to adsorb an extremely small amount of serum proteins as compared to a bare silica surface but the PGA ending films do not reduce bacterial adhesion. On the other hand, the adhesion of Escherichia coli bacteria is reduced by 72% on films ending by one (PLL/PGA-g-PEG) bilayer and by 92% for films ending by three (PLL/PGA-g-PEG) bilayers compared to bare substrate. Thus, our results show the ability of PGA-g-PEG to be inserted into multilayer films and to drastically reduce both protein adsorption and bacterial adhesion. This kind of anti-adhesive films represents a new and very simple method to coat any type of biomaterials for protection against bacterial adhesion and therefore limiting its pathological consequences.

Enhancement of gene delivery by an analogue of alpha-MSH in a receptor-independent fashion.

In order to transfect melanoma specifically by receptor-mediated endocytosis we prepared dioctadecyl aminoglycylspermine (lipospermine)--DNA complexes with [Nle(4),D-Phe(7)]-alpha-MSH(4--10), a pseudo-peptide analogue of alpha-melanocyte stimulating hormone (alpha-MSH) linked to a thiol-reactive phospholipid. With these complexes we obtained an up to 70-fold increase of transfection with B16-F1 melanoma cells. However when B16-G4F, an alpha-MSH receptor negative melanoma cell line was transfected, an up to 700-fold increased transfection efficiency was observed. The peptide hormone analogue was equally efficient when it was only mixed with lipospermine--DNA complexes without covalent coupling. In addition to melanoma cells we also obtained up to 30-fold increased transfection with BN cells (embryonic liver cells). Our data show that an alpha-MSH analogue increased transfection independently of the MSH receptor expression but reaches efficiencies approaching those obtained with peptides derived from viral fusion proteins. The absence of targeting of constructs containing [Nle(4),D-Phe(7)]-alpha-MSH(4-10) can probably be attributed due to the relatively modest number of MSH receptors at the surface of melanoma. We suggest, however, that the peptide hormone analogue used in this study has membrane-active properties and could be of interest as helper agent to enhance non-viral gene delivery presumably by endosomal-destabilizing properties.

Lectin-mediated drug targeting: selection of valency, sugar type (Gal/Lac), and spacer length for cluster glycosides as parameters to distinguish ligand binding to C-type asialoglycoprotein receptors and galectins.

PURPOSE: Common oligosaccharides of cellular glycoconjugates are ligands for more than one type of endogenous lectin. Overlapping specificities to beta-galactosides of C-type lectins and galectins can reduce target selectivity of carbohydrate-ligand-dependent drug targeting. The purpose of this study is to explore distinct features of ligand presentation and structure for design of cluster glycosides to distinguish between asialoglycoprotein-specific (C-type) lectins and galectins. METHODS: Extent of binding of labeled sugar receptors to two types of matrix-immobilized (neo)glycoproteins and to cells was evaluated in the absence and presence of competitive inhibitors. This panel comprised synthetic mono-, bi-, and trivalent glycosides with two spacer lengths and galactose or lactose as ligand part. RESULTS: In contrast to C-type lectins of hepatocytes and macrophages, bi- and trivalent glycosides do not yield a notable glycoside cluster effect for galectins-1 and -3. Also, these Ca2+-independent galactoside-binding proteins prefer to home in on lactose-bearing glycosides relative to galactose as ligand, while spacer length requirements were rather similar. CONCLUSIONS: Trivalent cluster glycosides with Gal/GalNAc as ligand markedly distinguish between C-type lectins and galectins. Undesired side reactivities to galectins for C-type lectin drug delivery will thus be minimal.

Differential reactivity of maleimide and bromoacetyl functions with thiols: application to the preparation of liposomal diepitope constructs.

