ABSTRACT
The construction of enzyme delivery systems, which can control enzymatic activity at a target site, is important for efficient enzyme-prodrug therapy/diagnosis. Herein we report a facile technique to construct a systemically applicable ß-galactosidase (ß-Gal)-loaded ternary complex comprising tannic acid (TA) and phenylboronic acid-conjugated polymers through sequential self-assembly in aqueous solution. At physiological conditions, the ternary complex exhibited a hydrodynamic diameter of â¼40 nm and protected the loaded ß-Gal from unfavorable degradation by proteinase. Upon cellular internalization, the ternary complex recovered ß-Gal activity by releasing the loaded ß-Gal. The intravenously injected ternary complex thereby delivered ß-Gal to the target tumor in a subcutaneous tumor model and exerted enhanced and selective enzymatic activity at the tumor site. Sequential self-assembly with TA and phenylboronic acid-conjugated polymers may offer a novel approach for enzyme-prodrug theragnosis.
Subject(s)
Boronic Acids/metabolism , Nanoparticles/metabolism , Neoplasms/metabolism , Polymers/metabolism , Tannins/metabolism , beta-Galactosidase/metabolism , Animals , Boronic Acids/chemistry , Cell Line, Tumor , Female , Hydrodynamics , Mice , Mice, Inbred BALB C , Models, Molecular , Molecular Structure , Nanoparticles/chemistry , Neoplasms/diagnosis , Particle Size , Polymers/chemical synthesis , Polymers/chemistry , Surface Properties , Tannins/chemistry , beta-Galactosidase/administration & dosage , beta-Galactosidase/bloodABSTRACT
GM1 gangliosidosis is a rare neurodegenerative lysosomal storage disease caused by loss-of-function mutations in the gene encoding beta-galactosidase (ß-gal). There are no approved treatments for GM1 gangliosidosis. Previous studies in animal models have demonstrated that adeno-associated viral (AAV) vector-mediated gene transfer to the brain can restore ß-gal expression and prevent the onset of neurological signs. We developed an optimized AAV vector expressing human ß-gal and evaluated the efficacy of a single intracerebroventricular injection of this vector into the cerebrospinal fluid (CSF) of a murine disease model. The AAV vector administration into the CSF increased ß-gal activity in the brain, reduced neuronal lysosomal storage lesions, prevented the onset of neurological signs and gait abnormalities, and increased survival. These findings demonstrate the potential therapeutic activity of this vector and support its subsequent development for the treatment of GM1 gangliosidosis.
Subject(s)
Brain/metabolism , Cerebrospinal Fluid/metabolism , Dependovirus/genetics , Gangliosidosis, GM1/therapy , Genetic Therapy/methods , Genetic Vectors/administration & dosage , beta-Galactosidase/physiology , Animals , Brain/pathology , Cerebrospinal Fluid/cytology , Disease Models, Animal , Gangliosidosis, GM1/genetics , Gangliosidosis, GM1/metabolism , Gangliosidosis, GM1/pathology , Genetic Vectors/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout , beta-Galactosidase/administration & dosage , beta-Galactosidase/geneticsABSTRACT
Fabry disease is a rare X-linked lysosomal disease, in which mutations in the gene encoding α-galactosidase A result in progressive cellular accumulation of globotriaosylceramide (GL-3) in various organs including the skin, kidney, and heart, often leading to life-threatening conditions. Enzyme replacement therapy is currently the standard therapy for the disease, to which two α-galactosidase A formulations have been approved: agalsidase α (Replagal®, Shire) and agalsidase ß (Fabrazyme®, Sanofi). We have recently developed a biosimilar of agalsidase ß, JR-051, and investigated its pharmacokinetics and pharmacodynamics to assess its bioequivalence to agalsidase ß. In a randomized phase I study, healthy adult male volunteers were treated with JR-051 or agalsidase ß and the pharmacokinetics of the drugs were compared. The ratio of geometric means (90% confidence interval [CI]) of the AUC0-24 and Cmax for JR-051 over agalsidase ß were 0.91 (0.8294, 1.0082) and 0.90 (0.7992, 1.0125), respectively. In a 52-week, single-arm, phase II/III study, patients with Fabry disease switched therapy from agalsidase ß to JR-051 to evaluate its pharmacodynamics. The mean (95% CI) plasma GL-3 concentrations at weeks 26 and 52 relative to pre-JR-051 administration were 1.03 (0.91, 1.15) and 0.96 (0.86, 1.06), respectively, which were within the pre-determined bioequivalence acceptance range (0.70, 1.43). The mean (95% CI) plasma globotriaosylsphingosine (lyso-GL-3) concentrations at weeks 26 and 52 relative to pre-JR-051 administration were 1.07 (0.92, 1.23) and 1.13 (1.03, 1.22), respectively. Estimated glomerular filtration rate and left ventricular mass index, as renal and cardiac function indicators, showed no notable changes from baseline throughout the study period, and no new safety concerns were identified. In conclusion, these studies demonstrated bioequivalence of JR-051 to agalsidase ß in terms of its pharmacokinetics and pharmacodynamics. JR-051 offers a potential new treatment option for patients with Fabry disease.
