Targeted tumor detection: guidelines for developing biotinylated diagnostics

Article abstract of DOI:10.1039/c7cc00311k

The challenge in achieving precision medicine relies on how to advance and/or enhance new as well as old therapeutic strategies. Here, we highlight the significant role hydrophilicity of biotinylated fluorescent probe's plays on their cellular uptake behaviour.

Full text of DOI:10.1039/c7cc00311k

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Targeted Tumor Detection: Guidelines for Developing Biotinylated
Diagnostics
Joo Hee Jang,a,‡ Woo Ri Kim,b,‡ Amit Sharma,a,‡ Suk Hee Cho,b Tony D. James,c,* Chulhun Kang,b,*
and Jong Seung Kima,*
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
The challenge in achieving precision medicine relies on how to
advance and/or enhance new as well as old therapeutic strategies.
genomics, we require a better understanding of the mechanisms
responsible for better targeted diagnostics leading to improved
Here, we highlight the significant role hydrophilicity of therapeutics.
Biotin (vitamin H), a critical cofactor for carboxylase activity, is
biotinylated fluorescent probe’s plays on their cellular uptake
behaviour.
involved in fatty acid synthesis, branched amino acids catabolism
and gluconeogenesis5,6 and is preferentially delivered into rapidly
proliferating cells including cancer cells (ovarian, colorectral, etc.),
through the overexpressed sodium dependent multivitamin
transporter (SMVT) on the cell surface, whose activity is additionally
regulated by protein kinase C (PKC).6,7 Biotin-conjugation is one of
the most plausible choices in developing various cancer selective
prodrugs, polymeric carriers for drug delivery, and theranostic
systems.8,9 Recently, biotin has been extensively used as a protein
labeling tool in order to identify various protein interactions.10 In
theranostic biotin-conjugates, a biotin unit is linked through a self-
immolative linker to the drug molecule, which may vary from
lipophilic drug11 to highly polar peptide.12 In general, cellular uptake
of hydrophilic molecules may be aided by the corresponding
membrane proteins, whereas the hydrophobic molecules, for
instance, steroid hormones, simply diffuse through the
membrane.13
Cancer is a near-term objective of the precision medicine initiative
(2015) which aims to revolutionize current health programs by
tailoring therapeutics towards individual patients.1 The overarching
goal behind this initiative is for medicinal care providers to decrease
the cancer modalities and morbidities, regardless of the cancer
subtype, contingent on the results obtained from reliable assays for
relevant markers from patients, that are pertinent for disease
prevention.2 However, this monumental task presents numerous
biological challenges such as pathological and physiological
complications, non-targeted delivery due to genetic heterogeneity
within tumors, dynamic drug resistance, problems with cancer
subtype classification and associated off-target side effects. With
the revolution in cancer genomics, immense effort has been
directed towards the development of non-invasive biomarkers for
assessing and transferring genetic information into clinical practices
in order to facilitate diagnosis, monitor the therapeutic response
and patient stratifications.3 Generally, development programs are
endorsed under regulatory guidelines for better outcomes from
various clinical processes and implementations.4 However,
regardless of these huge endeavors and resources provided, the
real hurdles confronting the clinical development and endorsement
of current precision based regimens are selectivity, tissue
penetration with adequate uptake followed by pathways for drug
clearing. In order to take full advantage of advanced cancer
In this context, despite the incorporation of a biotin moiety for
SMVT targeting, one may ask whether or not the hydrophobicity of
biotin-conjugates plays an adverse role in their cellular uptake.
Moreover, the biological pathway responsible for theranostic
biotin-conjugate’s uptake still remains elusive. These factors are
critical for the development of smarter biotin-conjugates in order to
widen the tenets of precision medicine with an emphasis on disease
prevention.
In order to investigate the role of hydrophilicity in biotinylated
conjugate towards cellular uptake, we designed biotin-conjugated
fluorescent probes (4-6) (Scheme 1), possessing dodecyl, hexyl,
diethylene glycol to adjust their overall hydrophobicity and the
corresponding non-biotin analogs (1-3) were used as controls
(Scheme 1). The 4-amino-1,8-naphthalimide fluorophore was
chosen due to its strong emission. Additionally, the two photon
properties can afford better cell imaging efficiency with minimal
background and enhanced light penetration. The in vitro cellular
uptake behavior and mechanism were investigated against HeLa
kangch@khu.ac.kr
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cells. Compared to other conjugates, biotin fluorescent probe 5 hydrophilicity. Thereby, we measured the partition-coefficients
with log Poct ~1 exhibited preferential cell membrane uptake via (logPoct) of the probes according to the solubility in n-octanol and
SMVT proteins under PKC-mediation using intracellular ATPs.
MOPS buffer (Table S1 and Fig. 2). The logPoct values for 4-6 are 1.29,
Compounds 1-6 were synthesized according to scheme S1 (ESI †) 1.03, and 0.807, respectively. These results suggest an optimum
in good yield. Spectroscopic studies of the probes were undertaken hydrophobicity for the biotin-conjugates with a logPoct value of
using UV/Vis absorption and fluorescence spectroscopy. These about 1.0
probes exhibit a broad absorption band at 430 (in toluene), 433 (in
acetonitrile), and 440 nm (in PBS) respectively, (Fig. S1) with a
bandwidth of ~ 75 nm. The corresponding fluorescence emission
bands of the probes in toluene and acetonitrile were slightly blue-
shifted with enhanced intensities (505 and 524 nm respectively)
when compared to the PBS (545 nm) system. Since the internal
charge-transfer (ICT) excited state phenomenon in these probes
offers a substantial excited-state dipole moment, which can be
