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1.
Anal Chem ; 90(15): 9457-9464, 2018 08 07.
Article in English | MEDLINE | ID: mdl-29976062

ABSTRACT

Phosphorylated compounds and organic acids with multiple carboxylate groups are commonly observed to have poor peak shapes and signal in LC/MS experiments. The poor peak shape is caused by the presence of trace metals, particularly iron, contributed from a variety of sources within the chromatographic system. To ameliorate this problem, different solvent additives were investigated to reduce the amount of metal in the flow path to achieve better analytical performance for these metal-sensitive compounds. Here, we introduce the use of a solvent additive that can significantly improve the peak shapes and signal of metal-sensitive metabolites for LC/MS analysis. Moreover, the additive is shown to be amenable for other metal-sensitive applications, such as the analysis of phosphopeptides and polar phosphorylated pesticides, where the instruments could be used in either positive or negative analysis mode.

2.
Sci Rep ; 6: 24023, 2016 Apr 18.
Article in English | MEDLINE | ID: mdl-27087367

ABSTRACT

Sexually dimorphic gene expression is commonly found in the liver, and many of these genes are linked to different incidences of liver diseases between sexes. However, the mechanism of sexually dimorphic expression is still not fully understood. In this study, a pCAG-eGFP transgenic mouse strain with a specific transgene integration site in the Akr1A1 locus presented male-biased EGFP expression in the liver, and the expression was activated by testosterone during puberty. The integration of the pCAG-eGFP transgene altered the epigenetic regulation of the adjacent chromatin, including increased binding of STAT5b, a sexually dimorphic expression regulator, and the transformation of DNA methylation from hypermethylation into male-biased hypomethylation. Through this de novo sexually dimorphic expression of the transgene, the Akr1A1(eGFP) mouse provides a useful model to study the mechanisms and the dynamic changes of sexually dimorphic gene expression during either development or pathogenesis of the liver.


Subject(s)
Epigenesis, Genetic , Green Fluorescent Proteins/metabolism , Liver/metabolism , Transgenes , Animals , CpG Islands , Cytomegalovirus/genetics , DNA Methylation , Estradiol/pharmacology , Female , Gene Expression Regulation, Developmental , Green Fluorescent Proteins/genetics , Locus Control Region , Male , Mice, Transgenic , Non-alcoholic Fatty Liver Disease/metabolism , Promoter Regions, Genetic , RNA, Messenger/metabolism , Sex Characteristics , Testosterone/pharmacology
3.
J Control Release ; 220(Pt B): 608-16, 2015 Dec 28.
Article in English | MEDLINE | ID: mdl-26394062

ABSTRACT

This work presents a new concept in hybrid hydrogel design. Synthetic water-soluble N-(2-hydroxypropyl)methacrylamide (HPMA) polymers grafted with multiple peptide nucleic acids (PNAs) are crosslinked upon addition of the linker DNA. The self-assembly is mediated by the PNA-DNA complexation, which results in the formation of hydrophilic polymer networks. We show that the hydrogels can be produced through two different types of complexations. Type I hydrogel is formed via the PNA/DNA double-helix hybridization. Type II hydrogel utilizes a unique "P-form" oligonucleotide triple-helix that comprises two PNA sequences and one DNA. Microrheology studies confirm the respective gelation processes and disclose a higher critical gelation concentration for the type I gel when compared to the type II design. Scanning electron microscopy reveals the interconnected microporous structure of both types of hydrogels. Type I double-helix hydrogel exhibits larger pore sizes than type II triple-helix gel. The latter apparently contains denser structure and displays greater elasticity as well. The designed hybrid hydrogels have potential as novel biomaterials for pharmaceutical and biomedical applications.


Subject(s)
Acrylamides/chemistry , DNA/chemistry , Gene Transfer Techniques , Peptide Nucleic Acids/chemistry , Acrylamides/metabolism , DNA/metabolism , DNA/ultrastructure , Elasticity , Hydrogels , Hydrophobic and Hydrophilic Interactions , Microscopy, Atomic Force , Nucleic Acid Conformation , Peptide Nucleic Acids/metabolism , Peptide Nucleic Acids/ultrastructure , Porosity , Protein Conformation , Rheology
4.
Biomater Sci ; 3(7): 908-22, 2015 Jul.
Article in English | MEDLINE | ID: mdl-26191406