Subject(s)
Biomarkers/blood , Biosimilar Pharmaceuticals/administration & dosage , Enzyme Replacement Therapy/methods , Fabry Disease/therapy , Glycolipids/blood , Sphingolipids/blood , beta-Galactosidase/administration & dosage , Adolescent , Adult , Aged , Biosimilar Pharmaceuticals/pharmacokinetics , Biosimilar Pharmaceuticals/pharmacology , Case-Control Studies , Child , Double-Blind Method , Fabry Disease/enzymology , Female , Humans , Male , Middle Aged , Tissue Distribution , Young AdultABSTRACT
Lactose intolerance is a common condition caused by lactase deficiency and may result in symptoms of lactose malabsorption (bloating, flatulence, abdominal discomfort, and change in bowel habits). As current data is limited, the aim of our study was to assess the efficacy of probiotics with a ß-galactosidase activity on symptoms of lactose malabsorption and on the lactose hydrogen breath test (LHBT). The study group comprised eight symptomatic female patients with a positive LHBT. Patients were treated for 6 months with a probiotic formula with ß-galactosidase activity (Bio-25, Ambrosia-SupHerb, Israel). All patients completed a demographic questionnaire as well as a diary for the assessment of symptom severity and frequency at entry, every 8 weeks, and at the end of the treatment period. Measurements of hydrogen (H2) levels (parts per million, ppm) at each of these time points were also performed. End points were a decrease of 50% in symptom severity or frequency, and the normalization (decrease below cutoff point of 20 ppm) of the breath test. Mean age and mean body mass index (BMI) were 36.4 ± 18.6 years and 23.2 kg/m2, respectively. Compared to baseline scores, the frequency of most symptoms, and the severity of bloating and flatulence, improved after treatment. Normalization of LHBT was obtained in only two patients (25%). In this pilot study, Bio-25, a unique formulation of probiotics with ß-galactosidase activity, demonstrated symptom resolution in most patients with lactose malabsorption. A larger randomized trial is warranted to confirm these preliminary findings.
Subject(s)
Lactose Intolerance/drug therapy , Lactose/metabolism , Probiotics/administration & dosage , beta-Galactosidase/administration & dosage , Adolescent , Adult , Female , Humans , Middle Aged , Pilot Projects , Surveys and Questionnaires , Young AdultABSTRACT
The development of new effective vaccines strongly depends on adjuvants and formulations able to stimulate not only strong humoral responses against a certain pathogen but also effector as well as memory CD4+ and CD8+ T cells (Dubensky et al., 2013). However, the majority of vaccines licensed for human use or currently under clinical investigation fail to stimulate efficient cellular responses. For example, vaccines against hepatitis B virus (HBV), human papillomavirus (HPV), diphtheria, tetanus and influenza are usually administered by intramuscular (i.m.) injection and contain aluminum salts (alum) as adjuvant. Alum has been shown to stimulate Th2 immune cells resulting in increased production of antigen-specific antibodies but to be incapable of stimulating robust Th1 or cytotoxic responses. To overcome such limitations recent research has focused on the development of adjuvant combinations (e.g., MF59, AS03 or AS04) to not only further strengthen antigen-specific immune responses but to also allow their modulation. We have shown previously that bis-(3',5')-cyclic dimeric adenosine monophosphate (c-di-AMP) constitutes a promising adjuvant candidate stimulating both effective Th1/Th2 and cytotoxic immune responses when included in mucosal or parenteral vaccine formulations. In the present work we demonstrate that c-di-AMP can be also combined with other adjuvants like alum resulting in increases in not only humoral responses but more striking also in cellular immune responses. This leads to improved vaccine efficacy against intracellular pathogens.
