Nanoliposomes as drug delivery systems: safety concerns.

The review highlights the safety issues of drug delivery systems based on liposomes. Due to their small sizes (about 80-120 nm, sometimes even smaller), phospholipid nanoparticles interact intensively with living systems during parenteral administration. This interaction significantly affects both their transport role and safety; therefore, special attention is paid to these issues. The review summarises the data on the basic factors affecting the safety of nanoliposomes: composition, size, surface charge, stability, the release of an incorporated drug, penetration into tissues, interaction with the complement system. Attention is paid to the authors' own research of unique phospholipid nanoparticles with a diameter of 20-30 nm. The influence of technological processes of nanoliposome production on their properties is considered. The article also discusses the modern safety assessment criteria contained in the preliminary regulatory documents of the manufacturing countries for new nanoliposome-based drugs being developed or used in the clinic.

Bacteriophage MS2 As a Tool for Targeted Delivery in Solid Tumor Chemotherapy.

Bacteriophage MS2 was employed for targeted delivery of an apoptosis-inducing agent, Tl+, into a tumor tissue. The targeted delivery was ensured by iRGD peptide, a ligand of integrins presumably located on the surface of endotheliocytes of the tumor tissue neovasculature and certain tumor cells. The synthesized peptide was conjugated to MS2 capsid proteins. Tl+ ions from TlNO3 penetrated the phage particles and tightly bound to phage RNA. Peptide-modified MS2 preparations filled with Tl+ caused cell death in two types of cultivated human breast cancer cells and effected necrosis of these tumor xenografts in mice. Neither peptide-conjugated bacteriophage MS2 without Tl+ nor the phage filled with Tl+ but without the peptide or the same phage with the non-conjugated peptide in solution produced such effects. The preparation exhibited no acute toxicity at a therapeutic dose.

Effect of Cell-Penetrating Arginine Peptide on Interaction of Photosensitizer Chlorin e6 Incorporated into Phospholipid Nanoparticles with Tumor Cells.

We studied the possibility of increasing the efficiency of photodynamic therapy by improving delivery of photosensitizers chlorin e6 into tumor cells. Previous studies showed that incorporation of chlorin e6 onto phospholipid nanoparticles with a diameter <20 nm reduces its cytotoxicity due to accelerated elimination from organs [8]. A heptapeptide R7 synthesized and added to this combination promoted internalization of chlorin e6 into HepG2 cells in comparison with initial nanoparticles without peptide R7. The observed effect of peptide R7 can be explained by activation of endocytosis and/or macropinocytosis (bearing in mind the interaction of arginine with carboxyl groups of e6. The development of this transporting system is a promising trend in photodynamic therapy of cancer diseases.

[Prospects for the design of new therapeutically significant protease inhibitors based on knottins and sunflower seed trypsin inhibitor (SFTI 1)]. / Perspektivy sozdaniia novykh ingibitorov terapevticheski znachimykh serinovykh proteaz na osnove knottinov i peptidnogo ingibitora tripsina iz semian podsolnechnika (SFTI 1).

Plant seed knottins, mainly from the Cucurbitacea family, and sunflower seed trypsin inhibitor (SFTI 1) are the most low-molecular canonical peptide inhibitors of serine proteases. High efficiency of inhibition of various serine proteases, structure rigidity together with the possibility of limited variations of amino acid sequences, high chemical stability, lack of toxic properties, opportunity of production by either chemical synthesis or use of heterologous expression systems make these inhibitors attractive templates for design of new compounds for regulation of therapeutically significant serine protease activities. Hence the design of such compounds represents a prospective research field. The review considers structural characteristics of these inhibitors, their properties, methods of preparation and design of new analogs. Examples of successful employment of natural serine protease inhibitors belonging to knottin family and SFTI 1 as templates for the design of highly specific inhibitors of certain proteases are given.