The comparative reactivity of maleimide and bromoacetyl groups with thiols (2-mercaptoethanol, free cysteine, and cysteine residues present at the N-terminus of peptides) was investigated in aqueous media. These studies were performed (i) with water-soluble functionalized model molecules, i.e., polyoxyethylene-based spacer arms that could also be coupled to lipophilic anchors destined to be incorporated into liposomes, and (ii) with small unilamellar liposomes carrying at their surface these thiol-reactive functions. Our results indicate that an important kinetic discrimination (2-3 orders of magnitude in terms of rate constants) can be achieved between the maleimide and bromoacetyl functions when the reactions with thiols are performed at pH 6.5. The bromoacetyl function which reacts at higher pH values (e.g., pH 9.0) retained a high chemoselectivity; i.e., under conditions where it reacted appreciably with the thiols of, e.g., HS-peptides, it did react with other nucleophilic functions such as alpha- and epsilon-amino groups or imidazole, which could also be present in peptides. This differential reactivity was applied to design chemically defined and highly immunogenic liposomal diepitope constructs as synthetic vaccines, i.e., vesicles carrying at their surface two different peptides conjugated each to a specific amphiphilic anchor. This was realized by coupling sequentially at pH 6.5 and 9.0 two HS-peptides to preformed vesicles containing lipophilic anchors functionalized with maleimide and bromoacetyl groups [Boeckler, C., et al. (1999) Eur. J. Immunol. 29, 2297-2308].

High prevalence of trichomoniasis in rural men in Mwanza, Tanzania: results from a population based study.

OBJECTIVES: To measure the prevalence of urethral infections including trichomoniasis in rural Tanzanian men, to assess the prevalence of symptoms and signs among men with Trichomonas vaginalis, and to analyse the risk factors for trichomoniasis. DESIGN: A cross sectional study of 1004 men aged 15-54 years in a rural community in north west Tanzania. METHODS: Participants were interviewed about sexual behaviour and symptoms of sexually transmitted diseases. First fraction urine samples and urethral swabs were collected and used to test for T vaginalis by wet preparation and culture, Neisseria gonorrhoeae by culture, Chlamydia trachomatis by ligase chain reaction and non-specific urethritis by Gram stain. Urine was also tested for the presence of leucocytes using a leucocyte esterase dipstick. Men were re-interviewed 2 weeks later to document new symptoms and signs of urethritis. RESULTS: Complete laboratory results were available on 980 men. One in four men had laboratory evidence of urethritis. T vaginalis was found in 109 individuals (11%), gonorrhoea in eight (0.8%), and chlamydial infection in 15 (1.5%). Over 50% of men with urethritis were asymptomatic. The prevalence of signs and symptoms was similar among men with T vaginalis alone compared with men with other urethral infections. The sensitivity and specificity of the leucocyte esterase dipstick (LED) test for detecting T vaginalis were 80% and 48% respectively in symptomatic men and 60% and 68% in asymptomatic men. Factors associated with trichomoniasis included religion, type of employment, and marital status. CONCLUSIONS: A high prevalence of urethritis was found in men in this community based study. More than half of the urethral infections detected were asymptomatic. The most prevalent pathogen was T vaginalis. Studies are needed on the prevalence of trichomoniasis in men presenting to health services with complaints suggestive of urethritis since treatment for T vaginalis is not included in the syndromic management of urethritis in most countries. The performance of the LED test as a screening test for trichomoniasis was unsatisfactory in both symptomatic and asymptomatic men. Improved screening tests are urgently needed to identify urethral infections that are asymptomatic and which are not covered by current syndromic management algorithms.

Design of highly immunogenic liposomal constructs combining structurally independent B cell and T helper cell peptide epitopes.

We have designed liposomal diepitope constructs that allow the physical combination, within the same vesicle, of B and Th epitopes as structurally separate entities. The immune response against such constructs was explored using TPEDPTDPTDPQDPSS (TPE), a B cell epitope originating from a Streptococcus mutans surface adhesin and QYIKANSKFIGITEL (QYI), a "universal" Th epitope from tetanus toxin. The two peptides were linked to the outer surface of small (diameter approximately 100 nm) unilamellar liposomes by covalent conjugation to two different anchors. To that end we have developed a strategy that allows the controlled chemical coupling of TPE and QYI, functionalized at their N terminus with a thiol, to preformed liposomes containing thiol-reactive derivatives of phosphatidylethanolamine and the lipopeptide S-[2,3-bis (palmitoyloxy)-(2-RS)-propyl]-N-palmitoyl-(R)-cysteinyl-alanyl-gly cine (Pam3CAG), respectively. This synthetic construct (administered i.p. to BALB/c mice) induced highly intense (titers > 20,000), anamnestic and long-lasting (over 2 years) immune responses, indicating that this strategy is successful. Two parameters were of prime importance to elicit this response with our liposomal diepitope constructs: (1) the simultaneous expression of B and Th epitopes on the same vesicle, and (2) the lipopeptide Pam3CAG anchor of the Th epitope QYI could not be replaced by a phosphatidylethanolamine anchor (a lesser immune response was observed). Analysis of the antibody response revealed a complex pattern; thus, besides the humoral response (production of IgG1, IgG2a, IgG2b) a superposition of a T-independent (TI-2 type) response was also found (IgM and IgG3). These results indicate that liposomal diepitope constructs could be attractive in the development of synthetic peptide-based vaccines.