Subject(s)
Adjuvants, Immunologic/administration & dosage , Alum Compounds/administration & dosage , Dinucleoside Phosphates/administration & dosage , Drug Synergism , Immunity, Cellular , Immunity, Humoral , Animals , Injections, Intramuscular , Mice, Inbred BALB C , Th1 Cells/immunology , Th2 Cells/immunology , Vaccines, Synthetic/administration & dosage , Vaccines, Synthetic/immunology , beta-Galactosidase/administration & dosage , beta-Galactosidase/immunologyABSTRACT
Oral local delivery of therapeutic biologics is generally limited due to the multiple obstacles of the gastrointestinal (GI) tract, mainly represented by acidic stomach pH and digestive enzymes. In the present study, spray congealing was used to prepare solid lipid microparticles (SLMs) loaded with ß-galactosidase (lactase), an enzyme used for the treatment of lactose intolerance, to achieve a local drug delivery to the small intestine. Lactase was characterized in terms of activity at different pH, kinetic parameters and proteolytic degradation by digestive enzymes. Then, five lipid excipients were used to prepare unloaded SLMs, which were tested regarding lipase-induced digestion. The lipid with the best performance (glyceryl trimyristate) was used to prepare lactase-loaded SLMs. Spray congealed SLMs were spherical with very good encapsulation efficiency (>95%). The ability of the SLMs to protect the enzyme from the degradation in gastric environment was correlated with the particle size and the best formulation preserved the lactase activity up to 70%. Lactase was promptly released in simulated intestinal environment, and an in vitro positive food effect was observed. The present study demonstrated the potential of spray congealing for the preparation of solid lipid formulations able to achieve local oral delivery of a biologic drug.
Subject(s)
Drug Carriers/chemistry , Drug Delivery Systems , Lipids/chemistry , beta-Galactosidase/administration & dosage , Administration, Oral , Chemistry, Pharmaceutical/methods , Drug Liberation , Excipients/chemistry , Hydrogen-Ion Concentration , Intestine, Small/metabolism , Particle Size , beta-Galactosidase/pharmacokineticsABSTRACT
Intracellular delivery of proteins is a promising strategy of intervention in disease, which relies heavily on the development of efficient delivery platforms due to the cell membrane impermeability of native proteins, particularly for negatively charged large proteins. This work reports a vesicle supra-assembly approach to synthesize novel amine-functionalized hollow dendritic mesoporous silica nanospheres (A-HDMSN). An amine silica source is introduced into a water-oil reaction solution prior to the addition of conventional silica source tetraethylorthosilicate. This strategy favors the formation of composite vesicles as the building blocks which further assemble into the final product. The obtained A-HDMSN have a cavity core of ≈170 nm, large dendritic mesopores of 20.7 nm in the shell and high pore volume of 2.67 cm3 g-1 . Compared to the calcined counterpart without amine groups (C-HDMSN), A-HDMSN possess enhanced loading capacity to large negative proteins (IgG and ß-galactosidase) and improved cellular uptake performance, contributed by the cationic groups. A-HDMSN enhance the intracellular uptake of ß-galactosidase by up to 5-fold and 40-fold compared to C-HDMSN and free ß-galactosidase, respectively. The active form of ß-galactosidase delivered by A-HDMSN retains its intracellular catalytic functions.
Subject(s)
Amines/chemistry , Nanospheres/chemistry , Proteins/administration & dosage , Silicon Dioxide/chemistry , Animals , CHO Cells , Carbon-13 Magnetic Resonance Spectroscopy , Cricetinae , Cricetulus , Flow Cytometry , Immunoglobulin G/administration & dosage , Nanospheres/ultrastructure , Porosity , Time Factors , beta-Galactosidase/administration & dosageABSTRACT
We report a strategy for creating a new class of protein transfection materials composed of a functional protein core chemically modified with a dense shell of oligonucleotides. These materials retain the native structure and catalytic ability of the hydrolytic enzyme ß-galactosidase, which serves as the protein core, despite the functionalization of its surface with â¼25 DNA strands. The covalent attachment of a shell of oligonucleotides to the surface of ß-galactosidase enhances its cellular uptake of by up to â¼280-fold and allows for the use of working concentrations as low as 100 pM enzyme. DNA-functionalized ß-galactosidase retains its ability to catalyze the hydrolysis of ß-glycosidic linkages once endocytosed, whereas equal concentrations of protein show little to no intracellular catalytic activity.
