Novel lysosome targeted molecular transporter built on a guanidinium-poly-(propylene imine) hybrid dendron for efficient delivery of doxorubicin into cancer cells

Article abstract of DOI:10.1039/c4cc09829c

An efficient synthetic approach has been adopted to construct a new dendron-based octa-guanidine appended molecular transporter with a lysosomal targeted peptide-doxorubicin conjugate. The transporter alone (G8-PPI-FL) is found to be non-toxic, showed hig

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COMMUNICATION
Novel Lysosome Targeted Molecular Transporter Built On
GuanidiniumꢀPolyꢀ(propylene imine) Hybrid Dendron For Efficient
Delivery of Doxorubicin Into Cancer Cells
Jyothi B Nair1,# Saswat Mohapatra2,#, Surajit Ghosh*,2, Ж , Kaustabh K Maiti*,1,§
5
Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX
DOI: 10.1039/b000000x
plays active role in cancer invasion, which is up regulated in
several malignant tumor cells, while extremely low in normal
cells. Thus, Cat B considered as a vital candidate for targeted
50 cancer therapy. Herein, we have introduced a new class of
synthetic octaꢀguanidine molecular transporter built on polyꢀ
(propylene imine) dendron starting from aminocaproic acid as the
focal point, G8ꢀPPI (Scheme 1) for target specific delivery of
Doxorubicin (Dox), an anthracyclineꢀbased DNA intercalator, is
55 one of the most typical anticancer drugs commonly employed in
the clinic.
An efficient synthetic approach has been adopted to construct
new dendronꢀbased octaꢀguanidine appended molecular
transporter with lysosomal targeted peptideꢀdoxorubicin
10 conjugate. Transporter alone (G8ꢀPPIꢀFL) is found nonꢀtoxic,
showed higher cellular uptake compared to Argꢀ8ꢀmer,
exhibited excellent selectivity towards lysosome in Cathepsin
B
expressing HeLa cells, while Doxꢀconjugate showed
significant cytotoxicity to cancer cell without affecting the non
15 cancerous cell.
Over the last decades target specific drug delivery of
anticancer drugs have huge interest in pharma industries because
some of them successfully delivered at the right target with
required doses, which minimizes dose limiting toxicity1. Most of
20 the widely used anticancer drugs are highly hydrophobic (poor
bioavailability) in nature and lack of specificity to particular
tumor site, causes severe toxicity to normal tissues and organs. It
is therefore a challenging task of pharmaceutical and medicinal
chemistry researcher to come up with a unique targeted drug
25 delivery system (TDDS) which selectively ferried the cargo
molecule to the diseased cells and tissues in order to achieve
maximum therapeutic effect by possessing minimum pernicious
concomitant effects to normal tissues. Encouraged by wellꢀ
established cellꢀpenetrating peptides (CPPs) like Tat (49ꢀ57) and
30 arginineꢀoctamer (Argꢀ8ꢀmer)2 that crossways biological barriers
efficiently, research on molecular transporter significantly
progressing towards TDDS development that directed to deliver
intracellular organelles of the diseased cells and tissues3.
Probable mechanism emphasized that guanidine group of
35 arginine residue leads to the formation of biꢀdentate hydrogen
bond between cell surface phosphates, carboxylates and/or
sulphates, which facilitates cellular entry2. Therefore, extensive
work on guanidine appended synthetic molecular transporter4,5
has been progressed over the years for the development of new
40 delivery carriers with improved submissive targeting system that
can actively reach to the cancer cells after extravasations6.
Attempts have also been made for improving pharmacokinetic
60
65
70
75
Scheme 1. Thematic representation of the proposed mechanism of drug
delivery by TDDS based on the G8ꢀ PPI Dendron scaffold and chemical
structure of G8ꢀPPI, G8ꢀPPIꢀFL, G8ꢀPPIꢀFKꢀPABCꢀDOX.
The straightforward synthetic construction initiated with PPI
80 dendron growth by sequential Michael addition of acrylonitrile
followed by reduction with RaneyꢀNi. Next, eight unit of free
amine terminal is coupled with Bocꢀprotected guanidine residue
affording the G8ꢀPPI skeleton. Finally, fluorescein (FL) is
attached via amino propanol linker to the G8ꢀPPI. Similarly the
85 G8ꢀPPI dendron further conjugated to Cat B specific small
peptide substrate Phe(F)ꢀLys(K)11 for synthetic ease as well as
potential systemic stability, followed by 4ꢀaminobenzyloxy
carbonyl (PABC), a selfꢀimmolative spacer and DOX resulted the
profile of poor bio available drugs7ꢀ11
.
