Preservation of exosomes at room temperature using lyophilization.

The application of exosomes as a therapeutic reagent or drug delivery vehicle can be expanded by developing a method to preserve exosomes. Although exosomes are generally stored at -80 °C, this temperature is not suitable for their handling or transportation and, therefore, other storage methods are desirable. Lyophilization is a promising storage method that can be used to preserve various substances at room temperature. In this study, we sought to develop a room temperature preservation method for exosomes using lyophilization and compared the properties of the lyophilized exosomes with ones stored at -80 °C. Lyophilization without cryoprotectant resulted in the aggregation of B16BL6 melanoma-derived exosomes, while the addition of trehalose, a cryoprotectant, prevented aggregation during lyophilization. PAGE analysis revealed that the proteins and RNA of exosomes were protected following lyophilization in the presence of trehalose. Lyophilization had little effect on the pharmacokinetics of Gaussia luciferase (gLuc)-labeled exosomes after an intravenous injection into mice. Moreover, it was found that lyophilized exosomes retained the activity of loaded gLuc and immunostimulatory CpG DNA for approximately 4 weeks even when stored at 25 °C. In conclusion, lyophilization with trehalose is an effective method for the storage of exosomes for various applications.

Bioluminescence Imaging of an Immunocompetent Animal Model for Glioblastoma.

In contrast to commonly reported human glioma xenograft animal models, GL261 murine glioma xenografts recapitulate nearly all relevant clinical and histopathologic features of the human disease. When GL261 cells are implanted intracranially in syngeneic C57BL/6 mice, the model has the added advantage of maintaining an intact immune microenvironment. Stable expression of luciferase in GL261 cells allows non-invasive cost effective bioluminescence monitoring of intracranial tumor growth. We have recently demonstrated that luciferase expression in GL261 cells does not affect the tumor growth properties, tumor cell immunomodulatory cytokine expression, infiltration of immune cells into the tumor, or overall survival of animals bearing the intracranial tumor. Therefore, it appears that the GL261 luciferase glioma model can be useful in the study of novel chemotherapeutic and immunotherapeutic modalities. Here we report the technique for generating stable luciferase expression in GL261 cells and how to study the in vitro and in vivo growth of the tumor cells by bioluminescence imaging.

Secreted luciferase for in vivo evaluation of systemic protein delivery in mice.

A naturally secreted Gaussia luciferase (Gluc) has been utilized as a reporter for bioluminescence imaging (BLI) evaluation. However, the potential application of Gluc for in vivo monitoring of systemic protein delivery, as well as its natural biodistribution, has not been studied. To examine Gluc secretion and uptake profile, we injected Gluc-encoding plasmids into mice by hydrodynamic tail-vein injection. Whole-body BLI showed that imaging quantification obtained at pawpad was directly correlated to blood Gluc activities. When gene expression was restricted to the liver by the use of a hepatic promoter, in vivo Gluc biodistribution analysis revealed the kidney/bladder, stomach/intestine, and lung as the major uptake organs. Three-dimensional BLI identified liver/stomach and lung as the main internal luminescent sources, demonstrating the feasibility of detecting major uptake organs in live animals by 3D BLI with high-background signals in circulation. Notably, Gluc levels in capillary-depleted brain samples from Gluc-injected mice were comparable to controls, suggesting that Gluc may not cross the blood-brain barrier. Gluc uptake kinetics and intracellular half-life were assessed in various types of cell lines, implicating the involvement of non-specific pinocytosis. These results suggest that Gluc-based system may provide a useful tool for in vivo evaluation of protein/agent biodistribution following systemic delivery.

Bioluminescence resonance energy transfer using luciferase-immobilized quantum dots for self-illuminated photodynamic therapy.

