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1.
Int J Pharm ; 661: 124465, 2024 Jul 14.
Article in English | MEDLINE | ID: mdl-39004290

ABSTRACT

Liposomes are one of the most important drug delivery vectors, nowadays used in clinics. In general, polyethylene glycol (PEG) is used to ensure the stealth properties of the liposomes. Here, we have employed hydrophilic, biocompatible and highly non-fouling N-(2-hydroxypropyl) methacrylamide (HPMA)-based copolymers containing hydrophobic cholesterol anchors for the surface modification of liposomes, which were prepared by the method of lipid film hydration and extrusion through 100 nm polycarbonate filters. Efficient surface modification of liposomes was confirmed by transmission electron microscopy, atomic force microscopy, and gradient ultracentrifugation. The ability of long-term circulation in the vascular bed was demonstrated in rabbits after i.v. application of fluorescently labelled liposomes. Compared to PEGylated liposomes, HPMA-based copolymer-modified liposomes did not induce specific antibody formation and did not activate murine and human complement. Compared with PEGylated liposomes, HPMA-based copolymer-modified liposomes showed a better long-circulating effect after repeated administration. HPMA-based copolymer-modified liposomes thus represent suitable new candidates for a generation of safer and improved liposomal drug delivery platforms.

2.
Nanomaterials (Basel) ; 11(10)2021 Sep 27.
Article in English | MEDLINE | ID: mdl-34684950

ABSTRACT

The evaluation of carbon-based nanomaterials' (C-BNMs') interactions with the immune system, notably their ability to cause inflammation, is a critical step in C-BNM health risk assessment. Particular attention should be given to those C-BNMs that do not cause direct cytotoxicity or inflammation on their own. However, the intracellular presence of these non-biodegradable nanomaterials could dysregulate additional cell functions. This is even more crucial in the case of phagocytes, which are the main mediators of defensive inflammation towards pathogens. Hence, our study was focused on multi-walled carbon nanotubes (MWCNTs) and two different types of graphene platelets (GPs) and whether their intracellular presence modulates a proinflammatory response from human primary monocytes towards common pathogens. Firstly, we confirmed that all tested C-BNMs caused neither direct cytotoxicity nor the release of tumour necrosis factor α (TNF-α), interleukin (IL)-6 or IL-10. However, such pre-exposed monocytes showed increased responsiveness to additional bacterial stimuli. In response to several types of bacteria, monocytes pre-treated with GP1 produced a significantly higher quantity of TNF-α, IL-6 and IL-10. Monocytes pre-treated with MWCNTs produced increased levels of IL-10. All the tested C-BNMs enhanced monocyte phagocytosis and accelerated their differentiation towards macrophages. This study confirms the immunomodulatory potential of C-BNMs.

3.
Pathol Oncol Res ; 27: 614281, 2021.
Article in English | MEDLINE | ID: mdl-34257584

ABSTRACT

Background and aims: The majority of colorectal cancers arise from detectable adenomatous or serrated lesions. Here we demonstrate how deregulated alternative splicing of CD44 gene in diseased colon mucosa results in downregulation of standard isoform of CD44 gene (CD44s) and upregulation of variant isoform CD44v8-10. Our aim is to show that upregulation of CD44v8-10 isoform is a possible marker of precancerous lesion in human colon. Methods: We analysed pairs of fresh biopsy specimen of large intestine in a cohort of 50 patients. We studied and compared alternative splicing profile of CD44 gene in colon polyps and adjoined healthy colon mucosa. We performed end-point and qRT PCR, western blotting, IHC staining and flow cytometry analyses. Results: We detected more than five-fold overexpression of CD44v8-10 isoform and almost twenty-fold downregulation of standard isoform CD44s in colon polyps compared to adjoined healthy tissue with p = 0.018 and p < 0.001 in a cohort of 50 patients. Our results also show that aberrant splicing of CD44 occurs in both biologically distinct subtypes of colorectal adenoma possibly in ESRP-1 specific manner. Conclusion: 92% of the colon polyp positive patients overexpressed CD44v8-10 isoform in their colon polyps while only 36% of them had positive fecal occult blood test which is currently a standard non-invasive screening technique. Impact: We believe that our results are important for further steps leading to application of CD44v8-10 isoform as a biomarker of colorectal precancerosis in non-invasive detection. Early detection of colon precancerosis means successful prevention of colorectal carcinoma.


