ABSTRACT
The design of functional polymers coupled with stimuli-triggered drug release mechanisms is a promising achievement to overcome various biological barriers. pH trigger methods yield significant potential for controlled targeting and release of therapeutics due to their simplicity and relevance, especially upon cell internalization. Here, we introduce reactive polymers that conjugate primary or secondary amines and release potential drugs under acidic conditions. For that purpose, we introduced methacrylamide-based monomers with pendant 2-propionic-3-methylmaleic anhydride groups. Such groups allow the conjugation of primary and secondary amines but are resistant to radical polymerization conditions. We, therefore, polymerized 2-propionic-3-methylmaleic anhydride amide-based methacrylates via reversible addition-fragmentation chain transfer (RAFT) polymerization. Their amine-reactive anhydrides could sequentially be derivatized by primary or secondary amines into hydrophilic polymers. Acidic pH-triggered drug release from the polymeric systems was fine-tuned by comparing different amines. Thereby, the conjugation of primary amines led to the formation of irreversible imide bonds in dimethyl sulfoxide, while secondary amines could quantitatively be released upon acidification. In vitro, this installed pH-responsiveness can contribute to an effective release of conjugated immune stimulatory drugs under endosomal pH conditions. Interestingly, the amine-modified polymers generally showed no toxicity and a high cellular uptake. Furthermore, secondary amine-modified immune stimulatory drugs conjugated to the polymers yielded better receptor activity and immune cell maturation than their primary amine derivatives due to their pH-sensitive drug release mechanism. Consequently, 2-propionic-3-methylmaleic anhydride-based polymers can be considered as a versatile platform for pH-triggered delivery of various (immuno)drugs, thus enabling new strategies in macromolecule-assisted immunotherapy.
Subject(s)
Citraconic Anhydrides , Polymers , Polymers/chemistry , Amines/chemistry , Anhydrides , Hydrogen-Ion ConcentrationABSTRACT
We introduce a pH-sensitive amide bond, inspired by citraconic anhydride, for the reversible conjugation of polymers to the lysine residues of proteins and antibodies. The pH sensitivity arises from a conformation lock at the end of the polymer, which we introduce by means of a Diels-Alder reaction, that positions a carboxylic acid close to the amide after conjugation occurs. The amide is stable over weeks at pH 7.4 but sensitive to hydrolysis at pH 5.5 and below, returning the amine to its original state. The pH sensitivity can be tuned by positioning secondary amide groups nearby. We use this approach to PEGylate an antibody to human serum albumin at high dilution and demonstrate successful recovery of the activity after hydrolysis at pH 5.5. These results offer a convenient and traceless approach to protein and antibody functionalization.
Subject(s)
Citraconic Anhydrides , Polymers , Humans , Citraconic Anhydrides/chemistry , Hydrogen-Ion Concentration , Chemical Phenomena , Antibodies , AmidesABSTRACT
The efficacy of cancer chemotherapy can be generally restrained by the multiple drug resistance (MDR) of tumors, which is typically attributed to the upregulation of ATP-binding cassette (ABC) transporter proteins, such as P-glycoprotein (P-gp). There is an urgent need to present innovative strategies to reverse MDR and enhance the therapeutic efficacy of chemotherapeutic agents in biomedical applications. Here, we report a novel nanosystem of charge-reversal-functionalized PLGA nanobubbles (denoted as Dox-NBs/PPP/P-gp shRNA) for the co-delivery of Dox and P-gp shRNA for the reversal of drug resistance and for ultrasonic-imaging-guided tumor therapy. 1H NMR, FT-IR, SEM, DLS, and UV-vis spectroscopy characterizations were conducted to determine the structure, morphology, and composition of the as-prepared nanobubbles. Dox-NBs/PPP/P-gp shRNA exhibited an average diameter of about 300 nm with good dispersity, biocompatibility, and pH-responsive release properties through the charge-reversal process. The in vitro experiments showed that Dox-NBs/PPP/P-gp shRNA nanobubbles could co-deliver Dox and P-gp shRNA into tumor cells and could effectively suppress P-gp expression, leading to enhanced overall therapeutic effects against MCF-7/MDR cells by restraining the drug efflux. An in vivo antitumor assay revealed an approximately 65% inhibition rate of Dox-NBs/PPP/P-gp shRNA against MCF-7/ADR tumors in tumor-bearing nude mice. Both the in vitro and in vivo toxicity results indicated the Dox-NBs/PPP/P-gp shRNA are highly biocompatible with reducing side-effects and have negligible systemic toxicity in the in vivo therapy of resistant cancers by combining with a chemotherapeutic agent and P-gp knockdown. Furthermore, the in vivo imaging data substantiated that the functionalized nanobubbles could be used as an efficient contrast agent for the ultrasonic imaging of solid tumors. This works highlights the great potential of Dox-NBs/PPP/P-gp shRNA nanobubbles for enhanced imaging-guided combination therapy for overcoming MDR.
