Conjugation of paclitaxel on adeno-associated virus (AAV) nanoparticles for co-delivery of genes and drugs

Article abstract of DOI:10.1016/j.ejps.2012.02.022

We have investigated the use of adeno-associated virus (AAV) nanoparticles as platforms for the co-delivery of genes and drugs to cancer cells. With its regular geometry, nanoscale dimensions, lack of pathogenicity, and high infection efficiency in a wide

Full text of DOI:10.1016/j.ejps.2012.02.022

European Journal of Pharmaceutical Sciences 46 (2012) 167–172
Contents lists available at SciVerse ScienceDirect
European Journal of Pharmaceutical Sciences
journal homepage: www.elsevier.com/locate/ejps
Conjugation of paclitaxel on adeno-associated virus (AAV) nanoparticles
for co-delivery of genes and drugs
a
a
b
a,
⇑
Fang Wei , Kellie I. McConnell , Tse-Kuan Yu , Junghae Suh
a
Department of Bioengineering, Rice University, Houston, Texas, USA
Department of Radiation Oncology, The University of Texas M.D. Anderson Cancer Center, Houston, Texas, USA
b
a r t i c l e i n f o
a b s t r a c t
Article history:
We have investigated the use of adeno-associated virus (AAV) nanoparticles as platforms for the co-deliv-
ery of genes and drugs to cancer cells. With its regular geometry, nanoscale dimensions, lack of pathoge-
nicity, and high infection efficiency in a wide range of human cells and tissues, AAV is a promising vector
for such applications. We tested the covalent conjugation of paclitaxel onto surface-exposed lysine res-
idues present on the virus capsid. Immunoblotting results suggest successful attachment of drug mole-
cules to the virus nanoparticles. Favorably, the reaction conditions did not reduce the gene delivery
efficiency of the AAV vectors. Unfortunately, decrease in cancer cell viability was not observed with
our AAV–taxol conjugates. For future attempts at conjugating drugs to the AAV nanoparticle, we have
identified several improvements than can be considered to achieve the desired cytotoxicity in target cells.
Ó 2012 Elsevier B.V. All rights reserved.
Received 17 October 2011
Received in revised form 22 February 2012
Accepted 26 February 2012
Available online 3 March 2012
Keywords:
Taxol
Drug delivery
Gene delivery
Immunoblotting
Fluorescence microscopy
Cytotoxicity
1
. Introduction
A number of virus capsid-based nanoparticles are currently
and Flotte, 2008). It is considered to be one of the safest viral vec-
tors due to its nonpathogenic nature and limited immunogenicity.
AAV is a non-enveloped virus with a single-stranded DNA genome
that contains two genes: rep and cap. The virus capsid, 25 nm in
diameter, is composed of 60 subunits: VP1, VP2, and VP3 assem-
bled in a 1:1:10 ratio (Grieger and Samulski, 2005). The supramo-
lecular capsid assembly lends itself well to multivalent conjugation
of small molecules. Therefore, with its regular geometry, nanoscale
dimensions, lack of pathogenicity, and high infection efficiency in a
wide range of human cells and tissues, AAV is a highly promising
vector for biomedical applications requiring co-delivery of small
molecules and genes.
Thus far, small molecule drugs have not been conjugated to the
AAV capsid so it is unclear if AAV will be amenable for co-delivery
of drugs and genes from the same platform. Co-administration of
drugs and AAV (i.e. mixing drugs and AAV vectors in the same
injection without use of covalent conjugation) has been tested by
others (Hillgenberg et al., 1999; Jiang et al., 2011; Koppold et al.,
2006; Zhang et al., 2010). Interestingly, chemotherapeutic drugs
appear to increase gene delivery efficiencies of AAV vectors under
certain conditions through unknown mechanisms.
being investigated as drug delivery platforms (Franzen and
Lommel, 2009; Hughes, 2005; Manchester and Singh, 2006;
Steinmetz, 2010; Yoo et al., 2011). For example, the chemothera-
peutic drug paclitaxel (or taxol) has been covalently attached to
the capsid of bacteriophage MS2 through cysteine alkylation (Wu
et al., 2009). The virus capsid scaffold helped to increase the solu-
bility of taxol while maintaining similar cytotoxicity levels as free
drug. Several types of plant viruses have also been tested as drug
delivery platforms (Loo et al., 2008; Ren et al., 2007). An advantage
of using bacteriophage or plant viruses for drug delivery applica-
tions is the ability to obtain large quantities of virus material read-
ily. Unfortunately, these viruses are ineffective at delivering genes
to human cells, so therapeutic approaches desiring to co-deliver
drugs and genes via these delivery vectors may require substantial
re-engineering of the viruses to achieve target efficacies. To over-
come this problem, mammalian viruses can be used as alternatives
for applications requiring co-delivery of drugs and genes.