Clinical significance of bone marrow biopsy and plasma cell morphology in MM and MGUS.

Multiple myeloma (MM) is characterized by plasma cell infiltration in the bone marrow, suppression of normal haematopoiesis, destruction of bone, renal failure and recurrent infections. The extent of organ involvement, severity of complications, drug sensitivity and therefore the clinical course differ widely among patients. Specific and reliable laboratory examinations and especially morphology including both biopsy and aspirations are required to evaluate its aggressivity and extent. In this review of MM and its variants we emphasize the impact of morphology on the diagnostic and prognostic approach to MM and MGUS, and on the planning and monitoring of appropriate therapy.

Bone scan and bone biopsy in the detection of skeletal metastases.

This study provides the first quantitative indication of the limits of sensitivity of a bone scan with technetium (99Tc-MDP) in detecting skeletal metastases and thereby also helps to explain the fact that bone scans may be negative when metastases are present in the bone marrow. Since 99Tc-MDP remains the least noxious and most widely used isotope for bone scanning, these results have direct clinical relevance in the evaluation of patients with solid tumors and possible metastatic spread.

Design and synthesis of thiol-reactive lipopeptides.

Lipopeptides are potent adjuvants that trigger an immune response against covalently conjugated low molecular mass antigens. We report here the design and synthesis of thiol-reactive lipopeptides (6, 7) which can be incorporated into liposomes and react, under mild conditions, with synthetic peptides carrying a thiol function.

Synthetic lipopeptides incorporated in liposomes: in vitro stimulation of the proliferation of murine splenocytes and in vivo induction of an immune response against a peptide antigen.

Amphiphilic lipopeptides, such as Pam3CysAlaGly and Pam3CysSerSer, were synthesized and incorporated into liposomes, and their ability to induce the proliferation of BALB/c mouse splenocyte was tested in vitro. When compared to monophosphoryl lipid A (MPL) the following potency order was found: liposomal lipopeptides > liposomal MPL > free (emulsified) lipopeptides. These results strongly depend on the size of the vesicles used: a mitogenic effect was observed only with lipopeptides incorporated within vesicles of diameter < or = 100 nm while lipopeptides in larger vesicles (diameter approximately 300 nm) gave no response. This may be related to the necessity for the liposome-associated lipopeptides to be endocytosed to reach putative intracellular targets. As immunoadjuvanticity seems to be linked to B-lymphocyte activation, the lipopeptides represent attractive alternatives to MPL for the realization of completely synthetic liposome-based peptide vaccine formulations. This was borne out by showing that Pam3CysAlaGly and Pam3CysSerSer, when incorporated in small unilamellar vesicles carrying a covalently conjugated synthetic peptide of sequence IRGERA, corresponding to an epitope of the C-terminal region of histone H3, were able to induce a potent and long-lasting immune response.

Temporal and spatial expression patterns of transgenes containing increasing amounts of the Drosophila clock gene period and a lacZ reporter: mapping elements of the PER protein involved in circadian cycling.