Subject(s)
DNA/chemistry , beta-Galactosidase/administration & dosage , Catalysis , Cell Line , Humans , Spectrophotometry, Ultraviolet , beta-Galactosidase/chemistryABSTRACT
Non-eosinophilic asthma is characterized by infiltration of neutrophils into the lung and variable responsiveness to glucocorticoids. The pathophysiological mechanisms have not been characterized in detail. Here, we present an experimental asthma model in mice associated with non-eosinophilic airway inflammation and airway hyper-responsiveness (AHR). For this, BALB/c mice were sensitized by biolistic DNA immunization with a plasmid encoding the model antigen ß-galactosidase (pFascin-ßGal mice). For comparison, eosinophilic airway inflammation was induced by subcutaneous injection of ßGal protein (ßGal mice). Intranasal challenge of mice in both groups induced AHR to a comparable extent as well as recruitment of inflammatory cells into the airways. In contrast to ßGal mice, which exhibited extensive eosinophilic infiltration in the lung, goblet cell hyperplasia and polarization of CD4(+) T cells into Th2 and Th17 cells, pFascin-ßGal mice showed considerable neutrophilia, but no goblet cell hyperplasia and a predominance of Th1 and Tc1 cells in the airways. Depletion studies in pFascin-ßGal mice revealed that CD4(+) and CD8(+) cells cooperated to induce maximum inflammation, but that neutrophilic infiltration was not a prerequisite for AHR induction. Treatment of pFascin-ßGal mice with dexamethasone before intranasal challenge did not affect neutrophilic infiltration, but significantly reduced AHR, infiltration of monocytes and lymphocytes as well as content of IFN-γ in the bronchoalveolar fluid. Our results suggest that non-eosinophilic asthma associated predominantly with Th1/Tc1 cells is susceptible to glucocorticoid treatment. pFascin-ßGal mice might represent a mouse model to study pathophysiological mechanisms proceeding in the subgroup of asthmatics with non-eosinophilic asthma that respond to inhaled steroids.
Subject(s)
Asthma/drug therapy , Asthma/immunology , Dexamethasone/therapeutic use , T-Lymphocytes, Cytotoxic/immunology , Th1 Cells/immunology , Animals , Biolistics , Bronchoalveolar Lavage Fluid/immunology , DNA/administration & dosage , DNA/immunology , Disease Models, Animal , Eosinophils/immunology , Female , Goblet Cells/pathology , Interferon-gamma/biosynthesis , Interferon-gamma/metabolism , Lung/cytology , Lung/immunology , Lymphocyte Activation/immunology , Lymphocyte Depletion , Mice , Mice, Inbred BALB C , Neutrophil Infiltration/immunology , Neutrophils/immunology , Th17 Cells/immunology , Th2 Cells/immunology , beta-Galactosidase/administration & dosage , beta-Galactosidase/geneticsABSTRACT
A combinatorial design, physical adsorption of water-soluble chitosan (WSC) to particle surface and covalent conjugation of wheat germ agglutinin (WGA) to WSC, was applied to surface modification of poly(lactic acid) nanoparticles (NPs) for targeted delivery of ß-galactosidase to the intestinal mucosa. All the surface-engineered NPs in the size range of 500-600 nm were prepared by a w/o/w solvent diffusion/evaporation technique. ß-Galactosidase encapsulated in these NPs was well protected from external proteolysis and exerted high hydrolytic activity on the permeable lactose. The presence of WSC coating, whether alone or with WGA, highly improved the suspension stability of NPs and tailored the particle surface positively charged. In comparison to NPs modified with WGA or WSC alone, the synergistic action of WGA and WSC greatly enhanced the NP-mucin interactions in vitro. The highest amount of NPs was found in the small intestine at 24 h after oral administration in rats. Notably, calculated half-life of WGA-WSC-NPs in the small intestine was 6.72 h, resulting in 2.1- and 4.3-fold increase when compared to WGA-polyvinylalcohol (PVA)-NPs and WSC-NPs, much longer than that of control PVA-NPs (6.9-fold). These results suggest that NPs with the combined WGA and WSC coating represent promising candidates for efficient mucosal drug delivery as well as biomimetic treatment of lactose intolerance.
