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Avidin-coated baculoviral vectors-biotinylated ultra-small superparamagnetic iron oxide nanoparticles

, PhD
National Center for Biotechnology Information, NLM, NIH, Bethesda, MD, vog.hin.mln.ibcn@dacim

Created: ; Last Update: April 30, 2008.

Chemical name:Avidin-coated baculoviral vectors-biotinylated ultra-small superparamagnetic iron oxide nanoparticles
Abbreviated name:Baavi-bUSPIO
Agent category:Protein, nanoparticle (virus)
Target category:Other
Method of detection:Magnetic resonance imaging (MRI)
Source of signal/contrast:Iron oxides
  • Checkbox In vitro
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No structure is currently available in PubChem.



Gene therapy delivers a functional gene into a target cell to restore the physiological levels of the deficient gene (1, 2). This therapeutic methodology has been used in the clinic to treat diseases and cancers induced by genetic disorders (3). For example, a cytotoxic gene can destroy a specific type of cancer cell without excessive damage to normal tissue (4). Because viruses are inherently capable of packaging nucleic acids and delivering them to susceptible cells with high efficiency, they become a preferred system for natural gene therapy delivery (5). Viral vectors can be engineered with a gene expression cassette or converted to a targeted vector. Both methods allow delivery of the gene of interest (transgene) to the desired cells or tissues (3). As a gene therapy vector, the viruses need to meet three criteria: safety, high transfer efficiency, and reliable transgene expression. Assessment of the development of viral vectors in vivo becomes an important step in the implementation of gene therapy in the clinic. With its inherent high spatial resolution, magnetic resonance imaging (MRI) can be effectively used to examine the pharmacokinetics and development of viral vectors in vivo, especially their adsorption, distribution, biotransformation, and excretion.

Baculoviruses (BV) are a family of rod-shaped viruses with a circular, double-stranded genome ranging from 80 to 180 kbp that can encode ~100–200 proteins (6). BV mainly replicates in insect cells, not in mammalian cells, through the function of a glycoprotein gp64 (512 amino acids) located on the viral envelop (7). BV can efficiently mediate gene transfer in mammalian cells in the presence of active mammalian cell promoters such as the cytomegalovirus promoter. The inherent inability of BV to replicate in mammalian cells makes it an attractive candidate for gene therapy (7) and for protein expressions such as ion channels, G-protein–coupled receptors, nuclear receptors, and transporters (5). As a gene delivery vector, BV has several unique features, including extremely high levels of gene expression and accessibility and an excellent biosafety profile (5). A BV with avidin is a recombinant BV that has avidin inserted into its native envelope glycoprotein gp64 (8). The presence of avidin on the surface of the BV provides a versatile platform, the avidin displaying BV (Baavi), for binding a variety of biotinylated molecules, including imaging probes (9). The insertion of avidin also increases the transduction efficiency of BV by 5–26-fold (8).

Complexation of Baavi with biotinylated ultra-small superparamagnetic iron oxide nanoparticles (bUSPIO) forms an agent (Baavi-bUSPIO) used for MRI imaging of recombinant BV (9). The USPIOs contain an icosahedral core of superparamagnetic crystalline Fe3O4 (magnetite) (10). They possess a high magnetic susceptibility that results in a significant induced magnetization inside a magnetic field. This creates microscopic field gradients that diphase nearby protons and cause a shortening of T2 relaxation times (11). With a 50-nm USPIO and a virus of approximately 25 nm × 200 nm, each bUSPIO binds ~1–2 viruses. The Baavi includes a LacZ transgene, which is a gene for encoding of a common enzymatic reporter protein β-galactosidase (β-gal) (12). The presence of β-gal allows for quantitative measurement of transduction efficiency of Baavi-bUSPIO in vitro and/or histological evaluation of Baavi-bUSPIO in targeted tissues ex vivo.



Baavi-bUSPIO was obtained by conjugation of Baavi with bUSPIO (9). Baavi was prepared in several steps (8). First, a β-gal (LacZ) cassette was cloned into a PυuII site of modified pFastBac1 donor vector under a cytomegalovirus (CMV) promoter to serve as the Baavi backbone. The SrfI site of the produced plasmid (BVlacZ) was ligated to a linker with a StuI site. Second, the avidin-gp64 sequence was included in the BVlacZ under the control of the polyhedrin promoter. The resulting 408-bp fragment was digested and subcloned into the PstI site of a pBACSurf-1 vector. Third, the EcoRV/SnaBI fragment (2,173 bp) containing the avidin-gp64 cassette was cloned between the StuI sites of BVlacZ to produce the Baavi donor plasmid. Finally, the recombinant Baavi viruses were generated using the standard Bac-to-Bac method. Successful avidin-gp64 fusion protein expression was confirmed with immunoblot assays. At the same time, bUSPIOs were obtained by coating 50-nm USPIOs with d-biotin in the presence of O-benzotriazole N,N,N’,N’-tetramethyl-uronium-hexafluorophosphate as a coupling agent (9). Baavi (2.5 × 1010 infective particle/ml) was mixed with bUSPIOs at a ratio of ~100 spheres per infective virus particle and incubated overnight at 4ºC to produce Baavi-bUSPIO.

In Vitro Studies: Testing in Cells and Tissues


The complex of Baavi-bUSPIO was examined with atomic force microscopy (9). Approximately 1–2 Baavi particles (200–300 nm long and 24–27 nm wide) were found per bUSPIO (42 ± 7 nm). The cytotoxicity of Baavi-bUSPIO was assessed with 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) viability assay (no data were reported) (9). The transduction efficiency of Baavi-bUSPIO was assessed in HepG2 cells (9). The cells were stained with X-gal to visualize β-gal–expressing cells 24 hr after incubation with Baavi-bUSPIO. The presence of Baavi-bUSPIO appeared to increase the transduction efficiency and transgene expression level.

Animal Studies



Raty et al. studied the distribution of Baavi-bUSPIO in rats with MRI at 4.7 T (9). Doses of Baavi-bUSPIO were injected intraventrically into rat brains, and MRI images were collected 2 h after injection and 1, 3, 6 days (n = 7), and 14 days (n = 2). A 10-μl solution that contained bUSPIO in 500 ng Fe and 2.5 × 108 infective Baavi was injected into the right ventricle. The Baavi was expected to deliver the cargo bUSPIO to choroid plexus cells. MRI images revealed that Baavi-bUSPIO was located in the ipsilateral ventricle of rat brains as soon as 2 h after injection, and the signal remained detectable for 2 weeks. In comparison, injection of an equal amount of bUSPIO did not generate similar contrast. Two types of histological staining were used to further confirm the distribution of Baavi-bUSPIO (9). Rats were euthanized at different time points after MRI sessions. Iron content was examined with diaminobenzene-enhanced Prussian Blue staining. Consistent staining was found in the cuboid epithelial cells of the choroid plexus in tissue sections from the ipsilateral side, whereas the rest of the brain remained unstained. The expression of the nuclear-targeted LacZ transgene in the cryosectioned brain was examined with β-gal staining. Rats injected with Baavi-bUSPIO exhibited a large number of positively stained cells in the choroid plexus, which was comparable to those injected with wild-type Baavi that contained LacZ. These stained tests confirmed the MRI observations.

Other Non-Primate Mammals


No publication is currently available.

Non-Human Primates


No publication is currently available.

Human Studies


No publication is currently available.


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