Rhythmic oscillations of the PER protein, the product of the Drosophila period (per) gene, in brain neurons of the adult fly are strongly involved in the control of circadian rhythms. We analyzed temporal and spatial expression patterns of three per-reporter fusion genes, which share the same 4 kb regulatory upstream region but contain increasing amounts of per's coding region fused in frame to the bacterial lacZ gene. The fusion proteins contained either the N-terminal half (SG), the N-terminal-two-thirds (BG), or nearly all (XLG) of the PER protein. All constructs led to reporter signals only in the known per-expressing cell types within the anterior CNS and PNS. Whereas the staining intensity of SG files was constantly high at different Zeitgeber times, the in situ signals in BG and XLG files cycled with approximately 24 hr periodicity in the PER-expressing brain cells in wild-type and per01 loss of function files. Despite the rhythmic fusion-gene expression within the relevant neurons of per01 BG files, their locomotor activity in light/dark cycling conditions and in constant darkness was identical to that of per01 controls, uncoupling protein cycling from rhythmic behavior. The XLG construct restored weak behavioral rhythmicity to (otherwise) per01 files, indicating that the C-terminal third of PER (missing in BG) is necessary to fulfill the biological function of this clock protein.

Staining in the brain of Pachymorpha sexguttata mediated by an antibody against a Drosophila clock-gene product: labeling of cells with possible importance for the beetle's circadian rhythms.

Central nervous system ganglia within the head of the beetle Pachymorpha sexguttata were labeled using an antibody that recognizes an evolutionarily conserved region of the period (per) gene product of Drosophila melanogaster. per and the protein it encodes (PER) are believed to play a central role in the generation of endogenous circadian rhythms in flies; therefore anti-PER-mediated immunoreactivity may help to uncover cellular components of the circadian clock system in that insect and in others. In the beetle, application of this antibody led to the staining of a distinct set of neurons located in the optic lobes and the central brain, plus small numbers of putative glial cells in the optic lobes. Neuronal perikarya (including their nuclei in a few cases), the axons, and terminal regions of the neurons were stained. The network formed by these labeled cells and processes are candidates for the neuronal basis of the beetle's circadian clock system: the pacemaker region situated next to the medulla neuropil, its connection to the apparent site of Zeitgeber input, and putative efferent pathways projecting to control centers of various effector systems. Anti-PER-mediated labeling and that resulting from application to beetle specimens of an antiserum against pigment-dispersing hormone (PDH) were compared; in the Drosophila brain all "PDH cells" express the per gene as well. In the beetle, however, the set of "PER cells" and PDH ones is at least in part nonoverlapping. The hypothesis that neurons stained by application of anti-PER participate in the control of the beetle's circadian rhythms is discussed in the context of previous electrophysiological and immunohistochemical studies. Also considered are analogies to, and differences from, labeling of the PER protein in fruit flies and PER-like immunoreactivity in other animals.

Immunogenicity of new heterobifunctional cross-linking reagents used in the conjugation of synthetic peptides to liposomes.

We have investigated the immunogenicity of six thiol-reactive heterobifunctional cross-linking reagents that permit the conjugation of cysteine carrying peptides to the surface of liposome containing monophosphoryl lipid A. Such constructs elicit an immune response against short synthetic peptides and our aim was to find the least immunogenic linkers to limit potential carrier-induced epitopic suppression. For that purpose the properties of three new polyoxyethylene linkers of different lengths and thiol-reactive moieties (maleimide, bromoacetyl, dithiopyridine) were compared to known derivatives obtained by reacting the classical reagents SMPB and SPDP or N-succinimidyl bromoacetate with phosphatidylethanolamine. The least immunogenic linkers were the bromoacetate derivatives whereas those containing a maleimide group evoked a significant anti-linker immune response. In addition, using IRGERA as a model peptide, we found that all six liposomal constructs strongly elicited the production of anti-peptide IgG antibodies. This immune response was therefore independent of the length of the linkers (ranging between 0.3 and 1.6 nm) and of the nature of the linkage. between the peptide and the thiol-reactive moieties of the cross-linkers, i.e. stable thioether or bio-reducible disulfide bonds.

Synthesis of short polyoxyethylene-based heterobifunctional cross-linking reagents. Application to the coupling of peptides to liposomes.