Subject(s)
Antineoplastic Agents/administration & dosage , Doxorubicin/administration & dosage , Nanostructures/administration & dosage , RNA, Small Interfering/administration & dosage , ATP Binding Cassette Transporter, Subfamily B/genetics , Animals , Antineoplastic Agents/chemistry , Cell Cycle/drug effects , Cell Survival/drug effects , Citraconic Anhydrides/administration & dosage , Citraconic Anhydrides/chemistry , Combined Modality Therapy , Doxorubicin/chemistry , Drug Resistance, Neoplasm/drug effects , Female , Humans , MCF-7 Cells , Mice, Inbred BALB C , Mice, Nude , Nanostructures/chemistry , Neoplasms/drug therapy , Neoplasms/pathology , Polymers/administration & dosage , Polymers/chemistry , RNA, Small Interfering/chemistry , Theranostic Nanomedicine , Tumor Burden/drug effects , UltrasonographyABSTRACT
BACKGROUND: Anticancer drug-delivery systems (DDSs) capable of responding to the physiological stimuli and efficiently releasing drugs inside tumor cells are highly desirable for effective cancer therapy. Herein, pH-responsive, charge-reversal poly(allylamine hydrochlorid)-citraconic anhydride (PAH-cit) functionalized boron nitride nanospheres (BNNS) were fabricated and used as a carrier for the delivery and controlled release of doxorubicin (DOX) into cancer cells. METHODS: BNNS was synthesized through a chemical vapor deposition method and then functionalized with synthesized charge-reversal PAH-cit polymer. DOX@PAH-cit-BNNS complexes were prepared via step-by-step electrostatic interactions and were fully characterized. The cellular uptake of DOX@PAH-cit-BNNS complexes and DOX release inside cancer cells were visualized by confocal laser scanning microscopy. The in vitro anticancer activity of DOX@ PAH-cit-BNNS was examined using CCK-8 and live/dead viability/cytotoxicity assay. RESULTS: The PAH-cit-BNNS complexes were nontoxic to normal and cancer cells up to a concentration of 100 µg/mL. DOX was loaded on PAH-cit-BNNS complexes with high efficiency. In a neutral environment, the DOX@PAH-cit-BNNS was stable, whereas the loaded DOX was effectively released from these complexes at low pH condition due to amide hydrolysis of PAH-cit. Enhanced cellular uptake of DOX@PAH-cit-BNNS complexes and DOX release in the nucleus of cancer cells were revealed by confocal microscopy. Additionally, the effective delivery and release of DOX into the nucleus of cancer cells led to high therapeutic efficiency. CONCLUSION: Our findings indicated that the newly developed PAH-cit-BNNS complexes are promising as an efficient pH-responsive DDS for cancer therapy.
Subject(s)
Antibiotics, Antineoplastic/administration & dosage , Doxorubicin/administration & dosage , Drug Delivery Systems/methods , Nanospheres/administration & dosage , Antibiotics, Antineoplastic/chemistry , Antibiotics, Antineoplastic/pharmacokinetics , Boron Compounds/chemistry , Citraconic Anhydrides/chemistry , Doxorubicin/chemistry , Doxorubicin/pharmacokinetics , Drug Liberation , HeLa Cells , Humans , Hydrogen-Ion Concentration , Hydrolysis , MCF-7 Cells , Nanospheres/chemistry , Polyamines/chemistry , Polymers/chemistry , Static ElectricityABSTRACT
The design of highly efficient drug carriers, and the development of appropriate techniques to monitor their mechanism of action and therapeutic effect, are both critical for improving chemotherapy. Herein, a polymeric nanoparticle, PAH-Cit/DOX (poly(allylamine)-citraconic anhydride/doxorubicin), was synthesized and used as a nanodrug system for the efficient delivery and pH-responsive release of doxorubicin (DOX) into cancer cells. The PAH-Cit/DOX nanoparticles were stable at physiological pH but effectively released DOX under weakly acidic conditions. The release efficiency was 90.6% after 60 h of dialysis in phosphate-buffered saline at pH 5.5. Confocal images showed the rapid movement of the drug from the cytoplasm to the nucleus, indicating the effective drug release MCF-7 cells. Notably, the combination of fluorescence lifetime imaging microscopy (FLIM) and phasor analysis (phasor-FLIM) provides an approach to monitor the dynamic change of DOX fluorescence lifetime in intercellular environments. Phasor-differentiated lifetime pixel intensity in FLIM images was quantified and used to evaluate the DOX release from nanocarriers, making it possible to detect the dynamics of intracellular release and transport of DOX.