Adeno-associated virus (AAV) is a promising mammalian virus
vector commonly used for gene therapy applications (Mueller
In this study, we investigate the covalent conjugation of taxol as
a model drug onto the AAV scaffold. Taxol was first converted to
taxol-NHS ester and then conjugated to the approximately 300 sur-
face-exposed lysine residues on the AAV capsid (Fig. 1). Results
indicate the conjugation reaction does not adversely impact gene
delivery or cytotoxicity of the virus nanoparticles.
⇑
Corresponding author. Address: 6100 Main Street, Houston, Texas 77005, USA.
Tel.: +1 713 348 2853; fax: +1 713 348 5877.
E-mail address: jsuh@rice.edu (J. Suh).
0
928-0987/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved.
doi:10.1016/j.ejps.2012.02.022

1
68
F. Wei et al. / European Journal of Pharmaceutical Sciences 46 (2012) 167–172
was loaded with 1 ml virus iodixanol solution, washed with
0 mM Tris buffer and eluted with Tris buffer with high NaCl con-
1
centrations (1 M). Elution was dialyzed against 10 mM sodium
phosphate buffer using slide-a-lyzer dialysis cassette (Pierce) with
a MWCO of 10 kD. Titers were determined by quantitative real-
time PCR (QPCR) using SYBR Green PCR Master Mix (Applied Bio-
systems, Foster City, CA) with primers against the CMV promoter.
For this study, the recombinant AAV vector encoding GFP is called
‘
‘wtAAV’’ to indicate the capsid is wildtype.
2.3. AAV–taxol conjugations
Taxol-NHS ester dissolved in DMSO was added to AAV2 virus in
DPBS (10 mM sodium phosphate, 300 mM NaCl) at a molar ratio of
00 k taxol-NHS per virus capsid. The final concentration of DMSO
1
is less than 10% to ensure homogeneity of the solution. The reac-
tion was protected from light and rocked for over 12 h at 4 °C.
The mixture was transferred to a dialysis cassette (Piece, 10 kD
cutoff) and dialyzed against acetone:DPBS (50:50) for 1 h and then
dialyzed against three buffer exchanges of DPBS only. For this
study, the recombinant AAV vector encoding GFP and conjugated
with taxol is called ‘‘AAV–taxol’’.
Fig. 1. Surface-exposed lysines on AAV capsid. Approximately 300 of the 1080
lysine residues per virus capsid are surface-exposed (light blue). Surface-exposed
lysines were determined using VIPERdb, based on residue radius and solvent
accessible surface area (5 lysines per subunit ꢀ 60 virus subunits per capsid). Image
generated by Pymol. (For interpretation of the references to color in this figure
legend, the reader is referred to the web version of this article.)
2.4. Western blots
Western blots were performed by denaturing virus in NuPAGE
LDS buffer (Invitrogen, Carlsbad, CA) for 10 min at 70 °C. Samples
were loaded on NuPAGE 7% Tris–acetate gels (Invitrogen) and
run for 90 min. Samples were transferred to Hybond ECL nitrocel-
lulose membrane (GE Healthcare, Piscataway, NJ) and blocked
using 5% skim milk in PBS-T (137 mM NaCl, 2.7 mM KCl, 10 mM
2
. Methods
2.1. Synthesis of taxol-NHS ester
The synthesis followed a published procedure by Luo and Prest-
2 4 2 4
Na HPO , 2 mM KH PO , 0.1% Tween-20). Membranes were either
wich (1999). Briefly, dry pyridine (10-fold molar excess) was added
to a stirring solution of taxol (LC laboratories) and succinic anhy-
dride (1.2 equivalents) in dichloromethane at room temperature.
The reaction mixture was stirred for 3 days at room temperature
and then concentrated in a rotary evaporator. The residue was dis-
solved in dichloromethane, and taxol-2 -hemisuccinate was puri-
fied by recrystallization with water. Mass calculated for
C
9
incubated for 1 h with B1 antibody (American Research Products,
Belmont MA) or anti-taxol antibody (Santa Cruz Biotechnology,
Santa Cruz, CA), each at a 1:50 dilution in PBS-T. Samples were
rinsed with PBS-T and incubated with a 1:2000 dilution of HRP-
conjugated goat anti-mouse secondary antibody for 1 h (Jackson
Immunoresearch, West Grove, PA). Samples were rinsed with
PBS-T and incubated with Lumi-Light Western blotting substrate
0
+
+
+
51
H55NO17: 953.99. Found: [M] : 954.1, [M+Na] : 977, [M+K] :
92.7 (Fig. S1).