ABSTRACT

This review highlights a unique research area in polymer-based nanomedicine designs. Drug-free macromolecular therapeutics induce apoptosis of malignant cells by the crosslinking of surface non-internalizing receptors. The receptor crosslinking is mediated by the biorecognition of high-fidelity natural binding motifs (such as antiparallel coiled-coil peptides or complementary oligonucleotides) that are grafted to the side chains of polymers or attached to targeting moieties against cell receptors. This approach features the absence of low-molecular-weight cytotoxic compounds. Here, we summarize the rationales, different designs, and advantages of drug-free macromolecular therapeutics. Recent developments of novel therapeutic systems for B-cell lymphomas are discussed, as well as relevant approaches for other diseases. We conclude by pointing out various potential future directions in this exciting new field.


Subject(s)
Drug Carriers/chemistry , Drug Carriers/therapeutic use , Drug Delivery Systems/methods , Lymphoma, B-Cell/chemistry , Macromolecular Substances/chemistry , Polymers/chemistry , Apoptosis , Drug Design , Humans , Lymphoma, B-Cell/therapy , Macromolecular Substances/metabolism , Nanomedicine/methods , Neoplasms/drug therapy
5.
Chembiochem ; 16(12): 1725-9, 2015 Aug 17.
Article in English | MEDLINE | ID: mdl-26097072

ABSTRACT

Super-resolution imaging was used to quantify organizational changes in the plasma membrane after treatment with hybrid nanoconjugates. The nanoconjugates crosslinked CD20 on the surface of malignant B cells, thereby inducing apoptosis. Super-resolution images were analyzed by using pair-correlation analysis to determine cluster size and to count the average number of molecules in the clusters. The role of lipid rafts was investigated by pre-treating cells with a cholesterol chelator and actin destabilizer to prevent lipid raft formation. Lipid raft cluster size correlated with apoptosis induction after treatment with the nanoconjugates. Lipid raft clusters had radii of ∼ 200 nm in cells treated with the hybrid nanoconjugates. Super-resolution images provided precise molecule location coordinates that could be used to determine density of bound conjugates, cluster size, and number of molecules per cluster.


Subject(s)
Membrane Proteins/metabolism , Molecular Imaging , Nanoconjugates/chemistry , Apoptosis , Cell Membrane/metabolism , Humans , Models, Biological
6.
Theranostics ; 5(8): 834-46, 2015.
Article in English | MEDLINE | ID: mdl-26000056

ABSTRACT

The use of rituximab, an anti-CD20 mAb, in combination with chemotherapy is the current standard for the treatment of B-cell lymphomas. However, because of a significant number of treatment failures, there is a demand for new, improved therapeutics. Here, we designed a nanomedicine that crosslinks CD20 and directly induces apoptosis of B-cells without the need for toxins or immune effector functions. The therapeutic system comprises a pretargeting component (anti-CD20 Fab' conjugated with an oligonucleotide1) and a crosslinking component (N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer grafted with multiple complementary oligonucleotide2). Consecutive treatment with the two components resulted in CD20 clustering on the cell surface and effectively killed malignant B-cells in vivo. To enhance therapeutic efficacy, a two-step pretargeting approach was employed. We showed that the time lag between the two doses can be optimized based on pharmacokinetics and biodistribution of the Fab'-oligonucleotide1 conjugate. In a mouse model of human non-Hodgkin lymphoma (NHL), increasing the time lag from 1 h to 5 h resulted in dramatically improved tumor growth inhibition and animal survival. When the 5 h interval was used, the nanotherapy was more efficacious than rituximab and led to complete eradication of lymphoma cells with no signs of metastasis or disease recurrence. We further evaluated the nanomedicine using patient mantle cell lymphoma cells; the treatment demonstrated more potent apoptosis-inducing activity than rituximab hyper-crosslinked with secondary antibodies. In summary, our approach may constitute a novel treatment for NHL and other B-cell malignancies with significant advantages over conventional chemo-immunotherapy.