Subject(s)
Antibiotics, Antineoplastic/pharmacology , Citraconic Anhydrides/chemistry , Doxorubicin/pharmacology , Drug Carriers/chemistry , Nanoparticles/chemistry , Polyamines/chemistry , Polymers/chemistry , Polypropylenes/chemistry , Antibiotics, Antineoplastic/chemistry , Doxorubicin/chemistry , Drug Carriers/chemical synthesis , Drug Carriers/toxicity , Drug Liberation , Humans , Hydrogen-Ion Concentration , MCF-7 Cells , Microscopy, Confocal , Microscopy, Fluorescence , Nanoparticles/toxicity , Polymers/chemical synthesis , Polymers/toxicity , Polypropylenes/chemical synthesis , Polypropylenes/toxicityABSTRACT
Increasing attention has been paid to nasal delivery. Subunit vaccines based on antigenic proteins or polypeptides offer good safety. However, lack of delivery efficiency, particularly for nasal immunization, is a big issue. Here we designed a highly tunable polymeric hybrid micelle (PHM) system offering good vaccine efficacy after nasal administration. PHMs are formulated from two amphiphilic diblock copolymers, polycaprolactone-polyethylenimine (PCL-PEI) and polycaprolactone-polyethyleneglycol (PCL-PEG), the ratio of which determines PHM physicochemical properties. Citraconic anhydride-modified ovalbumin (Cit-OVA), as model antigen, was incorporated into PHMs via electrostatic interaction, giving antigen-loaded micelles of around 150nm in size. Their surface characteristics which are found closely related to their in vivo kinetics can be modulated by adjusting the mass ratio of PCL-PEG and PCL-PEI. PHM/Cit-OVA complexes containing PCL-PEI and PCL-PEG in a 1:1 mass ratio induced strong immune responses in nasal mucosa and serum in vivo without causing obvious toxicity, and Cit-OVA was efficiently internalized by dendritic cells. These results demonstrate the promise of this multifunctional polymeric delivery system for nasal vaccination.
Subject(s)
Antigens/administration & dosage , Citraconic Anhydrides/administration & dosage , Micelles , Ovalbumin/administration & dosage , Polymers/administration & dosage , Administration, Intranasal , Animals , Antigens/chemistry , Cell Line , Citraconic Anhydrides/chemistry , Dendritic Cells/drug effects , Dogs , Female , Immunoglobulin A/immunology , Immunoglobulin G/immunology , Lymph Nodes/cytology , Madin Darby Canine Kidney Cells , Mice, Inbred BALB C , Mucous Membrane/immunology , Ovalbumin/chemistry , Polymers/chemistryABSTRACT
A boronate ester crosslinked zwitterionic nanogel (NGCA) with ATP/pH-sensitivity has been developed with an inverse nanoprecipitation technique to achieve a two-stage charge conversion that responds to tumor extracellular conditions (pH 6.5-6.8) and an intracellular acidic environment (pH 5-6). Cationic cytochrome C (CC), a therapeutic protein, has been encapsulated into NGCA through inverse nanoprecipitation via electrostatic interactions to form protein-loaded nanogel (NGCA-CC). By adjusting the ratio of the amino and carboxyl groups in the nanogels, negatively charged nanogels that are safer under physiological conditions (pH 7.4) can convert their surface charge to positive at tumor extracellular pH, which enhance their cellular uptake efficiency. The citraconic amide formed from citraconic anhydride and amine can be cleaved in the intracellular acidic organelles to expose more amino groups and facilitate endosomal escape. The release of CC is accelerated in the presence of 5 mM ATP or under acidic conditions. Confocal laser scanning microscopy (CLSM) and flow cytometry have shown that NGCA-CC's cell uptake is higher at pH 6.5 than at pH 7.4. MTT and real-time cell analysis (RTCA) have illustrated that there is more toxicity at pH 6.5 than at pH 7.4. The apoptosis process induced by CC was determined by flow cytometry.