Recently, focus on TDDS construction increases considering
45 various cellular proteases as target sites. Among these proteases,
cathepsin B (Cat B)12, is a ubiquitous lysosomal cysteine protease
This journal is © The Royal Society of Chemistry [year]
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desired TDDS, G8ꢀPPIꢀFKꢀPABCꢀDOX. In this synthetic
construct the ratio of loading of drug to carrier is 1:1. Cellular
internalization study has been compared with fluorescein label
arginineꢀoctamer (Argꢀ8ꢀGꢀFL) as a standard cellꢀpenetrating
peptide obtained by solid phase peptide synthesise (SPPS) using
manual coupling of Rink amide resin (ESI scheme 6). The final
target transporter molecules G8ꢀPPIꢀFL and G8ꢀPPIꢀFKꢀPABCꢀ
Dox were transformed to HCl salt after deprotection of Boc group
on the guanidine moieties by treating with ethyl acetate saturated
corresponds to 20µm.
of G8ꢀPPIꢀFL increases with time compared to Argꢀ8ꢀGꢀFL
60 in HeLa cells (ESI Fig. S10). Therefore, both flow cytometer
analysis and microscopic images indicates that cellular uptake
ability of G8ꢀPPIꢀFL is significantly higher than that of Argꢀ8ꢀGꢀ
FL. Furthermore, we have investigated the intracellular
localization of transporter G8ꢀPPIꢀFL and compared with Argꢀ8ꢀ
65 GꢀFL in HeLa cells using intracellular staining dyes such as
lysotracker and mitotracker. G8ꢀPPIꢀFL was found to be highly
coꢀlocalized with lysotracker and extent of coꢀlocalization is
much higher than Argꢀ8ꢀGꢀFL which reflects the lysosomal
selectivity G8ꢀPPIꢀFL carrier representing better delivery system
70 of Dox (Fig 1, S11, S13, S14 and S15). Again, we have evaluated
the coꢀlocalization potency of G8ꢀPPIꢀFL and Argꢀ8ꢀGꢀFL in
mitochondria. From confocal data (Fig 1a, S13 and S14 ) of G8ꢀ
PPIꢀFL found no significant coꢀlocalization with mitotracker.
Therefore, above results clearly indicates that of G8ꢀPPIꢀFL
75 having significant selectivity towards lysosome of both normal
and cancer cell. However, we did not find noticeable localization
of G8ꢀPPIꢀFL into the mitochondria.
Flow cytometry data reveals that uptake of Dox conjugate was
maximum after 120 min incubation (Fig 2aꢀc). We have also
80 quantified this result and observed significant release of Dox is
occurred from conjugate compared to free Dox (Fig 2, ESI Fig.
S9). Microscopic images revealed red colour fluorescence
reaches to maximum intensity after 120 min incubation with G8ꢀ
PPIꢀFKꢀPABCꢀDOX conjugate compared to free Dox (Fig 2dꢀf).
85 However, the significant uptake of Doxꢀconjugate shown
compare to free Dox which starts from 30 mins (Fig 2d).
Moreover, merged pink colour microscopic images indicate that
Dox releases at lysosome and reaches up to nucleus where it
binds with DNA (Fig 2dꢀf merged). Therefore, above results
90 clearly demonstrate that G8ꢀPPIꢀFKꢀPABCꢀDOX conjugate
serves as an efficient drugꢀconjugate for the delivery of Dox in
cervical cancer cell line.
5
10 with HCl gas (ESI scheme 4). The key intermediates and aimed
products, G8ꢀPPIꢀFL, G8ꢀPPIꢀFKꢀPABCꢀDOX were
characterized by HPLC, NMR spectroscopy and MALDIꢀTOF
mass spectrometry (ESI Sec.No:2). The stability of the DOX
conjugated carrier has been checked by HPLC in different time
15 interval (1, 2, and 4 month) (ESI Fig. S1a (iv)), also in vitro
stability in PBS buffer (7.4) containg 10% FBS (ESI Fig S7b).