Subject(s)
Chitosan/chemistry , Lactic Acid/chemistry , Nanoparticles/chemistry , Polymers/chemistry , Water/chemistry , Wheat Germ Agglutinins/chemistry , beta-Galactosidase/chemistry , Administration, Oral , Adsorption , Animals , Chitosan/administration & dosage , Drug Carriers/administration & dosage , Drug Carriers/chemistry , Drug Delivery Systems/methods , Drug Stability , Excipients/chemistry , Half-Life , Intestinal Mucosa/metabolism , Intestine, Small/metabolism , Lactic Acid/administration & dosage , Nanoparticles/administration & dosage , Particle Size , Polyesters , Polymers/administration & dosage , Rats , Rats, Wistar , Suspensions/administration & dosage , Suspensions/chemistry , Water/administration & dosage , Wheat Germ Agglutinins/administration & dosage , beta-Galactosidase/administration & dosageABSTRACT
The use of enzymes as therapeutics is very promising because of their catalytic activity and specificity. However, intracellular delivery of active enzymes is challenging due to their low stability and large size. The production of protein-enzyme nanoparticles was investigated with the goal of developing a protein carrier for active enzyme delivery. ß-Galactosidase (ß-gal), an enzyme whose deficiency is the cause of some lysosomal storage disorders, was incorporated into enhanced green fluorescent protein nanoparticles prepared via desolvation. Particle size was found to be sensitive to the type of cross-linker, cross-linking time, and the presence of imidazole. The results indicate that ß-gal activity is highly retained (>70%) after particle fabrication and >85% of protein is incorporated in the particles. Protein-enzyme nanoparticles exhibited higher internalization in multiple cell lines in vitro, compared with the soluble enzyme. Importantly, ß-gal retained its activity following intracellular delivery. These data demonstrate that protein nanoparticles are a biocompatible, high-efficiency alternative for intracellular delivery of active enzyme therapeutics.
Subject(s)
Nanoparticles , Proteins/chemistry , beta-Galactosidase/administration & dosage , Particle SizeABSTRACT
Nanoparticles have been widely used for delivering various chemical and biomolecular drugs, such as anti-cancer drugs and therapeutic proteins. Among nanoparticles, protein nanoparticles have advantages of non-cytotoxicity and biodegradability. In this study, a recombinant 30Kc19 protein was applied to human serum albumin (HSA) nanoparticles to enhance cellular uptake and stability of a nanoparticle cargo enzyme. The 30Kc19 protein, which originates from silkworm, has cell-penetrating and enzyme-stabilizing abilities. Therefore, 30Kc19-HSA nanoparticles were expected to enhance cellular uptake and stability of an enzyme loaded on the nanoparticles. Here, nanoparticles loaded with ß-galactosidase were prepared using the desolvation method. The 30Kc19-HSA nanoparticles were uniformly spherical in shape, dispersed evenly in phosphate buffered saline and cell culture media, and released ß-galactosidase in a sustained manner. The 30Kc19-HSA nanoparticles had negligible toxicity to animal cells and exhibited enhanced cellular uptake and intracellular stability of ß-galactosidase in HeLa and HEK293 cells when compared with those of HSA nanoparticles. These results suggest that 30Kc19-HSA protein nanoparticles could be used as a versatile tool for drug delivery to various cells.
Subject(s)
Drug Carriers , Nanoparticles , Serum Albumin/chemistry , beta-Galactosidase/administration & dosage , HEK293 Cells , Humans , Microscopy, Electron, Scanning , Recombinant Proteins/chemistryABSTRACT
The intracellular delivery of enzymes is an essential methodology to extend their therapeutic application. Herein, we have developed dissociable supermolecule-enzyme polyelectrolyte complexes based on reduction-cleavable cationic polyrotaxanes (PRXs) for the reactivation of delivered enzymes. These PRXs are characterized by their supramolecular frameworks of a polymeric chain threading into cyclic molecules, which can form polyelectrolyte complexes with anionic enzymes while retaining their three dimensional structure, although their enzymatic activity is reduced. Upon the addition of a reductant, the PRXs dissociate into their constituent molecules and release the enzymes, resulting in a complete recovery of enzymatic activity. Under the intracellular environment, the PRX-based enzyme complexes showed the highest intracellular enzymatic activity and efficient activation of anticancer prodrugs to induce cytotoxic effects in comparison with the non-dissociable complexes and the commercial cell-penetrating peptide-based reagents. Thus, the intracellularly dissociable supermolecules are an attractive system for delivering therapeutic enzymes into living cells.
