Nanocarriers for biomedical applications.
Li, Shuyi ; Nguyen, Lynsa ; Xiong, Hairong 等
Introduction
Conventional drug delivery relies on oral ingestion or injection.
These methods do not allow delivery to specific anatomic sites. In
addition, macromolecular drugs (peptides, proteins, antibodies, DNA, and
RNA) are often poor candidates for delivery by these routes due to their
instability, limited tissue penetration, or rapid turnover. The global
drug market has witnessed a significant growth in development of
macromolecular drugs, yet delivery remains a limiting factor.
Bioactive glass materials, or composites of bioactive glass and
polymers, have shown promise as carriers for both small molecules and
protein therapeutics, such as vascular endothelial growth factor and
bone morphogenetic proteins [1-5]. Glass materials have also been
developed as a therapeutic radiation delivery system [6-8].
Currently, use of glass materials for delivery of therapeutic
agents requires either bonding to organic polymers or the deposition of
chemical substances directly into the glass matrix. We recently
described a more general approach for drug delivery using glass
materials [9]. The approach is based on porous-wall hollow glass
microspheres (PW-HGMs), which are micron-scale glass
"balloons" with an internal cavity bounded by a 1 (im-thick
mesoporous wall [10]. PW-HGMs are generally fabricated in sizes ranging
from 10 to 100 [micro]m. The hollow central cavity can be loaded with a
high concentration of a soluble biological therapeutic, for example a
small RNA, protein, or antibody fragment. In addition, the walls are
characterized by worm-like, interconnected channels in a silica-rich
matrix [10]. Together, these characteristics allow them to function as
carriers for diverse materials.
We hypothesized that PW-HGMs might be useful for controlled
delivery of macromolecular therapeutics. We therefore tested their
ability to interact with proteins, carbohydrates, and nucleic acids [12]
In this chapter, we summarize the results of our studies. These results
suggest that PW-HGMs may be useful as a controlled release delivery
vehicle for both protein and oligonucleotide therapeutics.
Materials and Methods
Fabrication of PW-HGMs
Details of the process have been described elsewhere, along with
methodologies for loading or filling these materials [11-14]. Briefly,
the feed for producing PW-HGMs was a 20-40 [micro]m sodium borosilicate
glass powder, similar to commercial Vycor, and containing a sulfate
blowing agent. The powder was fed into a hot zone produced by a
controlled gas-air flame, which softens the glass to allow formation of
spherical particles. The material was quenched, and a flotation process
was used to retrieve the desired initial products. These were
heat-treated to produce two phases in the thin outer walls, one rich in
silica and the other in sodium and boron. The hollow microspheres were
treated with 4 M HCl, which preferentially leaches the sodium and
boron-rich phase, leaving interconnected channels in the silica-rich
phase.
Fluorescently-labeled biopolymers
Fluorescein-labeled dextrans were obtained from Sigma-Aldrich (St.
Louis, MO). A 55 base pair 5'-Alex Fluor 546labeled oligonucleotide
was prepared as described [9]. Cy 3-labeled GAPDH siRNA was from Applied
Biosystems (Austin, TX). Alexa Fluor 594-labeled goat anti-rabbit IgG
was obtained from Invitrogen.
Biopolymer loading
Dry PW-HGMs (2-3 mg) were suspended in 50-100 [micro]l of PBS
containing 200 [micro]g/ml of biopolymer and incubated at room
temperature for 5-10 min. A portion was used for direct observation, and
the remainder was collected by gentle centrifugation, washed with 0.5 ml
of PBS or fetal bovine serum, and centrifuged again. The pellet was
resuspended in 50-100 [micro]l of PBS or fetal bovine serum for imaging.
Microscopy was performed using a Zeiss LSM 510 laser scanning confocal
microscope with a 40X or a 63X oil objective or an Applied Precision
Deltavision microscope with a 20X or a 60X oil objective.
Mouse imaging
Animal experiments were performed at the Medical College of Georgia
according to an Institutional Animal Care and Use Committee-approved
protocol. PW-HGMs (3.3 mg) were incubated with fluorescein-labeled 70
kDa dextran (200 [micro]g/ml) in 100 [micro]l PBS. Just before use,
PW-HGMs were washed twice with PBS and resuspended in 500 [micro]L of
PBS. Fluorescence images were collected using a Xenogen IVIS Imaging
System equipped with 445-490 nm bandpass filter for excitation and a
515-575 nm bandpass filter for emissions. Images were acquired with a 1
s exposure, and LivingImage 2.60 Software was used to perform a
fluorescent overlay, which allowed the subtraction of background to
produce the final images.
Results and Discussion
Interaction of PW-HGMs with biological macromolecules
The diameters of PW-HGMs ranged from 10 to 100 [micro]m. with a
typical wall thickness of 1 [micro]m (Fig. 1A). Scanning electron
microscopy (not shown, see ref. [9]) reveals the presence of ink-bottle
shaped pores in the outer shell, with diameters ranging from about 10
nm, at the narrowest point, to about 300 nm. These pores, which connect
the exterior space with the interior volume of the microspheres, are the
distinguishing characteristic of PW-HGMs.