Subject(s)
Antineoplastic Agents/therapeutic use , Cross-Linking Reagents/therapeutic use , Lymphoma, Non-Hodgkin/drug therapy , Nanomedicine/methods , Rituximab/therapeutic use , Theranostic Nanomedicine , Animals , Antineoplastic Agents/pharmacokinetics , Cross-Linking Reagents/pharmacokinetics , Disease Models, Animal , Female , Humans , Mice, SCID , Rituximab/pharmacokinetics , Survival Analysis , Treatment Outcome
7.
Adv Healthc Mater ; 4(7): 1054-65, 2015 May.
Article in English | MEDLINE | ID: mdl-25612325

ABSTRACT

Two complementary coiled-coil peptides CCE/CCK are used to develop a "drug free" therapeutic system, which can specifically kill cancer cells without a drug. CCE is attached to the Fab' fragment of anti-CD20 1F5 antibody (Fab'-CCE), and CCK is conjugated in multiple grafts to poly[N-(2-hydroxypropyl)methacrylamide] (P-(CCK)x ). Two conjugates are consecutively administered: First, Fab'-CCE coats peptide CCE at CD20 antigen of lymphoma cell surface; second, CCE/CCK biorecognition between Fab'-CCE and P-(CCK)x leads to coiled-coil formation, CD20 crosslinking, membrane reorganization, and ultimately cell apoptosis. To prove that two conjugates can assemble at cell surface, multiple fluorescence imaging studies are performed, including 2-channel FMT, 3D confocal microscopy, and 4-color FACS. Confocal microscopy shows colocalization of two fluorescently labeled conjugates on non-Hodgkin's lymphoma (NHL) Raji cell surface, indicating "two-step" targeting specificity. The fluorescent images also reveal that these two conjugates can disrupt normal membrane lipid distribution and form lipid raft clusters, leading to cancer cell apoptosis. This "two-step" biorecognition capacity is further demonstrated in a NHL xenograft model, using fluorescent images at whole-body, tissue and cell levels. It is also found that delaying injection of P-(CCK)x can significantly enhance targeting efficacy. This high-specificity therapeutics provide a safe option to treat NHL and other B cell malignancies.


Subject(s)
Immunoglobulin Fab Fragments/immunology , Lymphoma/drug therapy , Lymphoma/immunology , Peptides/immunology , Peptides/therapeutic use , Acrylamides/immunology , Animals , Antigens, CD20/immunology , Apoptosis/drug effects , Apoptosis/immunology , Cell Line, Tumor , Female , Fluorescence , Humans , Lipids/immunology , Membrane Lipids/immunology , Mice, Nude , Mice, SCID , Multimodal Imaging/methods
8.
Biomaterials ; 35(22): 5886-96, 2014 Jul.
Article in English | MEDLINE | ID: mdl-24767787

ABSTRACT

A two-component CD20 (non-internalizing) receptor crosslinking system based on the biorecognition of complementary coiled-coil forming peptides was evaluated. Exposure of B cells to Fab'-peptide1 conjugate decorates the cell surface with peptide1; further exposure of the decorated cells to P-(peptide2)x (P is the N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer backbone) results in the formation of coiled-coil heterodimers at the cell surface with concomitant induction of apoptosis. The aim of this study was to determine the potential immunogenicity of this therapeutic system that does not contain low molecular weight drugs. Enantiomeric peptides (L- and D-CCE and L- and D-CCK), HPMA copolymer-peptide conjugates, and Fab' fragment-peptide conjugates were synthesized and the immunological properties of peptide conjugates evaluated in vitro on RAW264.7 macrophages and in vivo on immunocompetent BALB/c mice. HPMA copolymer did not induce immune response in vitro and in vivo. Administration of P-peptide conjugates with strong adjuvant resulted in antibody response directed to the peptide. Fab' was responsible for macrophage activation of Fab'-peptide conjugates and a major factor in the antibody induction following i.v. administration of Fab'-conjugates. There was no substantial difference in the ability of conjugates of D-peptides and conjugates of L-peptides to induce Ab response.


Subject(s)
Acrylamides/immunology , Immunoglobulin Fab Fragments/immunology , Peptides/immunology , Acrylamides/administration & dosage , Acrylamides/chemistry , Amino Acid Sequence , Animals , Antibody Formation/drug effects , Cell Line , Immunoglobulin Fab Fragments/administration & dosage , Immunoglobulin Fab Fragments/chemistry , Macrophages/drug effects , Macrophages/immunology , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Peptides/administration & dosage , Peptides/chemistry , T-Lymphocytes/drug effects , T-Lymphocytes/immunology
9.
ACS Nano ; 8(1): 719-30, 2014 Jan 28.
Article in English | MEDLINE | ID: mdl-24308267