Next, N-hydroxysuccinimido diphenyl phosphate (SDPP) was
(
Roche Diagnostics, Indianapolis, IN). Samples were then digitally
imaged using a FluorChem FC2 imager.
prepared from diphenylphosphoryl chloride, N-hydroxy-
succinimide, and triethylamine in dichloromethane as previously
described (Ogura et al., 1980). Crude SDPP was washed with ether,
dissolved in ethyl acetate, washed with water, dried with magne-
sium sulfate, and concentrated in vacuo. Triethylamine (4 equiva-
2
.5. Dot blots
Dot blots were performed by diluting virus in PBS (137 mM
2 4 2 4
NaCl, 2.7 mM KCl,10 mM Na HPO , 2 mM KH PO ) and then apply-
0
ing the virus samples to a Bio-Dot microfiltration apparatus (Bio-
Rad, Hercules, CA). Membranes were blocked as for Western blot-
ting and incubated with A20, A1, B1, or anti-taxol antibodies
lents) was added to a solution of taxol-2 -succinate and SDPP (1.5
equivalents) in acetonitrile. The reaction was stirred for 6 h at
room temperature and then concentrated in vacuo. The residue
was dissolved in ethyl acetate and hexane and purified on silica
(
1:200 dilutions). Secondary antibody application and visualization
were performed as with Western blots.
f
gel. Thin Layer Chromatography (TLC) showed R of 0.52. Mass cal-
+
+
culated for C55
H
58
N
2
O
19: 1051.07. Found: [M] : 1050.9, [M+Na] :
+
2.6. Transmission electron microscopy (TEM)
1
073.6, [M+K] : 1089.7 (Fig. S2).
5
–10 ll of wtAAV or AAV–taxol samples were applied to contin-
2
.2. Virus production
uous carbon-coated copper grids (Ted Pella) and stained with
freshly prepared 0.75% uranyl formate solution. JOEL 2010 electron
microscope operating at 120 kV was used to image the samples.
Virus was produced as described elsewhere (Xiao et al., 1998).
Briefly, pXX2 (AAV2 rep and cap genes) and AV-GFP (CMV pro-
moter and GFP gene between viral ITRs) were transfected with
pXX6 (adenoviral helper genes) into HEK 293T cells using poly-
ethylenimine. All three plasmids were generously provided by
Dr. R. Jude Samulski (University of North Carolina at Chapel Hill).
Virus was separated using ultracentrifugation and an iodixanol
step gradient. Virus iodixanol solution was further purified by
using HiTrap Heparin HP columns (GE Healthcare). The column
2.7. Gene expression analysis
HeLa cells were seeded on glass coverslips in 24 well plates
such that cell confluency would reach 75% at 24 h post seeding.
Virus samples (both unconjugated AAV and AAV–taxol) were ap-
plied at an MOI of 10,000 in duplicate wells in 200
media. Additional media (1 ml complete media) was added 4 h
lL complete

F. Wei et al. / European Journal of Pharmaceutical Sciences 46 (2012) 167–172
169
0
post-infection. After 48 h, samples were fixed with 4% paraformal-
dehyde in PBS and mounted onto glass slides. Samples were im-
aged using a Zeiss LSM 5 LIVE confocal microscope.
we have chosen to modify the hydroxyl group at the 2 position.
An ester bond was used to create a succinic linker, which was then
converted to an NHS ester for conjugation to AAV. Ester bonds are
known to easily hydrolyze in weak basic or acid conditions (Dosio
et al., 1997). Once an AAV–taxol conjugate enters a cell, the weakly
acidic conditions should induce the breakage of the ester bond,
releasing taxol molecules into the cell. Our AAV–taxol conjugates
were created through three synthesis steps (Fig. 2). First, taxol
2.8. MTT viability assay
HeLa cells were plated so that cell density would be 40-50% at
2
4 h post seeding. Virus (wtAAV2 and AAV2-taxol) was added at
an MOI of 50,000. Three days later, media was removed and MTT
5 mg/ml, Sigma Aldrich) was added in a 1:2 ratio with fresh med-
ia. Three hours later, media was removed and samples were dis-
solved in 250 L DMSO. For each sample, the absorbance was
measured at 570 nm using a Magellen plate reader.