Photodynamic therapy (PDT) is an innovative method for cancer treatment that involves the administration of a photosensitizing agent followed by exposure to visible light. An appreciable amount of a particular light source is a key to activate photosensitizers in PDT. However, the external excitation light source is a problem for clinical application because of the limitation of tissue-penetrating properties. Additionally, the wavelength of laser emission should match the absorption wavelength of each photosensitizer for efficient generation of reactive oxygen species and cell killing. In this study, Renilla luciferase-immobilized quantum dots-655 (QD-RLuc8) was used for bioluminescence resonance energy transfer (BRET)-mediated PDT to resolve these problems. The bioluminescent QD-RLuc8 conjugate exhibits self-illumination at 655 nm after coelenterazine addition, which can activate the photosensitizer, Foscan(®)-loaded micelles for PDT. Our results show that BRET-mediated PDT by QD-RLuc8 plus coelenterazine (20 µg/mL) successfully generated reactive oxygen species (40.8%), killed ~ 50% A549 cells at 2 µg/mL equivalent Foscan(®)in vitro and significantly delayed tumor growth in vivo due to cell apoptosis under TUNEL analysis without obvious weight loss. Based on immunohistochemical observations, the proliferating cell nuclear antigen (PCNA)-negative area of tumor sections after BRET-mediated PDT was obviously increased compared to the PDT-untreated groups without an external light source. We conclude that this nanotechnology-based PDT possesses several clinical benefits, such as overcoming light penetration issues and treating deeper lesions that are intractable by PDT alone.

Targeted delivery of proteins into the central nervous system mediated by rabies virus glycoprotein-derived peptide.

PURPOSE: Delivery of therapeutic proteins across the blood-brain barrier (BBB) is severely limited by their size and biochemical properties. Here we showed that a 39-amino acid peptide derived from the rabies virus glycoprotein (RDP) was exploited as an efficient protein carrier for brain-targeting delivery. METHODS: Three proteins with different molecular weight and pI, ß-galactosidase (ß-Gal), luciferase (Luc) and brain-derived neurotrophic factor (BDNF), were fused to RDP and intravenously injected into the mice respectively. The slices of different tissues with X-Gal staining were used to examine whether RDP could deliver ß-Gal targeted into the CNS. The time-course relationship of RDP-Luc was studied to confirm the transport efficiency of RDP. The neuroprotective function of RDP-BDNF was examined in mouse experimental stroke to explore the pharmacological effect of RDP fusion protein. RESULTS: The results showed that the fusion proteins rapidly and specific entered the nerve cells in 15 min, and the t(1/2) was about 1 hr. Furthermore, RDP-BDNF fusion protein showed the neuroprotective properties in mouse experimental stroke including reduction of stroke volume and neural deficit. CONCLUSIONS: RDP provides an effective approach for the targeted delivery of biological active proteins into the central nervous system.

Bone morphogenetic protein-2 used in spinal fusion with spinal cord injury penetrates intrathecally and elicits a functional signaling cascade.