Subject(s)
Boronic Acids/chemistry , Citraconic Anhydrides/chemistry , Cytochromes c/chemistry , Cytochromes c/therapeutic use , Nanoparticles/chemistry , Polyethylene Glycols/chemistry , Polyethyleneimine/chemistry , Apoptosis/drug effects , Cytochromes c/metabolism , Drug Carriers/chemistry , Flow Cytometry/methods , Humans , Hydrogen-Ion Concentration , Nanogels , NanotechnologyABSTRACT
Polyampholyte-based films can be efficiently self-assembled onto a surface in a one-pot manner. By using a gradient of protons, morphogens, generated at an electrode surface, a charge-shifting polyelectrolyte present in solution can be transformed into a polyampholyte, leading to the continuous buildup of a film based on polyelectrolyte complexation.
Subject(s)
Citraconic Anhydrides/chemistry , Electrolytes/chemistry , Polyamines/chemistry , Polymers/chemistry , Molecular Structure , Polyelectrolytes , Static Electricity , Surface PropertiesABSTRACT
Multifunctional nanocomplexes (NCs) consisting of urocanic acid-modified galactosylated trimethyl chitosan (UA-GT) conjugates as polymeric vectors, poly(allylamine hydrochloride)-citraconic anhydride (PAH-Cit) as charge-reversible crosslinkers, and vascular endothelial growth factor (VEGF) siRNA as therapeutic genes, were rationally designed to simultaneously overcome the extracellular, cellular, and intracellular barriers for siRNA delivery. The strong physical stability of UA-GT/PAH-Cit/siRNA NCs (UA-GT NCs) at pH 7.4 and 6.5 endowed protection from massive dilution, competitive ions, and ubiquitous nucleases in the blood and tumorous microenvironment. Their internalization into hepato-carcinoma cells was facilitated through the recognition of galactose receptors, followed by effective escape from endosomes/lysosomes owing to the strong buffering capacity of imidazole residues. At the meantime, the endosomal/lysosomal acidity triggered the charge reversal of PAH-Cit in UA-GT NCs, thus evoking their structural disassembly and subsequently accelerated release of siRNA in the cytosol. As a result, robust in vivo performance in terms of both gene silencing and tumor inhibition was achieved by UA-GT NCs at a low siRNA dose. Moreover, neither histological nor hematological toxicity was detected following repeated intravenous administration. Therefore, UA-GT NCs potentially served as an efficient and safe candidate in the treatment of hepatocellular carcinoma through knocking down the overall barriers for siRNA delivery.
Subject(s)
Antineoplastic Agents/pharmacology , Gene Transfer Techniques , Nanoparticles/chemistry , RNA, Small Interfering/metabolism , Animals , Buffers , Cell Line, Tumor , Chitosan/chemical synthesis , Chitosan/chemistry , Citraconic Anhydrides/chemical synthesis , Citraconic Anhydrides/chemistry , Endocytosis , Endosomes/metabolism , Female , Galactose/chemistry , Gene Silencing/drug effects , Humans , Liver/drug effects , Liver/metabolism , Mice, Inbred BALB C , Mice, Nude , Nanoparticles/toxicity , Particle Size , Polyamines/chemical synthesis , Polyamines/chemistry , Proton Magnetic Resonance Spectroscopy , Static Electricity , Urocanic Acid/chemical synthesis , Urocanic Acid/chemistry , Vascular Endothelial Growth Factor A/metabolismABSTRACT
Therapeutic platforms such as chemotherapy that respond to physical and biological stimuli are highly desirable for effective cancer therapy. In this study, pH-responsive charge-reversal, polymer-coated mesoporous silica nanoparticles [PAH-cit/APTES-MSNs; PAH-cit refers to poly(allylamine)-citraconic anhydride; APTES refers to (3-aminopropyl)triethoxysilane] were synthesized for application as drug-delivery systems for the treatment of malignant cells. Confocal laser scanning microscopy (CLSM) revealed that the PAH-cit/APTES-MSNs nanocomposite effectively delivered and released doxorubicin hydrochloride to the nucleus of HeLa (human cervical carcinoma) cells. Additionally, the real-time dynamic drug-release process was monitored by CLSM. The current pH-controlled-smart-release platform holds promise in drug-delivery and cancer therapy-related applications.