[NERVE GROWTH FACTOR IN THE URINE OF PATIENTS WITH IDIOPATHIC DETRUSOR OVERACTIVITY AND OVERACTIVE BLADDER WITHOUT DETRUSOR OVERACTIVITY].

The purpose was to determine the concentration of the neurotrophin nerve growth factor in urine to assess its possible role as a marker in the diagnosis of various forms of overactive bladder. The study included patients with urinary frequency and urgency: 21 patients with idiopathic detrusor overactivity, 18--with overactive bladder without detrusor overactivity and 11 healthy volunteers (control group). The level of nerve growth factor in the urine was determined in all participants of the study by the enzyme immunoassay (ELISA). In the control group the average ratio of nerve growth factor level to the level of urine creatinine was 0.2 ± 0.06, in patients with overactive bladder without detrusor overactivity -0.33 ± 0.06 (p > 0.05). In patients with idiopathic detrusor overactivity the rate was significantly higher and amounted to 6.04 ± 0.9 (p < 0.05). Therefore, measurement of the concentration of nerve growth factor in the urine may be used for differential diagnosis of the presence or absence of detrusor overactivity in patients with overactive bladder.

[Way to the peptide vaccine against hepatitis C].

In order to surpass the problem of genetic variability of hepatitis C virus envelope proteins during vaccine development, we used the so-called "reverse vaccinology"approach--"from genome to vaccine". Database of HCV protein sequences was designed, viral genome analysis was performed, and several highly conserved sites were revealed in HCV envelope proteins in the framework of this approach. These sites demonstrated low antigenic activity in full-size proteins and HCV virions: antibodies against these sites were not found in all hepatitis C patients. However, two sites, which contained a wide set of potential T-helper epitope motifs, were revealed among these highly conserved ones. We constructed and prepared by solid-phase peptide synthesis several artificial peptide constructs composed of two linker-connected highly conserved HCV envelope E2 protein sites; one of these sites contained a set of T-helper epitope motifs. Experiments on laboratory animals demonstrated that the developed peptide constructs manifested immunogenicity compared with one of protein molecules and were able to raise antibodies, which specifically bound HCV envelope proteins. We succeeded in obtaining antibodies reactive with HCV from hepatitis C patient plasma upon the immunization with some constructs. An original preparation of a peptide vaccine against hepatitis C is under development on the basis of these peptide constructs.

[Preparation of monospecific antibodies against isoform 2 of translation elongation factor 1A (eEF1A2)].

Amino acid sequences of eukaryotic translation elongation factor isoform 1 (eEF1A1) and 2 (eEF1A2) were compared and two peptide fragments of eEF1A2 were chosen as linear antigenic determinants for generation of monospecific antipeptide antibodies. Selected peptides were synthesized, conjugated to bovine serum albumin (BSA) and used for mice immunizations. Antibodies, produced against the eEF1A2 fragment 330-343 conjugated to BSA, specifically recognized this isoform in the native and partially denatured states but did not interact with the eEF1A1 isoform. It was shown that these monospecific anti-eEF1A2 antibodies could be employed for eEF1A2 detection both by enzyme-linked immunosorbent assay and by immunoblotting.

[Synthetic peptide vaccines].

This review considers the stages of the development of synthetic peptide vaccines against infectious agents, novel approaches and technologies employed in this process, including bioinformatics, genomics, proteomics, large-scale peptide synthesis, high-throughput screening methods, the use of transgenic animals for modelling human infections. An important role for the development and selection of efficient adjuvants for peptide immunogens is noted. Examples of synthetic peptide vaccine developments against three infectious diseases (malaria, hepatitis C, and foot-and-mouth disease) are given.

[Antigenicity and B-epitope mapping of hepatitis C virus envelope protein E2].