BACKGROUND CONTEXT: The use of recombinant human bone morphogenetic protein-2 (rhBMP-2) and its indications for spinal fusion continue to be expanded with recent reports citing spinal trauma application. However, there are no data establishing the effects of rhBMP-2 on the injured spinal cord. PURPOSE: The purpose of this study was to evaluate the extent of bone morphogenetic protein (BMP)-specific intrathecal signaling after application to the spine at various time points after a spinal cord injury (SCI). STUDY DESIGN: This is an in vivo rat study using a combination of the dorsal hemisection SCI and the posterolateral arthrodesis animal models. METHODS: Sixty-five female Sprague-Dawley rats underwent either a T9-T10 dorsal hemisection SCI (n=52) or laminectomy only (n=13). Spinal cord injury animals were further subdivided into four follow-up groups (n=13/group): 30 minutes, 24 hours, 7 days, and 21 days, at which time one of two secondary surgeries were performed: Eight rats per time point received either 43 microg of rhBMP-2 per side or sterile water control over T9-T11 on absorbable collagen sponges (ACSs). Animals were perfused after 24 hours, and spinal cords were immunohistochemically analyzed. Sections of the lesion were stained with BMP-specific pSmad 1, 5, 8 antibody and costained with cell-specific markers. pSmad-positive cells were then counted around the lesion. The remaining five rats (n=5/time point) had luciferase (blood spinal cord barrier [BSCB] permeability marker) injected through the jugular vein. Subsequently, spinal cords were collected and luciferase activity was quantified around the lesion and in the cervical samples (controls) using a luminometer. RESULTS: After injury, a significant increase in the number of pSmad-positive cells was observed when rhBMP-2 was implanted at the 30-minute, 24-hour, and 7-day time points (p<.05). Costaining revealed BMP-specific signaling activation in neurons, glial cells, macrophages, and fibroblasts. Spinal cord permeability to luciferase was significantly increased at 30 minutes, 24 hours, and 7 days post lesion (p<.05). A significant linear regression was established between the extent of BSCB permeability and pSmad signaling (r(2)=0.66, p=.000). CONCLUSIONS: Our results indicate that rhBMP-2 use around a spinal cord lesion elicits a robust signaling response within the spinal cord parenchyma. All CNS cell types and the invading fibroblasts are activated to the extent dependent on the integrity of the meningeal and BSCB barriers. Therefore, in the presence of a SCI and/or dural tear, rhBMP-2 diffuses intrathecally and activates a signaling cascade in all major CNS cell types, which may increase glial scarring and impact neurologic recovery.

Bootstrap method-based estimation of the minimum sample number for obtaining pharmacokinetic parameters in preclinical experiments.

Empirically, 3-6 samples at each sampling time point have been used for most preclinical one-point sampling experiments without any theoretical justification. The purpose of the present study is to propose a practical approach to determine the minimum sample number (N(min)) based on Monte Carlo simulation and a bootstrap resampling. A computer program MOMENT(BS), in which a bootstrap resampling algorithm is used to estimate mean and standard deviations of pharmacokinetic parameters, such as area under the curve and mean residence time, was applied to estimate N(min). A new simulation program, MONTE1, was developed to generate simulated data for bootstrap resampling using the model parameters including inter- and/or intra-individual variations. Then, an index, S(2)CV calculated as the sum of the squared coefficient of variation is proposed to determine the N(min). The proposed approach was applied to the actual data in preclinical experiments, and the usefulness of the approach was suggested. An issue that one-point sampling data cannot separately assess inter- and intra-individual variability is discussed.

[Real time ex vivo detection and dynamic monitoring of in vivo expression of secreted luciferase gene injected by hydrodynamic method].

We chose Gaussia luciferase (Gluc), a secreted luciferase gene as reporter to real-time detect and dynamically monitor hydrodynamic injection gene expression. First, we constructed an expression vector pAAV2neo-Gluc. Then Huh7 and HepG2 cells were transfected with pAAV2neo-Gluc and the activity of Gluc in the supernatant and cell lysates were assayed. Results showed that the Gluc activity in the supernatant was about 100 higher than that in cell lysates, indicating the expressed Gluc existing mainly as a secreted form as reported. Live bioluminescence imaging of mice hydrodynamic injected pAAV2neo-Gluc showed whole body distribution, while the pAAV2neo-Fluc primarily located in the liver. Then we injected different doses of pAAV2neo-Gluc into mice by tail-vein hydrodynamic injection, took minor amount of blood from mice tails at different time points and measured the luciferase activity to investigate dynamic changes of Gluc expression and secretion in vivo. The results suggested that the time courses of Gluc expression were highly consistent among each dose groups. The luciferase activity in blood could be detected as early as 2 h after injection, reached the peak at about 10 h and gradually decreased from then on. The expression level of Glue was positively correlated with the dose of injected plasmid DNA. To further detect the assay sensitivity of the ex vivo Gluc measurement method, we investigated three additional groups of mice injected with lower doses of 0.001 microg, 0.01 microg and 0.1 microg pAAV2neo-Gluc respectively. Results revealed that activity of Gluc in blood could be detected even at dose as low as 0.001 microg DNA, suggesting the assay sensitivity was extremely high. In conclusion, a real-time ex vivo detection method of dynamically monitoring of gene expression in vivo by hydrodynamic injection can be a valuable means for the study of gene expression regulation in vivo.