Subject(s)
Coated Materials, Biocompatible/chemistry , Delayed-Action Preparations/pharmacology , Drug Liberation , Intracellular Space/chemistry , Nanoparticles/chemistry , Polymers/chemistry , Silicon Dioxide/chemistry , Cell Death/drug effects , Cell Nucleus/drug effects , Cell Nucleus/metabolism , Citraconic Anhydrides/chemistry , Doxorubicin/pharmacology , HeLa Cells , Humans , Hydrogen-Ion Concentration , Hydrolysis , Microscopy, Confocal , Nanoparticles/ultrastructure , Polyamines/chemistry , Porosity , Propylamines , Silanes/chemistry , Spectroscopy, Fourier Transform InfraredABSTRACT
Our previous work showed that a charge-reversal layer-by-layer nanosystem, PEI/PAH-Cit/AuNP-CS, effectively facilitates cellular uptake of siRNA and enhances the silencing efficacy of MDR1 siRNA. Here, the plasmid loading capacity of this vehicle was examined using EGFP-N1, and the plasmid release profile was determined in response to pH changes. The cytotoxicity of the EGFP-N1/PEI/PAH-Cit/AuNP-CS complex against HeLa and 293T cells was almost negligible. PEI/PAH-Cit/AuNP-CS efficaciously delivered the plasmids EGFP-N1 (encoding green fluorescent protein) and pGL3.0 (encoding luciferase) into 293T and HeLa cells, thus verifying the universality of PEI/PAH-Cit/AuNP-CS as a gene carrier. The results of an inverted fluorescence microscopy, flow cytometry (FCM) and western blotting methods demonstrated that PC-3 prostate cancer cells treated with EGFP-p53/PEI/PAH-Cit/AuNP-CS expressed higher levels of GFP than cells treated with EGFP-p53/PEI. Furthermore, PC-3 cells treated with EGFP-p53/PEI/PAH-Cit/AuNP-CS showed reduced cellular viability and increased nuclear fragmentation, consistent with elevated p53 expression. Propidium iodide (PI) flow cytometric assays were conducted to demonstrate that EGFP-p53/PEI/PAH-Cit/AuNP-CS elevated the level of apoptosis in PC-3 cells. Western blotting and caspase activation studies revealed that EGFP-p53/PEI/PAH-Cit/AuNP-CS complexes may induce PC-3 apoptosis via the mitochondria-mediated signaling pathway by up-regulation of Bax, down-regulation of Bcl-2, and activation of caspase-3.
Subject(s)
Prostatic Neoplasms/genetics , Prostatic Neoplasms/therapy , Transfection , Tumor Suppressor Protein p53/metabolism , Cell Proliferation , Cell Survival , Chitosan/chemistry , Citraconic Anhydrides/chemistry , Gold/chemistry , Green Fluorescent Proteins/metabolism , HeLa Cells , Humans , Male , Metal Nanoparticles/chemistry , Metal Nanoparticles/ultrastructure , Microscopy, Fluorescence , Plasmids/metabolism , Polyamines/chemistry , Polyethyleneimine/chemistry , Prostatic Neoplasms/pathology , bcl-2-Associated X Protein/metabolismABSTRACT
An antigen retrieval method for immunohistochemical staining of glucagon-like peptide (GLP)-2-immunoreactive cells was investigated in the chicken small intestine. GLP-2-immunoreactive cells were observed as open-typed endocrine cells in the villous epithelium and crypts on both antigen retrieval agent-treated and untreated preparations. No obvious differences were detected in morphological features of GLP-2-immunoreactive cells between treated and untreated preparations. The frequencies of occurrence of GLP-2-immunoreactive cells, however, were significantly different in treated and untreated preparations: in the proximal and distal regions of jejunum and ileum obtained from untreated preparations, the frequencies of occurrence were 0.5 ± 0.2, 0.7 ± 0.1, 0.9 ± 0.2 and 1.5 ± 0.3, respectively (cell numbers per mucosal area: cells/mm(2), mean ± SD), whereas those from treated sections were 14.7 ± 2.3, 19.8 ± 2.3, 23.5 ± 4.7 and 34.6 ± 4.9 cells/mm(2), respectively. These data indicate that this antigen retrieval method is able to make immunoreactive GLP-2 available for detection and that GLP-2 may act as one of the common hormones secreted by L cells in the chicken small intestine.
