A biodegradable adamantane polymer with ketal linkages in its backbone for gene therapy

Article abstract of DOI:10.1039/c5cc05242d

In this report we present a polyketal, termed pADK, which has adamantane groups embedded in its backbone, and degrades into neutral excretable compounds. pADK was synthesized via click chemistry and had a M_W of 49472 and a PDI of 1.74. We demonstrate here that pADK can increase the transfection efficiency of CD1800 (PEI of 1800 modified β-cyclodextrin) 60 fold, yet cause no increase in toxicity.

Full text of DOI:10.1039/c5cc05242d

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COMMUNICATION)
A biodegradable adamantane polymer with ketal
linkages in its backbone for gene therapy
Santanu Maitya, Priya Choudharyb, Manu Manjunathb, Aditya Kulkarnib, Niren Murthya*!
!
Abstract. In this report we present a polyketal, termed
OH
O
PEI1800
O
pADK, which has adamantane groups embedded in its
backbone, and degrades into neutral excretable compounds.
pADK was synthesized via click chemistry and had a MW of
49,472 and a PDI of 1.74. We demonstrate here that pADK
can increase the transfection efficiency of CD1800 (PEI of
1800 modified β-cyclodextrin) by 60 fold, yet causes no
increase in toxicity.
O
N
N
O
O
N
N
N
N
OH
OH
O
n
+"
O
6
O
OH
OH
Adamantane grafted
polyketal (pADK)
PEI 1800 modified cationic
β-cyclodextrin (CD1800)
i)
O
N
N
O
O
O
N
N
N
N
β-Cyclodextrin (β-CD) based cationic drug and gene delivery
vehicles have found numerous applications in gene therapy because
of their lower toxicity in comparison to other poly-cations.1-12 Most
β-CD based gene delivery vehicles are composed of either cationic
β-CDs supramolecularly complexed with non-degradable guest
polymers or directly conjugated with non-degradable polymers. 2-6, 8,
n
PEI 1800
ii)
9
For example, Thompson and co-workers have developed poly-
Nucleic acid
vinyl alcohol (PVA) based drug delivery vehicle that have numerous
cholesterol or adamantane groups pendant on to the PVA, via an
acid degradable acetal linkage,13 which can efficiently deliver
siRNA. The cholesterol or adamantane embedded PVA reversibly
binds to cationic-β-CD and the resulting host-guest complex has a
transient high charge density on the surface and therefore has
minimal toxicity in-vitro.14-17 The high surface charge density
complex binds with siRNA with high affinity to form a polyplex,
which is easily internalized by cells via endocytosis. After
endocytosis, the polyplex is trafficked to the late endosome, where
the acetal linkage hydrolyzes and the resultant complex degrades
into monomeric cationic-β-CD, cholesterol and non-degradable
PVA.
therapeutic
!
Nucleic acid
therapeutic complex
!
iii)
Late endosome
The non-degradable PVA can be problematic for translational
studies due to its accumulation within cells in vivo and long term
persistence.18 To address this limitation, in this report we present a
poly-ketal that has adamantane units on its backbone, termed pADK,
which degrades into low molecular weight excretable compounds
and can self-assemble with cationic-β-CD and deliver DNA
efficiently to cells. The chemical structure of pADK is shown in
Figure 1, is composed of a polyketal that has adamantane groups
embedded in its backbone, flanked by triazole groups, and in the
presence of acid degrades into low molecular weight diols and
acetone, both of which are membrane permeable and potentially
excretable. pADK is designed to complex cationic-β-CD, generating
a multivalent high density polycation that can complex DNA and
deliver it into cells. However, after endocytosis and trafficking into
the late endosome, pADK hydrolyzes into small molecules, due to
hydrolysis of the ketal linkage, and should cause endosomal
disruption via the colloid osmotic mechanism.19-26 Importantly, the
cellular degradation products of pADK are an adamantane diol and
acetone, both of which should cause minimal toxicity. For example
iv)
N
N
N
N
HO
OH
N
N
O
+"
Figure 1: Polyadamantane-ketal (pADK), an adamantane
grafted biodegradable polymer for nucleic acid delivery: i)
pADK forms an inclusion complex with cationic-β-CD; ii) the
host-guest complex electrostatically binds to nucleic acid
therapeutics to form nanoparticles; iii) the pADK polyplex
enters cells via endocytosis; iv) the acidic environment of the
endosome degrades pADK into small molecules, causing the
dissociation of nucleic acids and release into the cytoplasm,
pADK degrades into membrane permeable small molecules
that are excreted.