Immunogenicity for laboratory animals (rabbits and mice) of the whole hepatitis C virus envelope proteins and their conserved as well as hypervariable HVR1 sites has been investigated. Rabbit immune responses to HCV envelope proteins (both single E2 and E1E2 heterodimer) were shown to be much more efficient than murine immune responses. Upon the immunization of the rabbit with E2 protein, antibodies to several highly conserved linear B-epitopes of this protein as well as to the N-terminal fragment of the hypervariable region HVRI were formed. Epitopes in the CR2 region were determined for the first time. Cross-reactivity was revealed between the N-terminal fragment of the protein E2 hypervariable region HVRI and the octapeptide fragment of the protein E1 conserved region CR1, which shared four identical amino acid residues.

[Putative hepatitis C virus cell receptors].

Penetration of a virus into a host cell comprises the first step of the viral life cycle. Blockage of this process can stop or prevent the rise of the infection. In order to develop substances that show directed blocking activity, one should know which host cell and viral molecules are involved in the reciprocal recognition and interaction leading to the virus entry into the cell. This review is devoted to the problems of the identification of cell outer membrane molecules that participate in the hepatitis C virus binding and its transfer inside the cells. The putative role of these molecules as hepatitis C virus receptors and coreceptors in the beginning and development of the infection is discussed.

[Synthesis of beta-amyloid fragment 5RHDSGY10 and its isomers].

Peptide RHDSGY that represents the fragment of human beta-amyloid Zn-binding site and its isomers RH(D-Asp)SGY and RH(beta-Asp)SGY have been prepared by solid-phase synthesis and analysed by HPLC and various mass-spectrometric methods. The problem of low yield of peptide RHDSGY and its isomers attributed to 9-fluorenylmethoxycarbonyl (Fmoc)-amino acids and/or formation of side-products as RH(Asp-imide)SGY and RHDSGY (instead of RH(beta-Asp)SGY) was solved via selection of reagents for the removal of Fmoc groups from the growing peptide chain.

Identification of glycosaminoglycan-binding sites within hepatitis C virus envelope glycoprotein E2*.

Heparan sulphate is one of the candidate receptors for hepatitis C virus (HCV). Envelope glycoproteins of HCV have been proposed to be responsible for recognition and binding with cell receptors. They are characterized by great genetic polymorphism. In this study the mapping of regions with glycosaminoglycan-binding properties within HCV envelope proteins has been undertaken. We prepared a set of overlapping peptides corresponding to conserved regions of these envelope proteins and analysed them by solid phase heparin-binding assay. The search for established glycosaminoglycan-binding motifs in the HCV envelope proteins showed the absence of the sites corresponding to the glycosaminoglycan-binding patterns in consensus sequence. We identified one highly conserved and two less conserved heparin-binding sequences within the envelope protein E2 based on solid phase assay results. We did not find any differences in binding efficiency of these peptides with heparin, heparan sulphate or dextran sulphate. Our data supported the specific association between HCV envelope protein E2 and cell surface glycosaminoglycans. We hypothesize that identified regions from E2 can contribute to HCV binding to cell surface glycosaminoglycans.

[Antigenic map of the cytochrome b5 cytosol domain]. / Antigennaia karta tsitozol'nogo domena tsitokhroma b5.

Because of the high conservativity of cytochrome b5 cytosolic domain (t-cytochrome b5) among mammalian species the antibodies against bovine t-cytochrome b5 were successfully obtained only from evolutionary distant species (chicken). Antigenic mapping of t-cytochrome b5 by the peptide scanning method showed the presence of a large set of linear B-epitopes of this protein. This is well consistent with high content of water-accessible amino acid residues and polypeptide chain turns in its molecule. Linear B-epitopes of t-cytochrome b5 are presumably located in sites with species specificity of the amino acid sequence, despite of the high-degree surface exposure of the highly conservative site, which is probably responsible for the interaction with cytochrome P450. It can be explained by the absence of the effective T-helper support of the antibody response to amino acid sequence sites that are identical to own proteins of an organism. Nevertheless, antibodies against bovine t-cytochrome b5 possess high cross-reactivity with regard to rabbit full-sized cytochrome b5 because of the presence of one identical linear B-epitope and cross-interactions between other antigenic sites with minor structural differences.