Subject(s)
Biomarkers, Tumor/metabolism , Colon/pathology , Colonic Polyps/pathology , Colorectal Neoplasms/diagnosis , Hyaluronan Receptors/metabolism , Biomarkers, Tumor/genetics , Colon/metabolism , Colonic Polyps/metabolism , Colorectal Neoplasms/genetics , Colorectal Neoplasms/metabolism , Humans , Hyaluronan Receptors/genetics , Prognosis , Protein Isoforms
4.
Virulence ; 12(1): 1271-1287, 2021 12.
Article in English | MEDLINE | ID: mdl-33993840

ABSTRACT

One of the proposed strategies for the development of a more efficient HIV-1 vaccine is based on the identification of proteins binding to a paratope of chosen broadly neutralizing antibody (bNAb) that will mimic cognate HIV-1 Env (glyco)protein epitope and could be used as potent immunogens for induction of protective virus-neutralizing antibodies in the immunized individuals. To verify this "non-cognate ligand" concept, we developed a highly complex combinatorial library designed on a scaffold of human myomesin-1 protein domain and selected proteins called Myomedins specifically binding to variable regions of HIV-1 broadly neutralizing antibody 10E8. Immunization of mice with these Myomedin variants elicited the production of HIV-1 Env-specific antibodies. Hyperimmune sera bound to Env pseudotyped viruses and weakly/moderately neutralized 54% of tested clade A, B, C, and AE pseudotyped viruses variants in vitro. These results demonstrate that Myomedin variants have the potential to mimic Env epitopes and could be used as potential HIV-1 vaccine components.


Subject(s)
HIV Infections , HIV-1 , Animals , Antibodies, Neutralizing , Broadly Neutralizing Antibodies , Epitopes , HIV Antibodies , HIV Infections/prevention & control , HIV-1/genetics , Mice , Viral Pseudotyping , env Gene Products, Human Immunodeficiency Virus/genetics
6.
Nanomaterials (Basel) ; 10(3)2020 Feb 27.
Article in English | MEDLINE | ID: mdl-32120988

ABSTRACT

Carbon-based nanomaterials (C-BNM) have recently attracted an increased attention as the materials with potential applications in industry and medicine. Bioresistance and proinflammatory potential of C-BNM is the main obstacle for their medicinal application which was documented in vivo and in vitro. However, there are still limited data especially on graphene derivatives such as graphene platelets (GP). In this work, we compared multi-walled carbon nanotubes (MWCNT) and two different types of pristine GP in their potential to activate inflammasome NLRP3 (The nod-like receptor family pyrin domain containing 3) in vitro. Our study is focused on exposure of THP-1/THP1-null cells and peripheral blood monocytes to C-BNM as representative models of canonical and alternative pathways, respectively. Although all nanomaterials were extensively accumulated in the cytoplasm, increasing doses of all C-BNM did not lead to cell death. We observed direct activation of NLRP3 via destabilization of lysosomes and release of cathepsin B into cytoplasm only in the case of MWCNTs. Direct activation of NLRP3 by both GP was statistically insignificant but could be induced by synergic action with muramyl dipeptide (MDP), as a representative molecule of the family of pathogen-associated molecular patterns (PAMPs). This study demonstrates a possible proinflammatory potential of GP and MWCNT acting through NLRP3 activation.

7.
Sci Rep ; 10(1): 4780, 2020 03 16.
Article in English | MEDLINE | ID: mdl-32179785

ABSTRACT

Gadolinium (Gd)-based contrast agents are extensively used for magnetic resonance imaging (MRI). Liposomes are potential nanocarrier-based biocompatible platforms for development of new generations of MRI diagnostics. Liposomes with Gd-complexes (Gd-lip) co-encapsulated with thrombolytic agents can serve both for imaging and treatment of various pathological states including stroke. In this study, we evaluated nanosafety of Gd-lip containing PE-DTPA chelating Gd+3 prepared by lipid film hydration method. We detected no cytotoxicity of Gd-lip in human liver cells including cancer HepG2, progenitor (non-differentiated) HepaRG, and differentiated HepaRG cells. Furthermore, no potential side effects of Gd-lip were found using a complex system including general biomarkers of toxicity, such as induction of early response genes, oxidative, heat shock and endoplasmic reticulum stress, DNA damage responses, induction of xenobiotic metabolizing enzymes, and changes in sphingolipid metabolism in differentiated HepaRG. Moreover, Gd-lip did not show pro-inflammatory effects, as assessed in an assay based on activation of inflammasome NLRP3 in a model of human macrophages, and release of eicosanoids from HepaRG cells. In conclusion, this in vitro study indicates potential in vivo safety of Gd-lip with respect to hepatotoxicity and immunopathology caused by inflammation.