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Br
(A)%
(a)
(b)
+
O
O
R
R
2
O
O
3
7: R = N3
4
(d)
(c)
O
6: R = Br
5: R = OH
(e)
O
O
+
OTMS
10
8
9
100
90
80
70
60
50
40
30
20
10
0
(B)%
Scheme 1: Synthesis of pADK monomers: (a) AIBN, Bu3SnH,
o
Toluene, 100 C, 61%; (b) LiAlH4, THF, rt, 94%; (c) CBr4, PPh3,
With Cyclodextrin%
DCM, 0 oC to rt, 84%; (d) NaN3, DMF-water (6:1), 50 oC, 97%; (e)
TMS-OTf, DCM, -78 oC, 63%.
the released acetone should be metabolized to pyruvate and used
for ATP production, and the 3-adamantaine-1,5-pentadiol should
get excreted on the time scale of hours to days based on its small
size and anticipated membrane permeability.20,27,28
The synthesis of pADK is shown in scheme 2 and was
accomplished via a click polymerization between the diazide
With PBS%
0
10
20
30
adamantane (7) and di-ketal alkyne (10).
Diazide 7 was
Time (hour)%
synthesized from 5,6-dihydro-2H-pyran-2-one 2, via a 1,4-addition
with the adamantane radical 3, followed by reduction with lithium
aluminium hydride, bromination and nucleophilic substitution with
sodium azide. The monomer 10 was synthesized in one step via
Figure 2: pADK microparticles respond to methyl-β-cyclodextrin (A)
SEM image of rhodamine encapsulated in pADK microparticle (500
µM scalebar); (B) pADK microparticles respond to 5 mM methyl-β-
cyclodextrin.
trimethylsilyl
trifluoromethanesulphonate
catalysed
ketal
formation of acetone (Scheme 1). pADK was synthesized via the
click reaction between 7 and 10. A key challenge in the synthesis
of pADK was its solubility, which prevented successful
polymerization in a wide variety of solvents, such as DMF,
chloroform, tetrahydrofuran and dichloromethane. We identified a
mixture of 1:1 tetrahydrofuran and toluene as a solvent, which has
the proper balance between hydrophobicity and polarity to
synthesize pADK. Using this solvent system a pADK (1) with
Mn=49,472 (PDI=1.74) was obtained (polymer 1 in table 1 of the
supporting information), which was used for further investigation.
pADK is designed to complex β-CD via supramolecular guest-
100
90
80
70
60
50
40
30
20
10
0
pADK
pADK:CD1800
pADK:CD1800:p
DNA
PEI1800
bPEI 25000
0
0.5
1
+
Concentration (mM)
O
O
Figure 3: PEI1800, CD1800, pADK cationic complexes and
pADK:CD1800:pDNA polyplexes have minimal toxicity
N3
N3
10
Cu(PPh3)3Br
THF-Toluene (1:1)
55oC
7
host interactions. To investigate if pADK can complex β-CD, we
formulated microparticles from pADK (polymer 1 in table 1 of the
supporting information), which encapsulated rhodhamine B (see
Figure 2A), and investigated if β-CD could stimulate release from
these microparticles. pADK microparticles were suspended into
solutions that contained 5 mM methyl-β-CD or PBS, and the release
of rhodamine-B was measured. Complexation of pADK
microparticles with methyl-β-CD should increase the hydrophilicity
of the particles, and catalyse their disassembly leading to release of
the dye. This can be attributed to the dissolution of surface pADK
polymers on the microparticles, and the creation of water channels in
N
N
O
O
N
N
O
N
N
O
1
n
the microparticles. Figure
stimulates the release of rhodhamine-B from pADK microparticles.