A bioluminescent assay for monoamine oxidase activity.

This article describes a novel two-step homogeneous bioluminescent assay for monoamine oxidase (MAO) that is simple, sensitive, and amenable to high-throughput screening. In the first step, MAO reacts with an aminopropylether analog of methyl ester luciferin. In the second step, a luciferin detection reagent inactivates MAO and converts the product of the first step into a luminescent signal. The amount of light produced is proportional to the amount of MAO and the time of incubation in the first step, but the luminescent signal is stable in the second step with a half-life greater than 5h. The assay has high precision, is more sensitive than current fluorescent methods, and can accurately measure the binding constants of known substrates and inhibitors. An automated screen of the Sigma-RBI Library of Pharmacologically Active Compounds (LOPAC(1280)) revealed a surprisingly high percentage of MAO inhibitors (16%) with a low false hit rate (0.9%). This implies that a significant number of compounds interact with the MAO enzymes and suggests that it is important to include MAO assays in drug metabolism studies. Other advantages of this bioluminescent assay over comparable fluorescent assays are discussed.

Poly(vinyl alcohol) nanofibers by electrospinning as a protein delivery system and the retardation of enzyme release by additional polymer coatings.

Protein-loaded (bovine serum albumin (BSA) or luciferase) poly(vinyl alcohol) (PVA) nanofibers were obtained by electrospinning. Poly(p-xylylene) (PPX, also coined as parylene) coated PVA/BSA nanofibers were prepared by chemical vapor deposition (CVD). The release of BSA from PVA nanofibers under physiological conditions was monitored by absorption spectroscopy. Burst release of BSA was noted with uncoated PVA nanofibers. In contrast, PPX-coated nanofibers exhibited a significantly retarded release of BSA depending on the coating thickness of PPX (ranging from 40 to 300 nm). Luciferase was used here as model enzyme, which after electrospinning retained its enzyme activity. This preservation of enzyme activity and the continuous release of the intact enzyme from the immersed fibers meets a fundamental prerequisite for the application of enzymes or other sensitive agents released from electrospun nanofibers under physiological conditions.

Hemin induces heme oxygenase-1 in spinal cord vasculature and attenuates barrier disruption and neutrophil infiltration in the injured murine spinal cord.

Heme oxygenase-1 (HO-1) has been shown to alter vascular function in part by attenuating inflammation. We induced HO-1 in blood vessels in the spinal cord by systemic administration of hemin. Twenty-four hours later, immediately prior to euthanasia, fluorescence conjugated Lycopersicon esculentum (tomato) lectin was given intravenously to label the vasculature. HO-1 was induced in blood vessels, particularly in the white matter, as evidenced by the immunolocalization of HO-1 in lectin positive vessels. Western blots confirmed the hemin-mediated induction of HO-1 in the uninjured spinal cord. We next examined the extent to which treatment with hemin or vehicle, 24 h prior to a moderate contusion injury, influenced early vascular dysfunction in the injured cord. All animals were euthanized 24 h after injury. Luciferase, a marker of barrier integrity, was given intravenously 30 min prior to euthanasia. The spinal cord was either prepared for quantification of luciferase activity or fixed by vascular perfusion and prepared for the immunolocalization of neutrophils. There was a significant attenuation of barrier permeability to luciferase and a significant reduction in the number of neutrophils in hemin treated animals as compared to the vehicle treated group. Together, these findings demonstrate that vascular induction of HO-1 modulates barrier function and neutrophil infiltration and suggest that this protein may be useful for limiting the early vascular dysfunction and inflammation that occurs in the acutely injured spinal cord.