Subject(s)
Contrast Media , Drug Carriers , Gadolinium DTPA , Hepatocytes/drug effects , Liposomes , Macrophages/drug effects , Magnetic Resonance Imaging , Phosphatidylethanolamines , Cells, Cultured , Fibrinolytic Agents , Gadolinium DTPA/adverse effects , Gadolinium DTPA/toxicity , Humans , Inflammasomes , NLR Family, Pyrin Domain-Containing 3 Protein , Nanoparticles , Phosphatidylethanolamines/adverse effects , Phosphatidylethanolamines/toxicity
8.
Pharmaceutics ; 11(12)2019 Dec 02.
Article in English | MEDLINE | ID: mdl-31810280

ABSTRACT

Development of tools for direct thrombus imaging represents a key step for diagnosis and treatment of stroke. Nanoliposomal carriers of contrast agents and thrombolytics can be functionalized to target blood thrombi by small protein binders with selectivity for fibrin domains uniquely formed on insoluble fibrin. We employed a highly complex combinatorial library derived from scaffold of 46 amino acid albumin-binding domain (ABD) of streptococcal protein G, and ribosome display, to identify variants recognizing fibrin cloth in human thrombus. We constructed a recombinant target as a stretch of three identical fibrin fragments of 16 amino acid peptide of the Bß chain fused to TolA protein. Ribosome display selection followed by large-scale Enzyme-Linked ImmunoSorbent Assay (ELISA) screening provided four protein variants preferentially binding to insoluble form of human fibrin. The most specific binder variant D7 was further modified by C-terminal FLAG/His-Tag or double His-tag for the attachment onto the surface of nanoliposomes via metallochelating bond. D7-His-nanoliposomes were tested using in vitro flow model of coronary artery and their binding to fibrin fibers was demonstrated by confocal and electron microscopy. Thus, we present here the concept of fibrin-targeted binders as a platform for functionalization of nanoliposomes in the development of advanced imaging tools and future theranostics.

9.
EBioMedicine ; 47: 247-256, 2019 Sep.
Article in English | MEDLINE | ID: mdl-31544770

ABSTRACT

BACKGROUND: The development of an effective vaccine preventing HIV-1 infection is hindered by the enormous antigenic variability and unique biochemical and immunological properties of HIV-1 Env glycoprotein, the most promising target for HIV-1 neutralizing antibody. Functional studies of rare elite neutralizers led to the discovery of broadly neutralizing antibodies. METHODS: We employed a highly complex combinatorial protein library derived from a 5 kDa albumin-binding domain scaffold, fused with support protein of total 38 kDa, to screen for binders of broadly neutralizing antibody VRC01 paratope. The most specific binders were used for immunization of experimental mice to elicit Env-specific antibodies and to test their neutralization activity using a panel of HIV-1 clade C and B pseudoviruses. FINDINGS: Three most specific binders designated as VRA017, VRA019, and VRA177 exhibited high specificity to VRC01 antibody. Immunized mice produced Env-binding antibodies which neutralize eight of twelve HIV-1 Tier 2 pseudoviruses. Molecular modelling revealed a shape complementarity between VRA proteins and a part of VRC01 gp120 interacting surface. INTERPRETATION: This strategy based on the identification of protein replicas of broadly neutralizing antibody paratope represents a novel approach in HIV-1 vaccine development. This approach is not affected by low immunogenicity of neutralization-sensitive epitopes, variability, and unique biochemical properties of HIV-1 Env used as a crucial antigen in the majority of contemporary tested vaccines. FUND: Czech Health Research Council 15-32198A, Ministry of Health, Czech Republic.


Subject(s)
Antibodies, Neutralizing/immunology , Antigens, Viral/immunology , Epitopes/immunology , HIV Antibodies/immunology , HIV Infections/immunology , HIV-1/immunology , AIDS Vaccines/immunology , Amino Acid Sequence , Animals , Antibodies, Neutralizing/blood , Antigens, Viral/chemistry , Disease Models, Animal , Epitopes/chemistry , HIV Antibodies/blood , HIV Envelope Protein gp120/immunology , HIV Infections/virology , Humans , Immunoglobulin G/blood , Immunoglobulin G/immunology , Mice , Models, Molecular , Protein Conformation
10.
Mol Pharm ; 16(8): 3441-3451, 2019 08 05.
Article in English | MEDLINE | ID: mdl-31184896

ABSTRACT

Nanodiamonds (ND), especially fluorescent NDs, represent potentially applicable drug and probe carriers for in vitro/in vivo applications. The main purpose of this study was to relate physical-chemical properties of carboxylated NDs to their intracellular distribution and impact on membranes and cell immunity-activation of inflammasome in the in vitro THP-1 cell line model. Dynamic light scattering, nanoparticle tracking analysis, and microscopic methods were used to characterize ND particles and their intracellular distribution. Fluorescent NDs penetrated the cell membranes by both macropinocytosis and mechanical cutting through cell membranes. We proved accumulation of fluorescent NDs in lysosomes. In this case, lysosomes were destabilized and cathepsin B was released into the cytoplasm and triggered pathways leading to activation of inflammasome NLRP3, as detected in THP-1 cells. Activation of inflammasome by NDs represents an important event that could underlie the described toxicological effects in vivo induced by NDs. According to our knowledge, this is the first in vitro study demonstrating direct activation of inflammasome by NDs. These findings are important for understanding the mechanism(s) of action of ND complexes and explain the ambiguity of the existing toxicological data.