Subject(s)
Cyclodextrins , Drug Carriers , Drug Delivery Systems , Enzymes/administration & dosage , Poloxamer , Rotaxanes , Antineoplastic Agents/metabolism , Cell Line , Cyclodextrins/chemistry , Drug Carriers/chemistry , Enzyme Activation , Humans , Macromolecular Substances/chemistry , Macromolecular Substances/metabolism , Oxidation-Reduction , Poloxamer/chemistry , Polymers/chemistry , Prodrugs/chemistry , Prodrugs/metabolism , Rotaxanes/chemistry , beta-Galactosidase/administration & dosageABSTRACT
Human red blood cells (RBCs) are emerging as a highly biocompatible microparticulate drug delivery system. So far, drugs have commonly been loaded into freshly isolated RBCs using rather disruptive methods based on hypotonic shock, and assessment of damage was restricted to hemolysis. Here, we investigated loading of RBCs from blood bank units with enzymes of various molecular weights using hypotonic dialysis (HD), pretreatment with chlorpromazine (CPZ) and fusion with liposomes. The latter two techniques have received little attention in RBC loading so far. Along with loading efficiency, all methods were tested for the induction of side effects. Very importantly, next to hemolysis, we also addressed morphological changes and phosphatidyl serine (PS) exposure, which has been recognized as a critical parameter associated with premature RBC removal and induction of transfusion-related pathologies. The efficiency of loading using hypotonic dialysis decreased with the molecular weight of the enzyme. For liposomes and chlorpromazine, loading efficiencies were higher and independent of enzyme molecular weights. While hypotonic dialysis always induced a high degree of hemolysis, irreversible modifications in the morphology of the cells and PS exposure, the side effects that were induced by loading using CPZ and liposomes were limited. In particular, PS exposure, although high immediately after treatment, returned to physiological levels after recovery. Retention and deformability studies using a spleen-mimicking device showed that RBCs treated with CPZ and liposomes behave like physiological RBCs, while HD led to very fragile and poorly deformable RBCs.
Subject(s)
Drug Carriers , Erythrocytes/metabolism , Antipsychotic Agents/administration & dosage , Chlorpromazine/administration & dosage , Dialysis , Erythrocytes/cytology , Erythrocytes/drug effects , Fluorescent Dyes/chemistry , Hemolysis/drug effects , Horseradish Peroxidase/administration & dosage , Horseradish Peroxidase/chemistry , Humans , Hypotonic Solutions , Phosphatidylserines/administration & dosage , Rhodamines/chemistry , Xanthine Oxidase/administration & dosage , Xanthine Oxidase/chemistry , beta-Galactosidase/administration & dosage , beta-Galactosidase/chemistryABSTRACT
Increasing protein expression enables researchers to better understand the functional role of that protein in regulating key biological processes(1). In the lung, this has been achieved typically through genetic approaches that utilize transgenic mice(2,3) or viral or non-viral vectors that elevate protein levels via increased gene expression(4). Transgenic mice are costly and time-consuming to generate and the random insertion of a transgene or chronic gene expression can alter normal lung development and thus limit the utility of the model(5). While conditional transgenics avert problems associated with chronic gene expression(6), the reverse tetracycline-controlled transactivator (rtTA) mice, which are used to generate conditional expression, develop spontaneous air space enlargement(7). As with transgenics, the use of viral and non-viral vectors is expensive(8) and can provoke dose-dependent inflammatory responses that confound results(9) and hinder expression(10). Moreover, the efficacy of repeated doses are limited by enhanced immune responses to the vector(11,12). Researchers are developing adeno-associated viral (AAV) vectors that provoke less inflammation and have longer expression within the lung(13). Using ß-galactosidase, we present a method for rapidly and effectively increasing protein expression within the lung using a direct protein transfection technique. This protocol mixes a fixed amount of purified protein with 20 µl of a lipid-based transfection reagent (Pro-Ject, Pierce Bio) to allow penetration into the lung tissue itself. The liposomal protein mixture is then injected into the lungs of the mice via the trachea using a microsprayer (Penn Century, Philadelphia, PA). The microsprayer generates a fine plume of liquid aerosol throughout the lungs. Using the technique we have demonstrated uniform deposition of the injected protein throughout the airways and the alveoli of mice(14). The lipid transfection technique allows the use of a small amount of protein to achieve effect. This limits the inflammatory response that otherwise would be provoked by high protein administration. Indeed, using this technique we published that we were able to significantly increase PP2A activity in the lung without affecting lung lavage cellularity(15). Lung lavage cellularity taken 24 hr after challenge was comparable to controls (27 ± 4 control vs. 31 ± 5 albumin transfected; N=6 per group). Moreover, it increases protein levels without inducing lung developmental changes or architectural changes that can occur in transgenic models. However, the need for repeated administrations may make this technique less favorable for studies examining the effects of long-term increases in protein expression. This would be particularly true for proteins with short half-lives.