0
was converted into 2 -taxol succinate following a previously pub-
lished method (Luo and Prestwich, 1999). The mass of the product
was confirmed by mass spectrometry (Fig. S1). Then, SDPP was re-
(
0
acted with 2 -taxol succinate to form taxol-NHS ester. The final
l
product was purified by column chromatography and taxol-NHS
ester mass was confirmed by mass spectrometry (Fig. S2). Finally,
taxol-NHS in DMSO was conjugated to AAV capsids in DPBS buffer.
The un-reacted taxol was removed by dialysis against 50% acetone/
DPBS, as dialysis with DPBS alone was unsuccessful in removing
free taxol (data not shown).
3
. Results and discussion
3.1. Synthesis of AAV–taxol conjugates
Taxol was chemically modified in preparation for conjugation to
the approximately 300 surface exposed lysine residues on the AAV
capsid (Fig. 1). Chemically modifying the hydroxyl group at posi-
tion 2 and 7 does not affect the drug potency (Deutsch et al.,
3.2. Dialysis against 50% acetone in DPBS does not affect capsid
structure
0
1
989; Lataste et al., 1984; Parness et al., 1982). Due to steric ef-
Fifty percent acetone in water has been used previously to pur-
ify macromolecules conjugated to taxol (Luo and Prestwich, 1999;
Luo et al., 2000). We evaluated the effects of exposure to 50% ace-
tone on virus capsid stability. One batch of virus was divided into
fects, hydroxyl groups at the 1 and 7 positions are not as active
as hydroxyl group at the 2 position (Fig. 2) (Deutsch et al., 1989;
Magri and Kingston, 1986; Mathew et al., 1992). Because of this,
0
AcO
O
OH
O
AcO
O
OH
7
O
Ph
NH
O
O
Ph
NH
O
Step 1
Ph
O
H
1
O
2
’
OAc
Ph
O
O
HO OCOPh
H
O
OAc
OH
HO OCOPh
1
2
O
OH
Step 2
AcO
O
OH
O
Ph
NH
O
O
Step 3
Ph
O
H
OAc
O
HO
OCOPh
O
O
O
O
N
O
3
4
Fig. 2. Synthesis of AAV–taxol. Step 1: succinic anhydride was added to taxol (1) in dichloromethane, pyridine was then added into the reaction, and the reaction was kept at
0
0
room temperature for 3 days to form 2 -taxol succinate (2). Step 2: SDPP was added to 2 -taxol succinate in acetonitrile, triethylamine was then added, and the reaction was
kept at room temperature for 3 h to form taxol-NHS ester (3). Step 3: Taxol-NHS ester in DMSO was added to DBPS containing AAV nanoparticles. The reaction was kept at
4
°C overnight to form AAV–taxol (4). SDPP: N-hydroxy-succinimido diphenyl phosphate. DMSO: Dimethyl Sulfoxide. DPBS: Dulbecco’s Phosphate Buffered Saline.

1
70
F. Wei et al. / European Journal of Pharmaceutical Sciences 46 (2012) 167–172
two portions – one portion was dialyzed against 50% acetone/
water and the other portion was dialyzed against 50% acetone/
DPBS. Dialysis solution was changed after 1 h followed by three
additional changes in DPBS. Dot blot analysis was used to evaluate
the integrity of the virus capsid after dialysis. Three antibodies
were used to characterize the capsid: A20 (binds to only intact
virus capsids), A1 (binds the N-terminus of VP1, inaccessible for in-
tact capsids), and B1 (binds C-terminus of all three VPs and is only
accessible when capsid has been denatured). Dot blot staining
B1
α-taxol
1
2
1
2
1
02 kDa
VP1
VP2
VP3
76 kDa
52 kDa
(Fig. S3) indicates that AAV capsids remain intact after dialysis
against 50% acetone in both DPBS and water. This is suggested by
positive A20 staining and the absence of A1 or B1 staining (de-
tected in denatured virus). Because virus dialyzed against 50% ace-
tone/water began to aggregate after 1 h, 50% acetone/DPBS was
used as dialysis solvent for removal of un-reacted taxol-NHS ester.
Overall, dialysis of AAV against 50% acetone in DPBS does not ad-
versely impact capsid stability.