Subject(s)
Inflammasomes/drug effects , Intravital Microscopy/methods , Lysosomes/drug effects , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , Nanodiamonds/administration & dosage , Cathepsin B/immunology , Cathepsin B/metabolism , Cell Membrane/drug effects , Cell Membrane/metabolism , Cell Membrane/ultrastructure , Dynamic Light Scattering , Fluorescence , Humans , Inflammasomes/immunology , Inflammasomes/metabolism , Lysosomes/immunology , Lysosomes/metabolism , Lysosomes/ultrastructure , Microscopy, Atomic Force , Microscopy, Confocal , Microscopy, Electron , NLR Family, Pyrin Domain-Containing 3 Protein/immunology , Nanodiamonds/chemistry , Pinocytosis , THP-1 Cells
11.
Carbohydr Polym ; 207: 521-532, 2019 Mar 01.
Article in English | MEDLINE | ID: mdl-30600036

ABSTRACT

New synthetic aminooxy lipid was designed and synthesized as a building block for the formulation of functionalised nanoliposomes (presenting onto the outer surface of aminooxy groups) by microfluidic mixing. Orthogonal binding of cellular mannan (Candida glabrata (CCY 26-20-1) onto the outer surface of functionalised nanoliposomes was modified by orthogonal binding of reducing termini of mannans to oxime lipids via a click chemistry reaction based on aminooxy coupling (oxime ligation). The aminooxy lipid was proved as a suitable active component for preparation of functionalised nanoliposomes by the microfluidic mixing method performed with the instrument NanoAssemblr™. This "on-chip technology" can be easily scaled-up. The structure of mannan-liposomes was visualized by transmission and scanning electron microscopy, including immunogold staining of recombinant mannan receptor bound onto mannosylated-liposomes. The observed structures are in a good correlation with data obtained by DLS, NTA, and TPRS methods. In vitro experiments on human and mouse dendritic cells demonstrate selective internalisation of fluorochrome-labelled mannan-liposomes and their ability to stimulate DC comparable to lipopolysaccharide. We describe a potentially new drug delivery platform for mannan receptor-targeted antimicrobial drugs as well as for immunotherapeutics. Furthermore, the platform based on mannans bound orthogonally onto the surface of nanoliposomes represents a self-adjuvanted carrier for construction of liposome-based recombinant vaccines for both systemic and mucosal routes of administration.


Subject(s)
Dendritic Cells/immunology , Lectins, C-Type/immunology , Liposomes/immunology , Mannans/immunology , Mannose-Binding Lectins/immunology , Nanoparticles/chemistry , Receptors, Cell Surface/immunology , Adjuvants, Immunologic/pharmacology , Animals , Antigens, Surface/metabolism , Candida glabrata/chemistry , Click Chemistry , Humans , Hydroxylamines/chemical synthesis , Hydroxylamines/chemistry , Lipids/chemical synthesis , Lipids/chemistry , Liposomes/chemistry , Liposomes/pharmacology , Mannans/chemistry , Mannans/pharmacology , Mannose Receptor , Mice, Inbred BALB C , Microfluidics/methods , Particle Size
12.
Bioconjug Chem ; 29(7): 2343-2356, 2018 07 18.
Article in English | MEDLINE | ID: mdl-29898364

ABSTRACT

New synthetic aminoxy lipids are designed and synthesized as building blocks for the formulation of functionalized nanoliposomes by microfluidization using a NanoAssemblr. Orthogonal binding of hyaluronic acid onto the outer surface of functionalized nanoliposomes via aminoxy coupling ( N-oxy ligation) is achieved at hemiacetal function of hyaluronic acid and the structure of hyaluronic acid-liposomes is visualized by transmission electron microscopy and cryotransmission electron microscopy. Observed structures are in a good correlation with data obtained by dynamic light scattering (size and ζ-potential). In vitro experiments on cell lines expressing CD44 receptors demonstrate selective internalization of fluorochrome-labeled hyaluronic acid-liposomes, while cells with down regulated CD44 receptor levels exhibit very low internalization of hyaluronic acid-liposomes. A method based on microfluidization mixing was developed for preparation of monodispersive unilamellar liposomes containing aminoxy lipids and orthogonal binding of hyaluronic acid onto the liposomal surface was demonstrated. These hyaluronic acid-liposomes represent a potentially new drug delivery platform for CD44-targeted anticancer drugs as well as for immunotherapeutics and vaccines.