[Computer-assisted vaccine design]. / Komp'iuternoe konstruirovanie vaktsin.

With the modern molecular biology techniques, it has been possible to detect, isolate and clone biological macromolecules, which could be used as immunogenes in artificial vaccine constructs. In the post-genomic era, the prospective immunogenic components are searched using bionformatic tools and proteomic technologies. Today it is quite realistic to combine the artificial vaccine constructs from the preselected molecular components. Existing computational methods are able to detect the potential immunogenes in genomic sequences, predict their characteristics and subcellular location. The set of methods is designed to predict the T- and B-epitopes that can be used as components of minimal vaccine constructs. The variety of systems for production and delivery of vaccines are developed and tested. These include transgenic plants, bacterial and viral vectors, DNA molecules etc. Several informational resources provide free access to molecular immunology data and deliver services on prediction of antigenic features. Several artificial vaccines have already been launched, but much more preparations are under preclinical and clinical trials. Computer-aided design of vaccines may significantly decrease time and costs required for their development. Modern bioinformatic technologies are now employed for discovery of more effective and potent vaccine.

[Introduction of biologically active fragments of interferon-alpha2 and insulin into the artificial protein albebetin affects immunogenicity of the final construct]. / Vvedenie biologicheski aktivnykh fragmentov interferona-al'fa2 i insulina v sostav iskusstvennogo belka al'bebetina vliiaet na immunogennost' itogovoi konstruktsii.

The immunogenicity of the artificial protein albebetin and its derivatives with active peptide fragments was investigated. We also studied the influence of the peptides on the immunogenicity of the whole construct and contribution of each component to the immunogenicity. Two of three studied proteins contained active peptides from human IFN-alpha and insulin. Three continuous antigenic sites with different immunogenic potential were recognized in the chimerical proteins. The interferon fragment was the immunodominant site in the albeferon and albeferon-insulin molecules, while the insulin fragment displayed low immunogenic activity. All continuous B-epitopes are located at the boundaries of the secondary structure elements and at the predicted surface-located sites of albebetin molecule. Thus, peptide fragments attached to the artificial protein carrier can influence immunogenicity of the resulting construct.

Mapping and characterization of B cell linear epitopes in the conservative regions of hepatitis C virus envelope glycoproteins.

Forty-eight overlapping octapeptides covering highly conservative regions of E1 and E2 hepatitis C virus (HCV) envelope proteins were synthesized and tested by ELISA against different groups of sera obtained from HCV-infected patients. All sera from patients with acute infection, except a single case of serum reactivity with the region HINRTALN, were nonreactive with any peptide. Sera obtained from chronic patients reacted with 12 peptides from five selected regions. Two immunodominant B epitopes were found, one being the precisely mapped antigenic site RMAWDM positioned inside the earlier shown immunodominant epitope from E1, and the second site, PALSTGLIH from E2, detected for the first time. New minor antigenic site was determined as PTDCFRKH from E2. We found only minor seroreactivity for one of the putative sites involved in CD81 binding, PYCWHYAP.

[Analysis of hepatitis C virus proteins based on amino acid sequence data and literature data]. / Analiz belkov virusa gepatita C na osnove aminokislotnykh posledovatel'nostei i literaturnykh dannykh.

To analyze the interrelationships between the amino acid sequences of the proteins of hepatitis C virus and the functional characteristics of different variants of this virus, a database of protein functional mapping of hepatitis C virus was developed. The database contains amino acid sequences (both full-size and fragmentary) retrieved from accessible databases and experimental data published in literature. The database also contains the results of comparison and treatment of primary data, including alignments and functional regions. On the basis of these data, variable and conservative regions of envelope proteins of hepatitis C virus were revealed. Antigenic and functional maps of structural and nonstructural proteins of the virus were constructed. The most variable region of the envelope protein E2 (HVR1) was analysed. It is assumed that the conservatism of some amino acid positions of HVR1 is related to the functions of this region.