Subject(s)
Chickens/metabolism , Enteroendocrine Cells/metabolism , Glucagon-Like Peptide 2/metabolism , Immunohistochemistry/veterinary , Intestine, Small/metabolism , Animals , Citraconic Anhydrides , Enteroendocrine Cells/immunology , Glucagon-Like Peptide 2/immunology , Immunohistochemistry/methods , MaleABSTRACT
Tannerella forsythensis (Bacteroides forsythus), an anaerobic Gram-negative species of bacteria that plays a role in the progression of periodontal disease, has a unique bacterial protein profile. It is characterized by two unique protein bands with molecular weights of more than 200 kDa. It also is known to have a typical surface layer (S-layer) consisting of regularly arrayed subunits outside the outer membrane. We examined the relationship between high molecular weight proteins and the S-layer using electron microscopic immunolabeling with chemical fixation and an antigen retrieval procedure consisting of heating in a microwave oven or autoclave with citraconic anhydride. Immunogold particles were localized clearly at the outermost cell surface. We also used energy-filtering transmission electron microscopy (EFTEM) to visualize 3, 3'-diaminobenzidine tetrahydrochloride (DAB) reaction products after microwave antigen retrieval with 1% citraconic anhydride. The three-window method for electron spectroscopic images (ESI) of nitrogen by the EFTEM reflected the presence of moieties demonstrated by the DAB reaction with horseradish peroxidase (HRP)-conjugated secondary antibodies instead of immunogold particles. The mapping patterns of net nitrogen were restricted to the outermost cell surface.
Subject(s)
Antigens, Bacterial/analysis , Bacteroides/immunology , Bacteroides/ultrastructure , Citraconic Anhydrides/chemistry , Heating/instrumentation , Immunohistochemistry/methods , Membrane Glycoproteins/analysis , Microscopy, Energy-Filtering Transmission Electron/methods , Microwaves , 3,3'-Diaminobenzidine/chemistry , Bacterial Proteins/analysis , Bacteroides/chemistry , Horseradish Peroxidase/chemistryABSTRACT
Intracellular delivery of active proteins presents an interesting approach in research and therapy. We created a protein transduction shuttle based on a new traceless click linker that combines the advantages of click reactions with implementation of reversible pH-sensitive bonds. The azidomethyl-methylmaleic anhydride (AzMMMan) linker was found compatible with different click chemistries, demonstrated in bioreversible protein modification with dyes, polyethylene glycol, or a transduction carrier. Linkages were stable at physiological pH but reversible at the mild acidic pH of endosomes or lysosomes. We show that pH-reversible attachment of a defined endosome-destabilizing three-arm oligo(ethane amino)amide carrier generates an effective shuttle for protein delivery. The cargo protein nlsEGFP, when coupled via the traceless AzMMMan linker, experiences efficient cellular uptake and endosomal escape into the cytosol, followed by import into the nucleus. In contrast, irreversible linkage to the same shuttle hampers nuclear delivery of nlsEGFP which after uptake remains trapped in the cytosol. Successful intracellular delivery of bioactive ß-galactosidase as a model enzyme was also demonstrated using the pH-controlled shuttle system.
Subject(s)
Citraconic Anhydrides/chemistry , Drug Carriers/chemistry , Green Fluorescent Proteins/administration & dosage , beta-Galactosidase/administration & dosage , Azides/chemistry , Azides/metabolism , Cell Membrane Permeability , Citraconic Anhydrides/metabolism , Click Chemistry , Cytosol/metabolism , Drug Carriers/metabolism , Endosomes/metabolism , Green Fluorescent Proteins/chemistry , Green Fluorescent Proteins/pharmacokinetics , HeLa Cells , Humans , Hydrogen-Ion Concentration , beta-Galactosidase/chemistry , beta-Galactosidase/pharmacokineticsSubject(s)