Subject(s)
Antineoplastic Agents/administration & dosage , Drug Delivery Systems/methods , Hyaluronan Receptors/metabolism , Hyaluronic Acid/chemistry , Lipids/chemical synthesis , Liposomes/chemistry , Cell Line , Endocytosis , Fluorescent Dyes , Humans , Hyaluronan Receptors/analysis , Hyaluronic Acid/metabolism , Liposomes/therapeutic use , Microfluidics , Microscopy, Electron, Transmission , Neoplasms/drug therapy
13.
J Med Chem ; 60(18): 7745-7763, 2017 09 28.
Article in English | MEDLINE | ID: mdl-28829599

ABSTRACT

Fatty acyl analogues of muramyldipeptide (MDP) (abbreviated N-L18 norAbuGMDP, N-B30 norAbuGMDP, norAbuMDP-Lys(L18), norAbuMDP-Lys(B30), norAbuGMDP-Lys(L18), norAbuGMDP-Lys(B30), B30 norAbuMDP, L18 norAbuMDP) are designed and synthesized comprising the normuramyl-l-α-aminobutanoyl (norAbu) structural moiety. All new analogues show depressed pyrogenicity in both free (micellar) state and in liposomal formulations when tested in rabbits in vivo (sc and iv application). New analogues are also shown to be selective activators of NOD2 and NLRP3 (inflammasome) in vitro but not NOD1. Potencies of NOD2 and NLRP3 stimulation are found comparable with free MDP and other positive controls. Analogues are also demonstrated to be effective in stimulating cellular proliferation when the sera from mice are injected sc with individual liposome-loaded analogues, causing proliferation of bone marrow-derived GM-progenitors cells. Importantly, vaccination nanoparticles prepared from metallochelation liposomes, His-tagged antigen rOspA from Borrelia burgdorferi, and lipophilic analogue norAbuMDP-Lys(B30) as adjuvant, are shown to provoke OspA-specific antibody responses with a strong Th1-bias (dominance of IgG2a response). In contrast, the adjuvant effects of Alum or parent MDP show a strong Th2-bias (dominance of IgG1 response).


Subject(s)
Acetylmuramyl-Alanyl-Isoglutamine/analogs & derivatives , Acetylmuramyl-Alanyl-Isoglutamine/pharmacology , Adjuvants, Immunologic/pharmacology , Antigens, Surface/pharmacology , Bacterial Outer Membrane Proteins/pharmacology , Bacterial Vaccines/pharmacology , Borrelia burgdorferi/immunology , Lipoproteins/pharmacology , Acetylmuramyl-Alanyl-Isoglutamine/chemistry , Acetylmuramyl-Alanyl-Isoglutamine/immunology , Adjuvants, Immunologic/chemistry , Animals , Antibody Formation , Antigens, Surface/chemistry , Antigens, Surface/immunology , Bacterial Outer Membrane Proteins/chemistry , Bacterial Outer Membrane Proteins/immunology , Bacterial Vaccines/chemistry , Bacterial Vaccines/immunology , Female , HEK293 Cells , Humans , Immunization , Lipoproteins/chemistry , Lipoproteins/immunology , Lyme Disease/immunology , Lyme Disease/microbiology , Mice , Mice, Inbred BALB C , NLR Family, Pyrin Domain-Containing 3 Protein/agonists , NLR Family, Pyrin Domain-Containing 3 Protein/immunology , RAW 264.7 Cells
14.
J Control Release ; 249: 183-195, 2017 03 10.
Article in English | MEDLINE | ID: mdl-27469472

ABSTRACT

Nanofibre-based mucoadhesive films were invented for oromucosal administration of nanocarriers used for delivery of drugs and vaccines. The mucoadhesive film consists of an electrospun nanofibrous reservoir layer, a mucoadhesive film layer and a protective backing layer. The mucoadhesive layer is responsible for tight adhesion of the whole system to the oral mucosa after application. The electrospun nanofibrous reservoir layer is intended to act as a reservoir for polymeric and lipid-based nanoparticles, liposomes, virosomes, virus-like particles, dendrimers and the like, plus macromolecular drugs, antigens and/or allergens. The extremely large surface area of nanofibrous reservoir layers allows high levels of nanoparticle loading. Nanoparticles can either be reversibly adsorbed to the surface of nanofibres or they can be deposited in the pores between the nanofibres. After mucosal application, nanofibrous reservoir layers are intended to promote prolonged release of nanoparticles into the submucosal tissue. Reversible adsorption of model nanoparticles as well as sufficient mucoadhesive properties were demonstrated. This novel system appears appropriate for the use in oral mucosa, especially for sublingual and buccal tissues. To prove this concept, trans-/intramucosal and lymph-node delivery of PLGA-PEG nanoparticles was demonstrated in a porcine model. This system can mainly be used for sublingual immunization and the development of "printed vaccine technology".