ABSTRACT

Hybrid nanomaterials composed of synthetic and biological building blocks possess high potential for the design of nanomedicines. The use of self-assembling nanomaterials as "bio-mimics" may trigger cellular events and result in new therapeutic effects. Motivated by this rationale, we designed a therapeutic platform that mimics the mechanism of immune effector cells to cross-link surface receptors of target cells and induce apoptosis. This platform was tested against B-cell lymphomas that highly express the surface antigen CD20. Here, two nanoconjugates were synthesized: (1) an anti-CD20 Fab' fragment covalently linked to a single-stranded morpholino oligonucleotide (MORF1), and (2) a linear polymer of N-(2-hydroxypropyl)methacrylamide (HPMA) grafted with multiple copies of the complementary oligonucleotide MORF2. We show that the two conjugates self-assemble via MORF1-MORF2 hybridization at the surface of CD20(+) malignant B-cells, which cross-links CD20 antigens and initiates apoptosis. When tested in a murine model of human non-Hodgkin's lymphoma, the two conjugates, either administered consecutively or as a premixture, eradicated cancer cells and produced long-term survivors. The designed therapeutics contains no small-molecule cytotoxic compounds and is immune-independent, aiming to improve over chemotherapy, radiotherapy and immunotherapy. This therapeutic platform can be applied to cross-link any noninternalizing receptor and potentially treat other diseases.


Subject(s)
Apoptosis , Morpholinos/chemistry , Nanoconjugates , Nucleic Acid Hybridization , Animals , Antigens, CD20/immunology , Cell Membrane/metabolism , Circular Dichroism , Drug Screening Assays, Antitumor , Female , Humans , Immunoglobulin Fragments/immunology , Lymphoma, B-Cell/immunology , Lymphoma, B-Cell/pathology , Methacrylates/chemistry , Mice , Mice, SCID
10.
Drug Deliv Transl Res ; 4(5-6): 389-94, 2014 Dec.
Article in English | MEDLINE | ID: mdl-25580376

ABSTRACT

A new drug-free nanotherapeutic approach for B-cell malignancies was developed. Exposure of B-cells to an anti-CD20 Fab'-morpholino oligonucleotide1 (MORF1) conjugate decorated the cell surface with MORF1; further exposure of the decorated cells to multivalent polymer-oligonucleotide2 conjugates (P-MORF2) resulted in CD20 clustering at the cell surface with induction of apoptosis. We evaluated this concept in chronic lymphocytic leukemia (CLL) cells isolated from 10 patients. Apoptosis and cytotoxicity were observed in eight samples, including 2 samples with the 17p13 deletion, which suggested a p53-independent mechanism of apoptosis induction. When compared to an anti-CD20 monoclonal antibody (mAb), the nanotherapeutic showed significantly more potent apoptosis-inducing activity and cytotoxicity. This was due to the multivalency effect (8 binding sites per polymer chain) of our design in comparison to the divalent mAb. In conclusion, we have developed a novel and potent therapeutic system against CLL and other B-cell malignancies with significant advantages over conventional chemo-immunotherapy.

11.
Biomaterials ; 33(29): 7174-81, 2012 Oct.
Article in English | MEDLINE | ID: mdl-22795544

ABSTRACT

A hybrid biomimetic system comprising high-molecular-weight, linear copolymer of N-(2-hydroxypropyl)methacrylamide (HPMA) grafted with multiple Fab' fragments of anti-CD20 monoclonal antibody (mAb) was synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization followed by attachment of Fab' fragments via thioether bonds. Exposure of human non-Hodgkin's lymphoma (NHL) Raji B cells to the multivalent conjugates resulted in crosslinking of CD20 receptors and commencement of apoptosis. Five conjugates with varying molecular weight and valence (amount of Fab' per polymer chain) were synthesized. One of the copolymers contained enzyme degradable peptide sequences (GFLG) in the backbone. The multivalency led to higher avidity and apoptosis induction compared to unconjugated whole mAb. Time-dependent studies showed that the cytotoxicity of conjugates exhibited a slower onset at shorter exposure times than mAb hyper-crosslinked with a secondary Ab; however, at longer time intervals the HPMA copolymer conjugates achieved significantly higher biological efficacies. In addition, study of the relationship between the structure of conjugates and Raji B cell apoptosis revealed that both valency and polymer molecular weight influenced biological activities, while insertion of peptide sequences into the backbone was not a factor in vitro.