We have initially assessed the cellular uptake efficiency of the
synthesized transporter G8ꢀPPIꢀFL in HeLa cells by flow
cytometery analysis and time dependent microscopy. In flow
20 cytometer analysis suspension of 1ꢀ5×105 HeLa cells were treated
with 2 µM G8ꢀPPIꢀFL and visually differentiated with Argꢀ8ꢀGꢀ
FL for various time periods (ESI Fig. S8a). After incubation at
37oC cells suspension was washed properly with PBS and reꢀ
suspended in colourless media and analyzed using BD
25 LSRFORTESA instrument in FITC channels with emission filters
at 530 nm. Flow cytometric analysis indicates that cellular uptake
of G8ꢀPPIꢀFL increases with time of incubation as we have
observed green colour histogram shifted towards higher intensity
(ESI Fig. S8a). Cellular uptake efficiency was further quantified
30 and found to be significantly higher uptake of both G8ꢀPPIꢀFL
and G8ꢀPPIꢀFKꢀPABCꢀDOX compared to Argꢀ8ꢀGꢀFL (ESI Fig.
S8b). Cellular uptake of G8ꢀPPIꢀFL and Argꢀ8ꢀGꢀFL in HeLa
cells using fluorescent microscope for various time periods (such
as 60, 120 and 240 min) reveals that green fluorescence intensity
35
40
45
50
Fig. 2 Flow cytometric analysis of intracellular DOX release kinetics of
55 Fig. 1 Coꢀlocalization studies by confocal microscopy: Confocal
G8ꢀPPIꢀFKꢀPABCꢀDOX (shown in pink colour) and bare Dox (shown in
images reveals coꢀlocalisation G8ꢀPPIꢀFL and (Arg)8ꢀGꢀFL (green) with 95 yellow colour) at 30 (a), 60 (b) and 120 (c) mins. Microscopic analysis of
mitotracker (red) (a) and lysotracker (red) (b) in HeLa cells. Scale bar
intracellular Dox release kinetics of G8ꢀPPIꢀFKꢀPABCꢀDOX and bare
2
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60 cytotoxicity (h).
Dox at 30 (d), 60 (e) and 120 (f) mins. Scale bar corresponds to 20µm.
We have further examined the drug release ability of Dox
from the conjugate only in cancer cell. Moreover, we have
checked the cellular morphology after treatment with 4 µM of
free doxorubicin and G8ꢀPPIꢀFKꢀPABCꢀDOX conjugate for 4 h
using Nikon TiꢀU microscope with a 40X objective in bright
65 field. The morphology of cells were compared with nonꢀtreated
cells and we have found that extensive damage of cellular
morphology in case of G8ꢀPPIꢀFKꢀPABCꢀDOX treated cells
compared to free Dox treated HeLa cells (Fig 3). Finally, we have
investigated type of cell death using flow cytometer by annexin V
70 and Propidium iodide (PI) method. Flow cytometry data clearly
indicates higher population of apoptotic death of HeLa cells
(66.99%) after treatment with G8ꢀPPIꢀFKꢀPABCꢀDOX
conjugate, while lesser population of apoptotic death of HeLa
cells (25.1%) after treatment with free Dox (Fig 3). This data
75 absolutely supports that Dox conjugate G8ꢀPPIꢀFKꢀPABCꢀDOX
is more potent compared to free Dox against HeLa cell.
In conclusion, we have designed and synthesized a polyꢀ
propylene imine) dendron based molecular transporter which
showed significantly higher cellular uptake than Argꢀ8ꢀmer and
80 have excellent selectivity towards lysosome. Further, we have
constructed TDDS and conjugated Dox through lysosomal
targeting Cat B (over expressed in cancer cell) specific peptide
sequence PheꢀLys with a selfꢀimmolative linker PABC (G8ꢀPPIꢀ
FKꢀPABCꢀDOX), which showed targeted killing of cancer cell
85 (HeLa) without affecting normal cell (WIꢀ38). G8ꢀPPIꢀFKꢀ
PABCꢀDOX shows equal uptake efficiency like G8ꢀPPIꢀFL and
enhanced cytotoxicity (IC50 value 0.40±0.01 µM) compared to
free Dox (IC50 value 1.15±0.10 µM) and apoptosis in HeLa cell
due to noticeable Dox release as shown by kinetic measurement
90 (>70%).
conjugated carrier by incubating G8ꢀPPIꢀFKꢀPABCꢀDOX in
NaOAc /EDTA buffer with Cat B enzyme in the ratio of 9:1.
Captivating the benefit of the intrinsic fluorescent property of
Dox, its cumulative release from G8ꢀPPIꢀFKꢀPABCꢀDOX was
judged by fluorescence measurement at 590 nm (ESI Fig. S7a).