Subject(s)
Drug Delivery Systems/methods , Nanofibers/chemistry , Pharmaceutical Preparations/administration & dosage , Vaccines/administration & dosage , Adhesives/chemistry , Administration, Buccal , Administration, Sublingual , Animals , Liposomes/chemistry , Lymph Nodes/metabolism , Mice , Mouth Mucosa/metabolism , Nanoparticles/chemistry , Polyethylene Glycols/chemistry , Polyglactin 910/chemistry , Swine , Vaccination/methods
15.
Cancer Immunol Immunother ; 66(2): 149-159, 2017 Feb.
Article in English | MEDLINE | ID: mdl-27864613

ABSTRACT

The natural adjuvant properties of bacterial ghosts (BGs) lie within the presence of intact pathogen-associated molecular patterns on their surface. BGs can improve the direct delivery, natural processing and presentation of target antigens within dendritic cells (DCs). Moreover, sensitization of human DCs by cancer cell lysate (oncolysate)-loaded BGs in the presence of IFN-α and GM-CSF enhanced DC maturation as indicated by an increased expression of maturation markers and co-stimulatory molecules, higher production of IL-12p70 and stimulation of significantly increased proliferation of both autologous CD4+ and CD8+ T cells compared to DCs matured in the presence of purified lipopolysaccharide. The induced T cells efficiently recognized oncolysate-derived tumor-associated antigens expressed by cancer cells used for the production of oncolysate. Our optimized one-step simultaneous antigen delivery and DC maturation-inducing method emerges as a promising tool for the development and implementation of next-generation cellular cancer immunotherapies.


Subject(s)
Dendritic Cells/immunology , Escherichia coli/immunology , Immunotherapy, Adoptive/methods , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Cell Differentiation/immunology , Cell Line, Tumor , Dendritic Cells/microbiology , Dendritic Cells/transplantation , Glioblastoma/immunology , Glioblastoma/therapy , Humans , Interleukin-12/biosynthesis , Interleukin-12/immunology , Lipopolysaccharides/pharmacology , Phenotype
16.
PLoS One ; 11(2): e0148497, 2016.
Article in English | MEDLINE | ID: mdl-26848589

ABSTRACT

Lyme disease, Borrelia burgdorferi-caused infection, if not recognized and appropriately treated by antibiotics, may lead to chronic complications, thus stressing the need for protective vaccine development. The immune protection is mediated by phagocytic cells and by Borrelia-specific complement-activating antibodies, associated with the Th1 immune response. Surface antigen OspC is involved in Borrelia spreading through the host body. Previously we reported that recombinant histidine tagged (His-tag) OspC (rOspC) could be attached onto liposome surfaces by metallochelation. Here we report that levels of OspC-specific antibodies vary substantially depending upon whether rOspC possesses an N' or C' terminal His-tag. This is the case in mice immunized: (a) with rOspC proteoliposomes containing adjuvants MPLA or non-pyrogenic MDP analogue MT06; (b) with free rOspC and Montanide PET GEL A; (c) with free rOspC and alum; or (d) with adjuvant-free rOspC. Stronger responses are noted with all N'-terminal His-tag rOspC formulations. OspC-specific Th1-type antibodies predominate post-immunization with rOspC proteoliposomes formulated with MPLA or MT06 adjuvants. Further analyses confirmed that the structural features of soluble N' and C' terminal His-tag rOspC and respective rOspC proteoliposomes are similar including their thermal stabilities at physiological temperatures. On the other hand, a change in the position of the rOspC His-tag from N' to C' terminal appears to affect substantially the immunogenicity of rOspC arguably due to steric hindrance of OspC epitopes by the C' terminal His-tag itself and not due to differences in overall conformations induced by changes in the His-tag position in rOspC variants.