[The immune response to artificial proteins containing biologically active fragments of human IFN-alpha2 and insulin]. / Immunnyi otvet pri vvedenii iskusstvennykh belkov, soderzhashchikh biologicheski aktivnye fragmenty IFN-alpha2 i insulina cheloveka.

A study was made of the humoral immune response of BALB/c mice to various doses of artificial proteins that contained biologically active fragments of human interferon alpha 2 (IFN-alpha 2) and insulin. The insulin fragment had no effect on the response to any protein construct. The IFN-alpha 2 fragment increased the titer of antibodies against the construct. Mapping of continuous B epitopes with immune sera revealed several antigenic determinants, the C end of the IFN-alpha 2 fragment with the adjacent de novo protein region being immunodominant. More effective binding of serum antibodies with the constructs containing the IFN-alpha 2 fragment was attributed to antibody interaction with the fragment and to a better recognition of the entire protein construct by the immune system.

[The role of single amino acid residues of the immunodominant continuous epitope of cytochrome P450CAM (CYP101) 312LKKGDQ317 in recognition by specific antibodies]. / Rol' otdel'nykh aminokislotnykh ostatkov immunodominantnogo nepreryvnogo épitopa tsitokhroma P450CAM (CYP101) 312LKKGDQ317 v raspoznavanii spetsificheskimi antitelami.

As it has been shown previously the site 311-318 of bacterial cytochrome P450cam (CYP101) contains an immunodominant continuous B-epitope. In order to investigate the role of single amino acid residues in antibody binding antigenic hexapeptide 312LKKGDQ317 analogues including single amino acid replacements were synthesized using Multipin technology. Antibodies from three anti-P450cam polyclonal rabbit sera interacted similarly to these peptides. The residue G315 was found to play a significant role in antibody recognition; any replacement leads there to considerable decrease of antibody binding. Residues L312, K313 and D316 occurred to be partly replaceable, whereas K314 and Q317 were not essential in recognition. These results correspond to known spatial structure of P450cam molecule. In its 312-317 site the polypeptide chain makes a turn and so some water-accessible atoms form a compact surface cluster including side chain atoms of K313 and D316, O-atom of K314 and C alpha-atom of G315, which present reliable Ig binding site. Side chain of K314 outlying from this cluster does not participate in the interaction. The received data permit to consider that the continuous epitope 312-317 of P450cam is conformationally dependent.

Comparative analysis of amino acid sequences from envelope proteins isolated from different hepatitis C virus variants: possible role of conservative and variable regions.

Sequences of the E1 and E2 envelope proteins of hepatitis C virus (HCV) (827 non-identical items) were collected from available sources and aligned. Analysis of the alignment identified regions with different sequence variability. It was found that 33% and 50% of positions within E1 and E2, respectively, were highly conservative. Such conservation can be considered as the minimum for maintaining stability of the three-dimensional structure and function of these proteins. Conserved cysteines in E1 and E2 (eight and 18 residues, respectively) were presumed to form intramolecular disulphide bonds. Both envelope proteins were predicted to contain 14 conservative glycosylation sites. Two additional glycosylation sites were predicted in 58% of E1 and 30% of E2 sequences within the corresponding regions. We describe the positions of six conservative regions in E1 and E2, which have several charged and aromatic residues known to participate frequently in protein-protein recognition. Peculiarities in the amino acid content of conservative fragments and putative differences in glycosylation were considered with regard to antigenic specificity and possible binding to surface structures of target cells. We also analysed the hypervariable region 1 (HVR1), located in the E2 protein. Aligned positions of HVR1 were described in relation to the maintenance of conformational stability and recognition of cell receptors.