Dendrimers/chemistry , Drug Carriers , Fluorescent Dyes/metabolism , Liver Neoplasms/metabolism , Oxazines/metabolism , Animals , Biological Transport , Chemistry, Pharmaceutical , Cholic Acid/chemistry , Citraconic Anhydrides/chemistry , Delayed-Action Preparations , Drug Compounding , Fluorescent Dyes/administration & dosage , Fluorescent Dyes/chemistry , Hep G2 Cells , Humans , Hydrogen-Ion Concentration , Hydrolysis , Injections, Intravenous , Male , Mice , Mice, Inbred BALB C , Micelles , Microscopy, Confocal , Oxazines/administration & dosage , Oxazines/chemistry , Spectroscopy, Near-Infrared , Technology, Pharmaceutical/methods , Time Factors , Tissue Distribution , Whole Body ImagingABSTRACT
We report a smart mesoporous silica nanoparticle (MSN) with a pore surface designed to undergo charge conversion in intracellular endosomal condition. The surface of mesopores in the silica nanoparticles was engineered to have pH-hydrolyzable citraconic amide. Solid-state nuclear magnetic resonance (NMR), Fourier-transform infrared (FT-IR) spectroscopy, and Brunauer-Emmett-Teller (BET) analyses confirmed the successful modification of the pore surfaces. MSNs (MSN-Cit) with citraconic amide functionality on the pore surfaces exhibited a negative zeta potential (-10 mV) at pH 7.4 because of the presence of carboxylate end groups. At cellular endosomal pH (approximately 5.0), MSN-Cit have a positive zeta potential (16 mV) indicating the dramatic charge conversion from negative to positive by hydrolysis of surface citraconic amide. Cytochrome c (Cyt c) of positive charges could be incorporated into the pores of MSN-Cit by electrostatic interactions. The release of Cyt c can be controlled by adjusting the pH of the release media. At pH 7.4, the Cyt c release was retarded, whereas, at pH 5.0, MSN-Cit facilitated the release of Cyt c. The released Cyt c maintained the enzymatic activity of native Cyt c. Hemolytic activity of MSN-Cit over red blood cells (RBCs) was more pronounced at pH 5.0 than at pH 7.0, indicating the capability of intracellular endosomal escape of MSN carriers. Confocal laser scanning microscopy (CLSM) studies showed that MSN-Cit effectively released Cyt c in endosomal compartments after uptake by cancer cells. The MSN developed in this work may serve as efficient intracellular carriers of many cell-impermeable therapeutic proteins.
Subject(s)
Drug Delivery Systems/methods , Nanoparticles/chemistry , Proteins/administration & dosage , Proteins/chemistry , Silicon Dioxide/chemistry , Adsorption , Cell Survival , Citraconic Anhydrides/chemistry , Citraconic Anhydrides/metabolism , Cytochromes c/chemistry , Cytochromes c/metabolism , HeLa Cells , Hemolysis , Humans , Hydrogen-Ion Concentration , Microscopy, Electron, Scanning , Microscopy, Fluorescence , Models, Chemical , Nitrogen , Porosity , Spectroscopy, Fourier Transform InfraredABSTRACT
BACKGROUND: Peritubular capillary complement C4d deposition is one of the criteria for diagnosis of acute humoral rejection in kidney allografts. Little information is provided for the effective technique to stain C4d protein. OBJECTIVE: To compare C4d staining results by using indirect immunofluorescence detection (IF) with immunoperoxidase methods (IP) in formalin-fixed, paraffin-embedded renal allograft tissue. MATERIAL AND METHOD: C4d protein was detected in renal allograft tissues by IF and IP methods. The antigen unmarking procedures were used including (i) heating with 0.05% citraconic anhydride in a water bath plus proteinase K digestion, (ii) heating with 10 mM citrate buffer in a microwave and (iii) digesting with proteinase K for comparing the deposition of C4d in paraffin-embedded tissues. RESULTS: The results showed that the unmarking solution containing 0.05% citraconic anhydride pH 7.4 and heating in a water bath revealed a signal enhancement in the IP method whereas the solution containing 0.05% citraconic anhydride pH 7.4 and heating in a water bath plus proteinase K digestion showed a greatly enhanced signal in the IF method. The prevalence of C4d staining detected in peritubular capillaries was 68% (17/25) and the results observed for both methods were similar. CONCLUSION: 0.05% citraconic anhydride in a water bath with or without proteinase K digestion is useful for unmasking C4d deposition in peritubular capillaries of renal allografts performed by IP and IF methods.