Subject(s)
Adjuvants, Immunologic , Antibodies, Bacterial/immunology , Antibody Formation/immunology , Antibody Specificity/immunology , Antigens, Bacterial/immunology , Bacterial Outer Membrane Proteins/immunology , Borrelia burgdorferi/immunology , Recombinant Fusion Proteins/immunology , Animals , Antigens, Bacterial/administration & dosage , Antigens, Bacterial/chemistry , Bacterial Outer Membrane Proteins/administration & dosage , Bacterial Outer Membrane Proteins/chemistry , Enzyme-Linked Immunosorbent Assay , Immunization , Lyme Disease/immunology , Mice , Models, Animal , Protein Stability , Protein Structure, Secondary , Proteolipids , Recombinant Fusion Proteins/administration & dosage , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/isolation & purification
17.
J Control Release ; 227: 45-57, 2016 Apr 10.
Article in English | MEDLINE | ID: mdl-26876783

ABSTRACT

Several plasminogen activators (PAs) have been found effective in treating different thromboembolic diseases. However, administration of conventional thrombolytic therapy is limited by a low efficacy of present formulations of PAs. Conventional treatments using these therapeutic proteins are associated with several limitations including rapid inactivation and clearance, short half-life, bleeding complications or non-specific tissue targeting. Liposome-based formulations of PAs such as streptokinase, tissue-plasminogen activator and urokinase have been developed to improve the therapeutic efficacy of these proteins. Resulting liposomal formulations were found to preserve the original activity of PAs, promote their selective delivery and improve thrombus targeting. Therapeutic potential of these liposome-based PAs has been demonstrated successfully in various pre-clinical models in vivo. Reductions in unwanted side effects (e.g., hemorrhage or immunogenicity) as well as enhancements of efficacy and safety were achieved in comparison to currently existing treatment options based on conventional formulations of PAs. This review summarizes present achievements in: (i) preparation of liposome-based formulations of various PAs, (ii) development of PEGylated and targeted liposomal PAs, (iii) physico-chemical characterization of these developed systems, and (iv) testing of their thrombolytic efficacy. We also look to the future and the imminent arrival of theranostic liposomal formulations to move this field forward.


Subject(s)
Fibrinolysin/administration & dosage , Fibrinolytic Agents/administration & dosage , Liposomes/chemistry , Metalloendopeptidases/administration & dosage , Streptokinase/administration & dosage , Tissue Plasminogen Activator/administration & dosage , Urokinase-Type Plasminogen Activator/administration & dosage , Animals , Fibrinolysin/therapeutic use , Fibrinolytic Agents/therapeutic use , Humans , Liposomes/ultrastructure , Metalloendopeptidases/therapeutic use , Nanostructures/chemistry , Nanostructures/ultrastructure , Streptokinase/therapeutic use , Thromboembolism/drug therapy , Thrombolytic Therapy/methods , Tissue Plasminogen Activator/therapeutic use , Urokinase-Type Plasminogen Activator/therapeutic use
18.
Vet Microbiol ; 184: 84-93, 2016 Feb 29.
Article in English | MEDLINE | ID: mdl-26854349

ABSTRACT

Pseudorabies virus (PrV), a causative agent of Aujeszky's disease, is deadly to most mammals with the exception of higher primates and men. This disease causes serious economic loses among farm animals, especially pigs, yet many European countries are today claimed to be Aujeszky's disease free because of the discovery of an efficient vaccination for pigs. In reality, the virus is still present in wild boar. Current vaccines are neither suitable for dogs nor are there anti-PrV drugs approved for veterinary use. Therefore, the disease still represents a high threat, particularly for expensive hunting dogs that can come into close contact with infected boars. Here we report on the anti-PrV activities of a series of synthetic diaminopurine-based acyclic nucleoside phosphonate (DAP-ANP) analogues. Initially, all synthetic DAP-ANPs under investigation are shown to exhibit minimal cytotoxicity by MTT and XTT tests (1-100µM range). Thereafter in vitro infection models are established using PrV virus SuHV-1, optimized on PK-15 and RK-13 cell lines. Out of the six DAP-ANP analogues tested, analogue VI functionalized with a cyclopropyl group on the 6-amino position of the purine ring proves the most effective antiviral DAP-ANP analogue against PrV infection, aided by sufficient hydrophobic character to enhance bioavailability to its cellular target viral DNA-polymerase. Four other DAP-ANP analogues with functional groups introduced to the C2'position are shown ineffective against PrV infection, even with favourable hydrophobic properties. Cidofovir(®), a drug approved against various herpesvirus infections, is found to exert only low activity against PrV in these same in vitro models.