Here we observed above 70% of Dox release occurred in the
presence of enzyme after 28 h as we know that Cat B activity
10 generally occurred in acidic pH which cleaves the specific
peptide substrate, subsequently releasing Dox12. Furthermore,
stability of the Cat B peptide substrate in TDDS was evaluated at
different pH conditions, which confirmed no significant drug
release even at physiological pH (ESI Fig. S7a(ii)). Next, we
15 have investigated in vitro cytotoxicity profiles of G8ꢀPPIꢀFKꢀ
PABCꢀDox conjugate using MTT assay. In this study G8ꢀPPIꢀFL
and free Dox has been treated as control. HeLa and WIꢀ38 cells
were incubated with various concentrations (0.5ꢀ4 µM) of G8ꢀ
PPIꢀFKꢀPABCꢀDox, G8ꢀPPIꢀFL and free Dox for 24 hours.
20 Interestingly, we found that novel G8ꢀPPIꢀFKꢀPABCꢀDOX
conjugate showed higher cytotoxicity with IC50 value 0.40±0.01
µM compared to that free Dox with IC50 value 1.15±0.10 µM (Fig
3a) and nonꢀcytotoxic against nonꢀcancerous cell WIꢀ38 (Fig 3h).
We did not observe any cytotoxicity after treatment with
25 transporter alone G8ꢀPPIꢀFL. Therefore, above data clearly
indicates that enhance cytotoxicity arises from release of Dox
5
30
Author (KKM) wish to thank DST (DST No: SR/S1/OCꢀ
67/2012), CSIR network project CSCꢀ0134, BSCꢀ0112 (KKM)
and BSCꢀ0113 (SG), DSTꢀRamanujan Fellowships (SG), CSIRꢀ
95 NIIST NMR, Mass Spectra and CSIRꢀIICB FACS facilities. We
thank anonymous referees for their invaluable comments during
review process of this manuscript.
35
40
Notes and references
1Chemical Sciences & Technology Division, CSIRꢀNational Institute for
100 Interdisciplinary Science & Technology (CSIRꢀNIIST), Industrial Estate,
Pappanamcode, Thiruvananthapuramꢀ 695019, Kerala, India; Eꢀmail :
§
kkmaiti@niist.res.in / kkmaiti29@gmail.com;
Assistant Professor,
45
Academy of Scientific and Innovative Research (AcSIR).
2Chemistry Division, CSIRꢀIndian Institute of Chemical Biology,
105 Jadavpur, Kolkataꢀ700032, India., Eꢀmail: sghosh@iicb.res.in;
ЖAssociate Professor, Academy of Scientific and Innovative Research
(AcSIR).
50 Fig. 3 Survival of HeLa cell line was assessed by MTT assay after
treatment with G8ꢀPPIꢀFL (transporter), doxorubicin (Dox) and G8ꢀPPIꢀ
FKꢀPABCꢀDOX (a). Cellular morphology of HeLa cells incubated in
absence of each drug (b), after treatment with doxorubicin (c) and after
treatment with G8ꢀPPIꢀFKꢀPABCꢀDOX (d). Scale bar corresponds to
55 20µm. FACS analysis of HeLa cells after treatment with annexinV and
propidium iodide. Control Cells (e), cells treated with 2µM doxorubicin
(f), cells treated with 2µM G8ꢀPPIꢀFKꢀPABCꢀDOX (g). MTT assay
indicates that G8ꢀPPIꢀFKꢀPABCꢀDOX conjugate nonꢀcytotoxic against
nonꢀcancerous cell (WIꢀ38) while free DOX shows significant
# These authors contributed equally to this work
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Graphical Abstract
Novel Lysosome Targeted Molecular Transporter Built On
GuanidiniumꢀPolyꢀ(propylene imine) Hybrid Dendron For
Efficient Delivery of Doxorubicin Into Cancer Cells
Jyothi B Nair1,# Saswat Mohapatra2,#, Surajit Ghosh*,2, Ж , Kaustabh K Maiti*,1,§
An efficient synthetic approach has been adopted to construct new dendronꢀbased octaꢀguanidine appended
molecular transporter with lysosomal targeted peptideꢀdoxorubicin conjugate. Transporter alone (G8ꢀPPIꢀFL) is
found nonꢀtoxic, showed higher cellular uptake compared to Argꢀ8ꢀmer, exhibited excellent selectivity towards
lysosome in cathepsin B expressing HeLa cells, while Doxꢀconjugate showed significant cytotoxicity.

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