Subject(s)
Complement C4b/metabolism , Graft Rejection/diagnosis , Kidney Transplantation/immunology , Peptide Fragments/metabolism , Staining and Labeling/methods , Biopsy , Citraconic Anhydrides , Endopeptidase K/immunology , Endopeptidase K/metabolism , Fluorescent Antibody Technique, Indirect , Graft Rejection/immunology , Humans , Immunoenzyme Techniques , Kidney Transplantation/pathology , Paraffin Embedding , Transplantation, Homologous/immunology , Transplantation, Homologous/pathologyABSTRACT
BACKGROUND: The introduction of heat-induced antigen retrieval has been a major milestone in diagnostic immunohistochemistry, enabling the application of many antibodies to fixed paraffin-embedded tissue sections. A number of important variables affect the preservation of tissue antigens, among which are analytical variables including the antigen retrieval methodology. Temperature of retrieval, duration of heating, source of heat, pH and nature of retrieval solution are among more important variables pivotal to results. Citrate buffer at pH 6.0 has become widely embraced as the universal fixative but some antibodies remain capricious and yield poor staining results. METHODS: This study examines the recent suggestion that citraconic anhydride may be a suitable universal retrieval reagent. Immunostaining of 65 commonly employed antibodies following microwave antigen retrieval in 0.05% citraconic anhydride for 10 minutes at 98 degrees C was compared with consecutive tissues sections subjected to antigen retrieval in citrate buffer at pH 6.0 at the same duration and temperature. RESULTS: Thirty-five of the 65 antibodies examined yielded more intense staining following antigen retrieval in citraconic anhydride, including some capricious antibodies such as MyoD1, myogenin, perforin, TIA-1, Tdt, RET and MiTF, confirming the efficacy of this retrieval solution. CONCLUSION: It is recommended that consideration be given to 0.05% citraconic anhydride as an antigen retrieval solution, particularly for antibodies that fail to work or stain weakly in fixed paraffin-embedded tissue sections.
Subject(s)
Antigen-Antibody Reactions , Antigens/chemistry , Citraconic Anhydrides/chemistry , Fixatives/chemistry , Immunoenzyme Techniques/methods , Tissue Fixation/methods , Biomarkers/chemistry , HumansABSTRACT
Studies on the aggregation of mutant proteins have provided new insights into the genetics of amyloid diseases and the role of the net charge of the protein on the rate, extent, and type of aggregate formation. In the present work, hen egg white lysozyme (HEWL) was employed as the model protein. Acetylation and (separately) citraconylation were employed to neutralize the charge on lysine residues. Acetylation of the lysine residues promoted amyloid formation, resulting in more pronounced fibrils and a dramatic decline in the nucleation time. In contrast, citraconylation produced the opposite effect. In both cases, native secondary and tertiary structures appeared to be retained. Studies on the effect of pH on aggregation suggested greater possibilities for amorphous aggregate formation rather than fibrillation at pH values closer to neutrality, in which the protein is known to take up a conformation more similar to its native form. This is in accord with reports in the literature suggesting that formation of amorphous aggregates is more favored under relatively more native conditions. pH 5 provided a critical environment in which a mixture of amorphous and fibrillar structures were observed. Use of Tango and Aggrescan software which describe aggregation tendencies of different parts of a protein structure suggested critical importance of some of the lysine residues in the aggregation process. Results are discussed in terms of the importance of the net charge in control of protein-protein interactions leading to aggregate formation and possible specific roles of lysine residues 96 and 97.
Subject(s)
Amyloid/biosynthesis , Amyloid/chemistry , Muramidase/chemistry , Muramidase/metabolism , Acetic Anhydrides , Acetylation , Amyloid/ultrastructure , Animals , Chickens , Citraconic Anhydrides , Female , Hydrogen-Ion Concentration , Kinetics , Lysine/chemistry , Microscopy, Electron, Transmission , Muramidase/ultrastructure , Protein Interaction Domains and Motifs , Protein Structure, Secondary , Spectrometry, FluorescenceABSTRACT
Insulin is a polypeptide hormone which is produced by the beta-cell of pancreas and controls the blood glucose level in the human body. Enzymatic modification of human proinsulin using trypsin and carboxypeptidase B generally causes high accumulation of insulin derivatives, leading to more complicated purification processes. A simple method including citraconylation and decitraconylation in the enzymatic modification process was developed for the reduction of a major derivative, des-threonine human insulin. Addition of 3.0 g citraconic anhydride per g protein into the reaction solution led to the citraconylation of lysine residues in human proinsulin and reduction of relative des-threonine insulin content from 13.5 to 1.0%. After the enzymatic hydrolysis of the citraconylated proinsulin, 100% of lysine residues can be decitraconylated and restored by adjusting pH to 2-3 at 25 degrees C. Combination of hydrogen peroxide addition and citraconylation of proinsulin expressed in recombinant Escherichia coli remarkably improved the conversion yield of insulin from 52.7 to 77.7%. Consequently, citraconylation of lysine residues blocked the unexpected cleavage of human proinsulin by trypsin, minimized the formation of des-threonine insulin and hence increased the production yield of active insulin.