Biodegradable triblock copolymer of PLGA-PEG-PLGA enhances gene transfection efficiency.

PURPOSE: A tri-block copolymer of PLGA-PEG-PLGA was used as an excipient to enhance the gene transfection efficiency of various cationic polymeric carriers. METHODS: Luciferase plasmid DNA was complexed with polyethylenimine for gene transfection. Various concentrations of PLGA-PEG-PLGA copolymer up to 0.5% were added in the transfection medium to explore whether the copolymer increased the level of gene expression. Pluronic F68 was used as a control. Various polyplexes and different cell lines were used to verify the effect of the triblock copolymer on gene transfection. The cellular uptake extent of radiolabeled plasmid was quantitatively determined as a function of PLGA-PEG-PLGA concentration. RESULTS: PLGA-PEG-PLGA copolymer significantly enhanced gene transfection efficiency at a concentration as low as 0.25% (w/v), which was more effective than Pluronic F68 at the same concentration range. The additive effect of the triblock copolymer in the transfection medium was clearly observed for various cationic polyplexes and cell lines, although the gene expression extents largely depended on polymers and cell lines used. Five- to 10-fold increment of gene transfection levels were attained in the presence of the PLGA-PEG-PLGA tri-block copolymer. The enhanced gene transfection efficiency was attributed to the increased cellular uptake of PEI/DNA complexes in the presence of the PLGA-PEG-PLGA tri-block copolymer. CONCLUSIONS: Biodegradable PLGA-PEG-PLGA tri-block copolymer that facilitates the endocytic process can be used as a novel additive in non-viral gene transfection.

Inducible expression of the megakaryocyte-specific gene glycoprotein IX is mediated through an Ets binding site and involves upstream activation of extracellular signal-regulated kinase.

Glycoprotein IX is a megakaryocyte-specific gene crucial for adequate and functional expression of the Glycoprotein Ib-IX complex. This study used phorbol 12-myristate 13-acetate (PMA) and thrombopoietin (TPO)-induced differentiation of Dami and UT-7 cells, respectively, to investigate the regulation of inducible Glycoprotein IX expression during megakaryocyte differentiation. PMA and TPO were able to modulate GPIX expression at mRNA and protein levels. Transient transfection studies using nested 5'-deletions and mutations of the GPIX promoter demonstrated the absolute requirement of an inverted Ets site 5'-ACTTCCT-3' for inducible reporter gene expression. The upstream signaling events associated with PMA and TPO-inducible expression of GPIX were also investigated. The mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase inhibitor PD98059 inhibited both PMA and TPO-inducible reporter activity in a dose-dependent manner, whereas inhibition of p38/MAPK had no significant effect. The protein kinase C inhibitor GF109203X failed to inhibit TPO-activation of the GPIX promoter in UT-7 cells. This study demonstrates that inducible expression in response to either PMA or TPO is mediated through the Ets site in the proximal promoter of GPIX and is dependent upon the upstream activation of MAPK/extracellular signal-regulated kinase.

Luciferase: a sensitive and quantitative probe for blood-brain barrier disruption.

A novel method for quantitative analysis of blood-brain barrier (BBB) disruption is described, using luciferase as a probe in a murine model system. Purified luciferase was delivered to mouse brain by osmotic BBB disruption with hypertonic mannitol; control animals received an intracarotid inoculation of saline prior to infusion of luciferase. Delivery of luciferase to brain tissue was then assessed by enzyme assay of tissue extracts, and by immunohistochemical staining. Luciferase activity in the brain of mannitol-treated animals was found to be significantly elevated (approx. sevenfold), when compared to activity in control (saline-treated) mice. This finding was confirmed by quantitative immunohistochemical staining of tissue sections, using a luciferase-specific antibody. These studies showed that there was an eight-fold elevation in the level of extravascular luciferase particles within the brain of mannitol-treated animals, as compared to controls. Taken together these data show that purified recombinant luciferase can be used as a sensitive probe, with which to study the integrity of the BBB.