Subject(s)
Lipids/administration & dosage , Lung/drug effects , Lung/metabolism , Transfection/methods , beta-Galactosidase/administration & dosage , Animals , Bronchoalveolar Lavage/methods , Liposomes/administration & dosage , Mice , Transfection/instrumentationABSTRACT
This study was carried out to determine the optimum conditions for water-in-oil-in-water (W/O/W) microencapsulated lactase (ß-galactosidase) in order to prevent the intolerance of lactose in milk. The core material was lactase and the coating materials were medium-chain triglyceride for W/O phase, and whey protein isolate (WPI), maltodextrin, gum arabic, and its mixtures for W/O/W phase. Polyglycerol polyricinoleate (PGPR) was used as a primary emulsifier, and polyoxyethylene sorbitan monolaurate (PSML) was selected as a secondary emulsifier based on emulsion stability index. To determine the most efficient conditions for the W/O/W-lactase microencapsulation, the ratio of core to coating materials and amounts of emulsifiers were investigated by response surface methodology. The optimum ratio of core to coating materials in W/O, amount of PGPR, ratio of core to coating material in W/O/W, and amount of PSML were found to be 0.5-9.5, 0.75% (w/v), 1.7-8.3, and 0.25% (w/v), respectively. The average size of the microcapsules was about 10 µm under optimum conditions. Microcapsules of 30% (w/v) WPI as a secondary coating material could evenly distribute the pocket of lactase. Based on the data obtained from this study, lactase microcapsules could effectively be produced by the method of W/O/W double emulsion.
Subject(s)
Emulsions/chemistry , Enzymes, Immobilized/administration & dosage , beta-Galactosidase/administration & dosage , Capsules , Drug Compounding , Enzymes, Immobilized/chemistry , Enzymes, Immobilized/metabolism , Glycerol/analogs & derivatives , Glycerol/chemistry , Gum Arabic/chemistry , Humans , Lactose/metabolism , Polysaccharides/chemistry , Polysorbates/chemistry , Ricinoleic Acids/chemistry , Triglycerides/chemistry , Water/chemistry , beta-Galactosidase/chemistry , beta-Galactosidase/metabolismABSTRACT
Brain-targeted delivery of drug or imaging agent is hard to achieve efficiently due to the infiltrative nature of the blood-brain barrier (BBB). Moreover, delivery of therapeutic proteins to brain tissue is further limited by the size and physic-chemical properties of proteins. In this work, we developed a chitosan-conjugated Pluronic-based nano-carrier with a specific target peptide for the brain (rabies virus glycoprotein; RVG29) and applied for the protein delivery to the brain. The in-vivo brain accumulation of the nano-carrier in mice followed i.v injection was optically monitored with Cy5.5-conjugation to the nano-carrier, and the result showed that the Pluronic-based nano-carrier conjugated with both chitosan and the peptide was very efficient for the accumulation in brain tissue and was remarkably better than the nano-carrier conjugated with the peptide only. ß-galactosidase, a model protein, was also delivered and accumulated efficiently in the brain by loading in the nano-carrier, analyzed by the bio-distribution of ß-galactosidase. The delivered protein in the brain also maintained its bioactivity. Therefore, RVG29- and chitosan-conjugated Pluronic-based nano-carrier could be potentially useful for the diagnosis and therapy of brain diseases.