Fig. 4. Western blot of AAV–taxol conjugates show successful conjugation of taxol
to VP subunits. B1 antibody binds to the C-terminus of all three capsid proteins and
a-taxol antibody binds to taxol. Lane 1: AAV–taxol conjugate. Lane 2: wtAAV.
intense VP3 band as compared to VP1 and VP2. Both wtAAV and
AAV–taxol conjugations yield similar B1 staining (Fig. 3, left). An
anti-taxol antibody was used to detect the presence of taxol conju-
gated on the AAV capsid subunits. As shown in Fig. 4 (right),
unconjugated wtAAV capsid does not yield positive taxol staining.
However, AAV–taxol conjugate shows positive anti-taxol staining
at the correct molecular weights of VP1, VP2, and VP3, suggesting
successful conjugation of taxol onto the AAV capsid.
Capsid integrity of the AAV–taxol conjugation was further con-
firmed by TEM. Fig. 5 displays wtAAV (left panel) and AAV–taxol
(right panel) virus images. We observe similar capsid morpholo-
gies in both samples, supporting the structural integrity of the con-
jugated virus. Overall, we have demonstrated successful
conjugation of taxol onto AAV capsids and the resulting conjugates
being structurally intact.
3.3. Confirmation of successful AAV–taxol conjugation
Dot blot analysis was performed to verify the successful conju-
gation of taxol-NHS onto AAV capsids (Fig. 3). Positive A20 staining
for AAV–taxol conjugate indicates that AAV capsids remain intact
after undergoing the reaction and subsequent dialysis against
5
0% acetone in DPBS. Notably, positive a-taxol staining indicates
the presence of taxol on the conjugated virus capsids. Mock conju-
gation reactions were performed to ensure the complete removal
of free taxol-NHS after 50% acetone/DPBS dialysis. These conjuga-
tions were performed as an exact parallel to the AAV–taxol conju-
gation, but without the addition of virus. A lack of positive a-taxol
staining for the dialyzed taxol-NHS control indicates that our 50%
acetone/DPBS dialysis protocol is successful for removing all free
taxol.
3
.4. Conjugation does not negatively impact gene delivery or
cytotoxicity of virus nanoparticles
Western blot analysis was performed to further confirm the
successful conjugation of taxol to the AAV capsid (Fig. 4). B1 anti-
body was used to detect the common C-terminus of the three cap-
sid proteins: VP1, VP2 and VP3. As subunits are incorporated into
the AAV capsid in a 1:1:10 ratio (VP1:VP2:VP3), we observe a more
We next evaluated if the conjugation reaction affected the func-
tional properties of AAV. First, the ability of AAV–taxol to deliver
genes to cells was tested via a transduction assay. Unconjugated
and conjugated virus nanoparticles were added to HeLa cervical
cancer cells and imaged for GFP transgene expression 48 h later
(Fig. 6A). No difference in gene expression is observed between
A20 A1 B1 α-taxol
the two samples, indicating the conjugation reaction did not nega-
tively impact the ability of AAV to deliver genes. Next, the cytotox-
AAV
icity of virus nanoparticles was tested via
a MTT assay.
Unconjugated and conjugated virus nanoparticles were added to
HeLa cervical cancer cells and probed for metabolic activity 72 h la-
ter (Fig. 6B). No difference in viability is observed between the two
samples, indicating the conjugation of taxol onto the virus capsid
did not impact the cytotoxicity of AAV. Overall, conjugation of tax-
PBS only
AAV-taxol after dialysis
Taxol-NHS after dialysis
Taxol-NHS only
AAV denatured
Fig. 3. Dot blot of AAV–taxol conjugates show successful conjugation of taxol to
intact virus capsids. Four antibodies were used to characterize the capsid: A20
binds intact virus capsids, A1 binds N-terminus of VP1 (for capsids that have been
activated by temperature shock (Musick et al., 2011)), B1 binds C-terminus of all
three VPs (only accessible when capsid has been denatured), and a-taxol binds to
taxol. The AAV–taxol after dialysis sample shows robust A20 and taxol signals,
indicating the virus capsids are intact and conjugated with taxol. Unconjugated AAV
nanoparticles are detected by A20 but not by any of the other antibodies, indicating
the capsids are intact and do not have any associated taxol. Free taxol-NHS dialyzed
against 50% acetone in DPBS is included as a control to show our dialysis method
effectively removes free taxol. Unconjugated AAV denatured via heat treatment at
Fig. 5. TEM images of wtAAV (left) and AAV–taxol (right). Both samples were
negatively stained with fresh 0.75% uranyl formate solution. We observe full
capsids (light center) and empty capsids (dark center) for both samples.