To investigate the ability of biological polymers to penetrate the
interior volume of the PW-HGMs, we performed experiments using
fluorescently-labeled dextrans, proteins, and nucleic acids. A 500 kDa
dextran, with an estimated diameter of about 14.4 nm, failed to enter
the microspheres (Fig. 1B), whereas a smaller, 70 kDa dextran, with an
estimated diameter of 6.0 nm, freely equilibrated with the interior
volume. Results with these and other dextrans [9] suggest that the
porous walls behave as molecular sieves, passing the smaller dextrans
and excluding the larger.
Interestingly, the 70 kDa dextran accumulated in the microsphere
walls to levels exceeding its concentration in solution, a behavior not
seen with any of the larger or smaller dextrans (Fig. 1B and data not
shown, see ref [9]). The 70 kDa dextran is about the same size as the
diameter of the pores themselves, and we hypothesize that it is of a
critical size that allows a large fraction of its surface area to
contact the wall material at any given time, stabilizing its
interaction.
A fluorescently-labeled Immunoglobulin G (IgG), which is about the
same size as the 70 kDa dextran (5.3 nm) behaved similarly, with
concentration and retention in the microsphere walls. By contrast,
several smaller proteins, including ovalbumin, carbonic anhydrase, and
RNase A, entered the PWHGMs freely but showed little retention after
washing (data not shown, see ref [9]). We suggest that they are too
small to make a sufficient number of contacts with the walls of the
pores, accounting for poor retention.
We also explored the interactions of PW-HGMs with nucleic acids. A
double-stranded 55-mer DNA behaved much like the smaller dextrans and
proteins, freely entering and exiting the interior volume, with some
retention within the porous walls after washing (data not shown, see ref
[9]). A double-stranded 21-mer small interfering RNA (siRNA) behaved
similarly to the DNA, freely equilibrating between the exterior medium
and the interior cavity (Fig. 1B).
[FIGURE 1 OMITTED]
SiRNAs are of particular interest because they are in widespread
development as therapeutic agents. Efficient delivery methods are the
limiting factor in many applications (reviewed in [15,16]). Based on the
idea that the 70 kDa dextran was about the same size as the diameter of
the pores, we investigated whether it could be used to "gate"
them in order to control the uptake or release of nucleic acid cargo. We
loaded the PW-HGMs with red fluorescently-labeled siRNA, then incubated
with green fluorescently-labeled 70 kDa dextran. Prior to washing, the
RNA was seen inside the PW-HGMs, and the dextran was enriched within the
walls (Fig. 1B). After washing, some PW-HGMs retained the siRNA
(although it leached out of others) (Fig. 1B). We performed time-lapse
studies of release of siRNA from individual PW-HGMs (data not shown, see
ref [9]). The signal density for siRNA was bright initially and declined
with time. These results suggest that, in principle, PW-HGMs may be
useful as a controlled release delivery vehicle for siRNA.
Visualization of PW-HGMs in vivo
The PW-HGMs are considerably larger than blood cells and are thus
too large for systemic administration by an intravenous route. We
envision their use in applications, such as tumor embolization or tissue
regeneration, where there is a need to retain the therapeutic molecules
at a localized site. In this application, it would be ideal to be able
to visualize the initial placement of the PW-HGMs and their retention at
the desired site.
As a first step toward determining whether PW-HGMs could be
visualized in vivo, we tested the ability to detect them using a
standard small-animal imaging system. We loaded PW-HGMs with
fluorescently-labeled 70 kDa dextran, transferred them to
microcentrifuge tube, and performed imaging as described in Materials
and Methods. The method proved to be very sensitive, as evidenced by the
ability to detect even very small droplets of PW-HGM slurry on the walls
of the tube (Fig. 2A, 2B). Quantitative image analysis, reported
elsewhere, reveals a linear relationship between the amount of material
loaded and the corresponding photon counts [9]. We also injected of the
same PW-HGMs into the liver of an anesthetized, laparotomized mouse. The
image shows clear localization at the site of injection (Fig. 2C), with
some signal also around the periphery of the liver, possibly following
leakage along the injection track. In experiments elsewhere, we have
also shown the ability to detect PW-HGMs in situ following intratumoral
injection [9].
[FIGURE 2 OMITTED]
Conclusions
The results suggest that PW-HGMs may be useful for delivery of
macromolecular therapeutics. Interaction of biomolecules is strongly
size dependent, and there are several examples of molecules of a
critical size that interact strongly with the porous walls. In the case
of 70 kDa dextran, we show that this molecule can actually be used to
gate the pores, so as to obtain controlled release of a small RNA from
the interior volume.
We show also that it is possible to visualize the initial placement
of PW-HGMs in vivo using standard small animal imaging approaches.
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Shuyi Li (a), Lynsa Nguyen (b), Hairong Xiong (a,c), Meiyao Wang
(d), Tom C.-C. Hub, Jin-Xiong She (d), Steven M. Serkiz (e), George G.
Wicks (e), and William S. Dynan * (a)
* Corresponding author. Tel:706-721-8756; E-mail:
[email protected].
(a) Institute of Molecular Medicine and Genetics, Georgia Health
Sciences University, Augusta, GA 30912
(b) Small Animal Imaging, Department of Radiology,, Georgia Health
Sciences University, Augusta, GA 30912
(c) Institute of Medical Virology, Wuhan University, Hubei
Province, China 430071
(d) Center for Biotechnology and Genomic Medicine,, Georgia Health
Sciences University, Augusta, GA 30912
(e) Savannah River National Laboratory, Aiken, South Carolina 29808