Subject(s)
Antiviral Agents/pharmacology , Herpesviridae/drug effects , Organophosphonates/pharmacology , 2-Aminopurine/analogs & derivatives , 2-Aminopurine/chemistry , 2-Aminopurine/pharmacology , Animals , Antiviral Agents/chemistry , Cell Line , DNA Replication/drug effects , Dogs , In Vitro Techniques , Madin Darby Canine Kidney Cells , Microscopy, Electron, Transmission , Organophosphonates/chemistry , Pseudorabies/drug therapy , Vero Cells
19.
J Control Release ; 207: 59-69, 2015 Jun 10.
Article in English | MEDLINE | ID: mdl-25861728

ABSTRACT

Pro-apoptotic analogues of vitamin E (VE) exert selective anti-cancer effect on various animal cancer models. Neither suitable formulation of α-tocopheryl succinate (α-TOS), representative semi-synthetic VE analogue ester, nor suitable formulations of the other VE analogues for clinical application have been reported yet. The major factor limiting the use of VE analogues is their low solubility in aqueous solvents. Due to the hydrophobic character of VE analogues, liposomes are predetermined as suitable delivery system. Liposomal formulation prevents undesirable side effects of the drug, enhances the drug biocompatibility, and improves the drug therapeutic index. Liposomal formulations of VE analogues especially of α-TOS and α-tocopheryl ether linked acetic acid (α-TEA) have been developed. The anti-cancer effect of these liposomal VE analogues has been successfully demonstrated in pre-clinical models in vivo. Present achievements in: (i) preparation of liposomal formulations of VE analogues, (ii) physico-chemical characterization of these developed systems and (iii) testing of their biological activity such as induction of apoptosis and evaluation of anti-cancer effect are discussed in this review.


Subject(s)
Antineoplastic Agents/administration & dosage , Drug Delivery Systems/methods , Lipids/chemistry , Neoplasms/drug therapy , Vitamin E/administration & dosage , Animals , Antineoplastic Agents/chemistry , Apoptosis/drug effects , Chemistry, Pharmaceutical , Humans , Liposomes , Neoplasms/pathology , Solubility , Vitamin E/analogs & derivatives , Vitamin E/chemistry , alpha-Tocopherol/administration & dosage
20.
Pharm Res ; 32(4): 1186-99, 2015 Apr.
Article in English | MEDLINE | ID: mdl-25630814

ABSTRACT

PURPOSE: The aim of this work was to demonstrate an immunostimulatory and adjuvant effect of new apyrogenic lipophilic derivatives of norAbuMDP and norAbuGMDP formulated in nanoliposomes. METHODS: Nanoliposomes and metallochelating nanoliposomes were prepared by lipid film hydration and extrusion methods. The structure of the liposomal formulation was studied by electron microscopy, AF microscopy, and dynamic light scattering. Sublethal and lethal γ-irradiation mice models were used to demonstrate stimulation of innate immune system. Recombinant Hsp90 antigen (Candida albicans) bound onto metallochelating nanoliposomes was used for immunisation of mice to demonstrate adjuvant activities of tested compounds. RESULTS: Safety and stimulation of innate and adaptive immunity were demonstrated on rabbits and mice. The liposomal formulation of norAbuMDP/GMDP was apyrogenic in rabbit test and lacking any side effect in vivo. Recovery of bone marrow after sublethal γ-irradiation as well as increased survival of mice after lethal irradiation was demonstrated. Enhancement of specific immune response was demonstrated for some derivatives incorporated in metallochelating nanoliposomes with recombinant Hsp90 protein antigen. CONCLUSIONS: Liposomal formulations of new lipophilic derivatives of norAbuMDP/GMDP proved themselves as promising adjuvants for recombinant vaccines as well as immunomodulators for stimulation of innate immunity and bone-marrow recovery after chemo/radio therapy of cancer.


Subject(s)
Acetylmuramyl-Alanyl-Isoglutamine/analogs & derivatives , Adaptive Immunity/drug effects , Adjuvants, Immunologic/pharmacology , Drug Carriers/chemistry , Immunity, Innate/drug effects , Acetylmuramyl-Alanyl-Isoglutamine/administration & dosage , Acetylmuramyl-Alanyl-Isoglutamine/chemistry , Acetylmuramyl-Alanyl-Isoglutamine/pharmacology , Acetylmuramyl-Alanyl-Isoglutamine/therapeutic use , Adjuvants, Immunologic/administration & dosage , Adjuvants, Immunologic/chemistry , Adjuvants, Immunologic/therapeutic use , Animals , Antibodies, Fungal/blood , Antigens, Fungal/immunology , Female , HSP90 Heat-Shock Proteins/immunology , Liposomes , Mice , Mice, Inbred ICR , Microscopy, Atomic Force , Microscopy, Electron, Scanning , Microscopy, Electron, Transmission , Molecular Structure , Nanoparticles , Rabbits , Radiation Injuries, Experimental/immunology , Radiation Injuries, Experimental/prevention & control , Recombinant Proteins/immunology , Survival Analysis
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