7
5 °C and PBS only samples are included as controls for the AAV antibodies. Taxol-
NHS only sample is included as a positive control for the -taxol antibody.
a

F. Wei et al. / European Journal of Pharmaceutical Sciences 46 (2012) 167–172
171
quantify the amount of taxol conjugated per virus capsid, our
experimental limit was based on virus MOI that is reasonable to
use in cell studies. This negative result can be due to a couple of
factors. First, again, since we do not know quantitatively how
much taxol we are delivering with each virus nanoparticle, we
may be well below the LD50 of taxol in our cell study. Alterna-
tively, the conjugated taxol may experience difficulty in releasing
from the virus nanoparticle. Although the previous work using
MS2 capsid utilized a similar ester bond for release of taxol, it
may be that in the context of our virus platform the cleavage is
not as efficient.
For future attempts to conjugate drugs to the AAV capsid, sev-
eral improvements should be considered. First, the virus should
be produced via a high-yield method, such as by using the baculo-
virus system (Aslanidi et al., 2009; Kotin, 2011; Negrete and Kotin,
2
008a,b; Urabe et al., 2002). The typical yield of such production
1
4
methods is reported to be 1L of 1 ꢀ 10 vector particles. With
the greater amount of virus material in hand, MS analysis of the
conjugations will be more easily obtainable. This quantitative
information will be critical in determining the efficacy of the
drug-virus nanoparticle designs. Second, other types of cleavable
linkers can be tested. For example, hydrazone linkers are highly
acid-labile and have been shown to be effective at releasing drugs
in cellular endosomes (Brunel et al., 2010; Dirksen and Dawson,
Fig. 6. Taxol conjugation to AAV nanoparticles does not negatively affect gene
delivery efficiency or cell viability. (A) Virus nanoparticles, unconjugated or
conjugated with taxol, were added to HeLa cells at an MOI of 10,000. At 48 h
post-addition, GFP expression was imaged using fluorescence microscopy. Scale bar
is 100 lm. Images are representative of three independent experiments. (B)
Cytotoxicity of virus nanoparticles, unconjugated or conjugated with taxol, was
tested via MTT viability assay. Virus nanoparticles were added to HeLa cells at an
MOI of 50,000. At 72 h post-addition, metabolic activity of cells was measured using
MTT. Data is normalized to the control samples.
2
008). With improvements in AAV–taxol production, we will be
able to quantify drug loading, drug release, as well as in vivo effi-
cacy and immunogenicity.
ol on AAV does not appear to affect gene delivery efficiency or
cytotoxicity of the virus nanoparticles.
Overall, although we were unable to show effective killing of
cancer cells using our virus-taxol nanoparticles, we have learned
important lessons that can be used in future attempts at conjugat-
ing drugs to AAV. With improvements in virus source and possible
alternatives in linker choice, we are optimistic of the AAV capsid
being able to withstand the conjugation reaction conditions re-
quired to attach taxol, or other types of hydrophobic drugs, for
co-delivery of drugs and genes.
3.5. Discussion
Due to low solubility issues, taxol is currently delivered clini-
cally with the aid of chemical solvents. Unfortunately, solvents
may lead to undesirable side effects. Conjugating taxol to delivery
platforms, such as virus nanoparticles, should improve the solubil-
ity of the drug as well as enhance the therapeutic payload.
We have tested the ability of AAV nanoparticles to be chemi-
cally reacted with drug molecules. Our results indicate that the
capsid is able to withstand the conjugation and purification condi-
tions, which included dialysis against 50% acetone in DPBS (Figs. 3
and S3). Our immunoblotting results show successful conjugation
of taxol to the virus capsid (Figs. 3 and 4). Furthermore, the conju-
gation process does not negatively impact gene delivery efficiency
or cytotoxicity properties of the AAV capsid (Fig. 6), supporting fu-
ture attempts at conjugating drugs to the virus.
Acknowledgments
The authors would like to thank Vamseedhar Rayaprolu and
Brian Bothner for help with ESI mass spectrometry analysis. The
work was supported by the Department of Defense Breast Cancer
Research Program (BC087034).
Appendix A. Supplementary data
During the course of our work, we were met with several chal-
lenges. The most considerable challenge was the difficulty in
obtaining large quantities of virus material through the common
virus preparation protocol involving the transfection of HEK293T
producer cells. An average virus preparation yields approximately
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.ejps.2012.02.022.
References
1
1
12
1
.5 mL of 5 ꢀ 10 –1 ꢀ 10 genomes/mL after iodixanol separa-
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