Subject(s)
Antigens, CD20/chemistry , Apoptosis , Immunoglobulin Fab Fragments/chemistry , Lymphoma, Non-Hodgkin/immunology , Methacrylates/chemistry , Polymers/chemistry , Antibodies, Monoclonal, Murine-Derived/pharmacology , Antigens, CD20/immunology , Biomimetics , Caspase 3/metabolism , Cell Line, Tumor , Cell Survival , Dose-Response Relationship, Drug , Humans , Hybridomas/metabolism , Lymphoma, Non-Hodgkin/therapy , Microscopy, Confocal/methods , Models, Chemical , Rituximab , Time Factors
12.
Biometals ; 23(3): 543-54, 2010 Jun.
Article in English | MEDLINE | ID: mdl-20148305

ABSTRACT

In this study, Lactobacillus casei was used to deliver and express human lactoferrin (hLF) to protect the host against bacterial infection. Full-length hLF cDNA was cloned into a Lactobacillus-specific plasmid to produce the L. casei transformants (rhLF/L. casei). Antimicrobial activity of recombinant hLF was examined in inhibition of bacteria growth in vitro. A mouse model was established to test in vivo antibacterial activity and protective effect of orally-administered probiotic L. casei transformant in the gastrointestinal tract. Trials were conducted in which animals were challenged with E. coli ATCC25922. E. coli colony numbers in duodenal fluid from the group fed with rhLF/L. casei were significantly lower than those of the group fed with wild-type L. casei or placebo (P < 0.01). Histopathological analyses of the small intestine, showed both decreased intestinal injury and increased villi length were observed in the mice fed with rhLF/L. casei as compared with the control groups (P < 0.01). Our results demonstrate that L. casei expressing hLF exhibited antibacterial activity both in in vitro and in vivo. It also provides a potentially large-scale production of hLF as applications for treatment of infections caused by clinically relevant pathogens.


Subject(s)
Anti-Bacterial Agents/metabolism , Gastrointestinal Tract/microbiology , Lacticaseibacillus casei/genetics , Lacticaseibacillus casei/metabolism , Lactoferrin/metabolism , Probiotics/metabolism , Animals , Anti-Bacterial Agents/administration & dosage , Escherichia coli/growth & development , Gastrointestinal Tract/pathology , Humans , Lacticaseibacillus casei/cytology , Lactoferrin/biosynthesis , Lactoferrin/genetics , Mice , Mice, Inbred ICR , Microbial Sensitivity Tests , Recombinant Proteins/biosynthesis , Structure-Activity Relationship
13.
Vaccine ; 26(23): 2882-9, 2008 Jun 02.
Article in English | MEDLINE | ID: mdl-18450335

ABSTRACT

Enterovirus 71 (EV71) is the most common etiological agent detected in cases of hand-foot-and-mouth disease (HFMD) resulting in incidences of neurological complications and fatality in recent years. The clinical data have already shown the significant increase in recent EV71 epidemic activity throughout the Asia-Pacific region. Due to the lack of an effective antiviral agent, primary prevention of the disease, including the development of an effective vaccine, has been the top priority in terms of control strategies. In this study, we first generated a transgenic animal system to produce the EV71 VP1 capsid protein under the control of alpha-lactalbumin promoter and alpha-casein leader sequences. A high level of recombinant VP1 protein (2.51 mg/ml) was expressed and secreted into the milk of transgenic mice. Mouse pups that received VP1-transgenic milk orally demonstrated relatively better health conditions after challenge with the respective virus as compared with the non-transgenic milk fed group; moreover, the mice fed with the VP1-milk had body weights similar to those of the PBS placebo control groups. According to the serum-neutralization assay and serum antibody detection, the littermates suckling VP1-milk generated antibodies specific to EV71. Our data suggest that EV71 VP1-containing milk is suitable for development as a potential oral vaccine.


Subject(s)
Enterovirus A, Human/immunology , Enterovirus Infections/immunology , Enterovirus Infections/prevention & control , Milk/chemistry , Viral Vaccines/therapeutic use , Administration, Oral , Aging/immunology , Animals , Antibodies, Viral/analysis , Antibodies, Viral/biosynthesis , Capsid Proteins/genetics , Capsid Proteins/immunology , Child, Preschool , DNA, Complementary/biosynthesis , DNA, Complementary/genetics , Enzyme-Linked Immunosorbent Assay , Female , Genotype , Humans , Immunoblotting , Lactalbumin/genetics , Mice , Mice, Inbred ICR , Mice, Transgenic , Neutralization Tests , Promoter Regions, Genetic/genetics , Viral Fusion Proteins/genetics , Viral Vaccines/administration & dosage , Viral Vaccines/genetics
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