We describe the synthesis of [2-[2-[2-[(2-bromoacetyl)amino]ethoxy]ethoxy]ethoxy acetic acid (7), [2-[2-(2,5-dioxo-2,5-dihydropyrrol-1-yl)ethoxy]ethoxy] acetic acid (11), and [2-[2-(pyridin-2-yldisulfanyl)-ethoxy]ethoxy] acetic acid (16), three new thiol-reactive heterobifunctional reagents, and the preparation of their corresponding dipalmitoylphosphatidylethanolamine derivatives (8, 12, and 17). Such phospholipid amide derivatives were aimed to be incorporated into the bilayers of liposomal constructs used for immunization with e.g. synthetic peptides. The spacer arms introduced by 8, 12, and 17 are hydrophilic polyoxyethylene chains of variable lengths that were expected to provide a good accessibility to their conjugates and have a lesser intrinsic immunogenicity than the spacer introduced by N-[4-(p-maleimidophenyl)butyryl]phosphatidylethanolamine (MPB-PE), a classical reagent used for conjugation of ligands to the surface of liposomes. Such an immunogenicity might be prejudicial (e.g. carrier-induced epitopic suppression) to the development of synthetic vaccination formulations. Moreover, the derivatives 8, 12, and 17 allowed the coupling of peptides, bearing a thiol function, to their liposomal carrier via two types of linkages, i.e. stable thio ether (8 and 12) and bioreducible disulfide (17) bonds; this might be of importance in the mechanism of antigen presentation by competent cells. Using CG-IRGERA as a model peptide, the rate of coupling to 8, 12, and 17 was assessed as a function of pH.

Efficient gene delivery with neutral complexes of lipospermine and thiol-reactive phospholipids.

The presence of thiol-reactive phospholipid derivatives, such as N-4-(p-maleimidophenyl)butyryl) dipalmitoylphosphatidylethanolamine (MPB-DPPE), in electrically neutral lipospermine/DNA particles results in more than a 100-fold increased transfection efficiency of human hepatoma HepG2 cells and murine 3T3 fibroblasts. These effects could be ascribed to the presence of thiol-reactive functions, such as maleimide, bromoacetamide and dithiopyridyl linkage, on the transfecting particles. We propose that such particles react with thiol groups present at the surface of the cells, leading to their covalent anchoring, a process that is probably followed by an endocytosis of the complex.

Selective induction of protection against influenza virus infection in mice by a lipid-peptide conjugate delivered in liposomes.

We have previously reported (Muller et al. Vaccine 1990, 8, 308) that two cyclic peptide analogues called D loop and K loop, corresponding to residues 139-147 in site A of the haemagglutinin (HA) of influenza A virus (strain X31), were both able to provide protective immunity to infected OF1 mice when administered in the form of peptide-ovalbumin conjugates. The predicted conformation of the D loop is nearly identical to that of the native loop known from the X-ray structure of HA, while the predicted conformation of the K loop differs significantly from the native one. In this study, the two peptides were conjugated to small unilamellar liposomes, thus creating a chemically defined immunogen, and OF1 mice were immunized with these liposomes containing monophosphoryl lipid A as adjuvant. Compared with protein carrier systems, the liposomal preparations are completely synthetic and avoid the use of Freund's adjuvant. By using liposomes associated with the D loop, we were able to achieve 70% protection of the mice against intranasal challenge with the influenza virus while no protection was obtained with the liposome-associated K loop. The difference in effect between the two liposome and ovalbumin carrier systems may result from the induction of different structures in the peptides when coupled to lipid anchors than when coupled to proteins.

Bone formation in coralline hydroxyapatite. Effects of pore size studied in rabbits.

We analyzed osseous reactions in the rabbit femoral condyle to coralline hydroxyapatite bone substitutes of various pore sizes by radiology and histology. The results were compared to bone repair of empty cavities and to integration of allografts. Spontaneous bone repair of the empty cavities took approximately 12 weeks, while integration of the cryopreserved allografts occurred after 9 weeks. However, no signs of new bone formation were found with the 200 microns pore size hydroxyapatite. In contrast, there was substantial production of bone within the 500 microns pore size implants at 12 and 26 weeks. Our results indicate that the pore size of the coralline hydroxyapatite influenced the development of bone in the implants in the cancellous bone bed of the rabbit femoral condyle. The results also show that spontaneous bone repair should be taken into consideration when the integration of implants is evaluated.