Subject(s)
Brain/metabolism , Chitosan/chemistry , Glycoproteins/pharmacokinetics , Nanocapsules/chemistry , Poloxamer/pharmacokinetics , Rabies virus/chemistry , beta-Galactosidase/pharmacokinetics , Animals , Brain/pathology , Diffusion , Male , Mice , Mice, Inbred C3H , Mice, Nude , Nanocapsules/administration & dosage , Peptides/pharmacokinetics , Tissue Distribution , beta-Galactosidase/administration & dosageABSTRACT
Botulinum toxin A (BT) is used therapeutically for the treatment of primary focal hyperhidrosis, a chronic debilitating condition characterised by over-activity of the eccrine sweat glands. Systemic toxicity concerns require BT to be administered by local injection, which in the case of hyperhidrosis means multiple painful intradermal injections by a skilled clinician at 6-monthly intervals. This study investigates the potential of a liquid-loaded pocketed microneedle device to deliver botulinum toxin A into the human dermis with the aim of reducing patient pain, improving therapeutic targeting and simplifying the administration procedure. Initially, ß-galactosidase was employed as a detectable model for BT to (i) visualise liquid loading of the microneedles, (ii) determine residence time of a liquid formulation on the device and (iii) quantify loaded doses. An array of five stainless steel pocketed microneedles was shown to possess sufficient capacity to deliver therapeutic doses of the potent BT protein. Microneedle-mediated intradermal delivery of ß-galactosidase and formaldehyde-inactivated botulinum toxoid revealed effective deposition and subsequent diffusion within the dermis. This study is the first to characterise pocketed microneedle delivery of a liquid formulation into human skin and illustrates the potential of such systems for the cutaneous administration of potent proteins such as BT. A clinically appropriate microneedle delivery system for BT could have a significant impact in both the medical and cosmetic industries.
Subject(s)
Botulinum Toxins, Type A/administration & dosage , Drug Delivery Systems/instrumentation , Skin/metabolism , Administration, Cutaneous , Botulinum Toxins, Type A/pharmacokinetics , Equipment Design , Humans , Needles , Skin/ultrastructure , beta-Galactosidase/administration & dosage , beta-Galactosidase/pharmacokineticsABSTRACT
Driven by constantly increasing knowledge about skin immunology, vaccine delivery via the cutaneous route has recently gained renewed interest. Considering its richness in immunocompetent cells, targeting antigens to the skin is considered to be more effective than intramuscular or subcutaneous injections. However, circumvention of the superficial layer of the skin, the stratum corneum, represents the major challenge for cutaneous immunization. An optimal delivery method has to be effective and reliable, but also highly adaptable to specific demands, should avoid the use of hypodermic needles and the requirement of specially trained healthcare workers. The P.L.E.A.S.E. (Precise Laser Epidermal System) device employed in this study for creation of aqueous micropores in the skin fulfills these prerequisites by combining the precision of its laser scanning technology with the flexibility to vary the number, density and the depth of the micropores in a user-friendly manner. We investigated the potential of transcutaneous immunization via laser-generated micropores for induction of specific immune responses and compared the outcomes to conventional subcutaneous injection. By targeting different layers of the skin we were able to bias polarization of T cells, which could be modulated by addition of adjuvants. The P.L.E.A.S.E. device represents a highly effective and versatile platform for transcutaneous vaccination.
Subject(s)
Antigens/administration & dosage , Lasers , Skin/immunology , Vaccination/instrumentation , Administration, Cutaneous , Animals , Cell Line , Cytokines/immunology , Dextrans/administration & dosage , Drug Delivery Systems , Female , Fluorescein-5-isothiocyanate/administration & dosage , Fluorescein-5-isothiocyanate/analogs & derivatives , Immunoglobulin E/blood , Immunoglobulin G/blood , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Ovalbumin/administration & dosage , Porosity , Skin/metabolism , Swine , T-Lymphocytes/immunology , Vaccination/methods , beta-Galactosidase/administration & dosageABSTRACT
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.