2 demonstrates that methyl-β-CD
Scheme 2: Synthesis of pADK.
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systems.13-16 Figure 3 demonstrates that pADK-cationic complexes
have minimal toxicity. For example at a 1 mM concentration, none
of the pADK formulations had any toxicity, whereas PEI 25,000 was
cytotoxic at 100 µM (amine content). pADK:CD1800 should have
the charge density of a high molecular weight polycation, yet its
toxicity is 2 orders of magnitude lower than PEI of 25,000. The
reduced cytotoxicity of pADK:CD1800 is presumably due to the
biodegradability of the polymer and the transient nature of its
cooperative cationic charges.
The ability of pADK:CD1800 complexes to deliver enhanced
green fluorescent protein-pDNA (EGFP-pDNA) into HeLa cells was
investigated and compared against CD1800. For these experiments
an N/P ratio of 30, between the CD1800 and EGFP-pDNA was used,
which was demonstrated to complex EGFP-pDNA as determined by
For example, in the presence of methyl-β-CD, pADK particles
released their contents with a half-life of 15 hours, in contrast, in
PBS, only 5% of the contents were released after 30 hours. Thus
methyl-β-CD dramatically accelerates the release of compounds
from pADK microparticles, demonstrating that pADK can complex
β-CD.
A major application envisioned for pADK is for pendant
polymer, cationic-β-CD mediated gene therapy. The toxicity of
polycations is a major challenge with cationic drug/gene delivery
vehicles, and we therefore investigated the in-vitro toxicity of
pADK, pADK complexed with bPEI-1800 modified cyclodextrin
(B)
(A)
a gel shift assay (see supporting information).
pADK was
complexed with CD1800 and then mixed with EGFP-pDNA and
added to HeLa cells. Two negative controls were used for this
experiment consisting of either EGFP-pDNA complexed with
PEI1800 (N/P=30/1) or EGFP-pDNA complexed with CD1800
(N/P=30/1). Lipofectamine 2K was used as a positive control. After
36 hours the cells were analysed for green fluorescent protein (GFP)
fluorescence via fluorescence microscopy. Figure 4 demonstrates
that pADK can dramatically improve the transfection efficacy of
CD1800. For example PEI1800 and CD1800 complexed with EGFP-
pDNA had transfection efficacies that were approximately 1% of
lipofectamine, whereas this increased to approximately 60% with the
addition of pADK. pADK:CD1800’s ability to increase the
transfection ability of EGFP-pDNA and its low toxicity are
presumably due to its high transient change density and ability to
disrupt the endosome via either a proton sponge effect or acid
catalyzed hydrolysis of pADK.19-21
(d)
(D)
(C)
(E)
Conclusions
In this report we present a new polymer, termed pADK, for gene
delivery, which is a polyketal that has adamantane groups
embedded on its backbone. pADK is capable of complexing with
β-CD or β-CD derivatives and was able to improve the gene
delivery efficacy of CD1800 by 60 fold. In addition, pADK
complexed with CD1800 and pADK:CD1800-pDNA had minimal
toxicity, even at a 1mM concentration (amine content). We
anticipate numerous drug and gene delivery applications for pADK
due to its ability to form host guest complexes with CD1800 and
degrade into neutral excretable compounds.
120
100
80
60
40
20
Notes and references
a University of California, Berkeley.
b Aten Biotherapeutics Pvt. Ltd., Bangalore, India.
0
†
Footnotes should appear here. These might include comments
Lipofectamine 2000 pADK:CD1800
PEI 1800
CD1800
relevant to but not central to the matter under discussion, limited
experimental and spectral data, and crystallographic data.
Electronic Supplementary Information (ESI) available: [details of any
supplementary information available should be included here]. See
DOI: 10.1039/c000000x/
Figure 4: pADK improves the transfection efficiency of cationic
cyclodextrins: (A) Bright field image of HeLa cells; (B) EGFP-
pDNA transfection with lipofectamine 2000 (relative
fluorescence intensity = 100) (C) EGFP-pDNA transfection with
pADK:CD1800 complexes (relative fluorescence intensity to
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