stabilized/destabilized depending upon the choice of solvent. The
lower fluorescent emission intensity of the probes in PBS is ascribed
to their hydrogen bond donor (HBD) or hydrogen bond acceptor
(HBA) ability.14
Scheme 1 Chemical structure of the non-biotin (1-3) and biotin-conjugated probes (4-
6).
Fig. 1 Confocal microscopy images of HeLa cells upon treatment with probes 2 and 5.
(a) The fluorescent images were obtained at variable time (2 μM each); (b) and at
variable probe’s concentration for 10 min. The cells were incubated in high glucose
To examine the cell uptake behavior of biotin-conjugates, we
performed fluorescent confocal microscopic experiments in HeLa serum free DMEM media at 37 ℃, λex = 458 nm, bandpath filter (505-550 nm). (c , d)
Fluorescence intensities (a.u.) per cell in the images of the panel (a) and (b)
cells for each conjugate (1-6). As shown in Fig. S2(a), probe 1 with a
respectively. The images were obtained using Image J software. The data are presented
dodecyl group displayed the strongest fluorescence intensity among
as mean ± SD (n=5).
the non-biotin probes (1-3) and its intensity is expected to keep on
increasing even after 60 min. Due to the more hydrophobic tail of
Subsequently, in order to identify the intracellular location of 2 and
5, a series of colocalization experiments were performed using
commercially available organelle selective markers. As seen in Fig.
S3, the green-channel fluorescence intensity of 5 showed an
excellent overlay with the red-channel fluorescence of ER
(Pearson’s correlation coefficient (PCC) is 0.9729), whereas the
overlap with other trackers were relatively poor (Lyso; 0.4617 and
Mito; 0.5576). Its preference for the ER could be explained by the
fact that the ER is the metabolic center of various lipophilic
compounds such as xenobitics and lipids with the largest membrane
area among the organelles. In contrast, the non-biotin-conjugate, 2
was localized in the ER as well as mitochondria (PCC = 0.9373 and
0.7809, respectively).
this molecule, it penetrates into the cell membrane via diffusion.
Conversely, among the biotin-conjugates (4-6), the largest intensity
was observed for the system with a hexyl moiety (5), while one with
a more hydrophobic dodecyl (4) and a hydrophilic hydroxylethyl-
oxyethyl moiety (6) do not display comparable fluorescence
intensity (Fig S2b). These results indicate that the biotin-conjugates
enter the cells through an alternate pathway from that of the non-
biotin counterparts.
In order to investigate the detailed cellular uptake mechanism of
the biotin-conjugates (4-6),
a time course analysis of the
fluorescence intensity in the cells was carried out for probes 2 and 5
at various concentrations. We have found that the enhancement of
fluorescence displays both time and concentration dependent
behavior (Fig. 1). And, probe 5 shows 6 times higher fluorescence
intensity than 2 incorporating the same alkyl chain, indicating that
the biotin moiety plays a significant role in the cellular uptake.
However, what makes the dramatic difference of uptake behavior
among the biotin-conjugates shown in Fig S2b. The most apparent
difference among the conjugate structures was the probes’
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Next, to demonstrate sodium ion-dependency for the uptake of 5,
its cellular uptake behavior was examined in the presence of
sodium ions and selective inhibitors for the sodium pump. As shown
in Figure S5, depletion of sodium ions by replacing the salts with
potassium salts in the media induced a drastic decrease in the
uptake of 5. This result indicates that the sodium gradient across
the plasma membrane is the driving force of the biotin-conjugate’s
uptake, which is further confirmed by the inhibition of uptake by
amiloride, a sodium ion transport inhibitor15 (Fig. S5 a, b). The
replacement of chloride to phosphate in the media did not show
any significant change in the uptake of probe 5. A gradual increase
in 5’s cellular uptake is observed according to the increase of
sodium ions in the range 0-100 mM (Fig. S5c). Similar experiments
for the corresponding non-biotin probe 2 did not show any
significant change (Fig. S5a, b, and S6).
Fig. 2 Lipophilicity of probes (1-6) according to logPoct value.
It is well known that biotin’s uptake is regulated through SMVT,
and it would be natural to ask whether biotin-conjugates enter the
cells via the same mechanism. To the best of our knowledge, this
has never been reported before. The uptake of probe 5 in Fig. 1 was
examined in the presence of biotin and pantothenic acid, a well-
known substrate for SMVT, and in the presence of folic acid and
ascorbic acid for comparison (Fig. 3). Biotin and pantothenic acid
induced significant inhibitory effects on the uptake of 5 in a
concentration dependent manner whereas ascorbic acid and folic
acid display no such effect. Similarly,
a series of parallel
experiments were also performed for the uptake of probe 2, the
Fig. 4 Effect of various signal transduction pathway modulators on the uptake of 5 in
HeLa cells such as genistein, forskolin, PMA and KN-62. (a) After preincubation of the
cells with inhibitors for 1 hr. the cells were further incubated with 2 μM of a probe for
10 min. under high glucose serum free DMEM media at 37 ℃. λex = 458 nm, bandpath
filter (505-550 nm). (b) Fluorescence intensity (F.I.) per cell in the images of the panel
(a). The images were obtained using Image J software. The data are presented as mean
± SD (n =5).
Considering that the sodium-dependent nutrient uptake as
shown in this study is a typical characteristic of the secondary pump,
biotin-conjugate uptake will depend on the cellular ATP level, which
is examined in Fig. S7. Indeed, the uptake is clearly inhibited by the
presence of ouabain,16 sodium azide17 or 2,4-dinitrophenol,18 which
are an Na+/K+-ATPase inhibitor, an oxidative phosphorylation
inhibitor and a mitochondrial uncoupler, respectively where all the
chemicals reduce the cellular ATP level. Taken together with the
results from Fig. 3 and Fig. S5, the results in Fig. S7 indicate that the
cellular uptake of the biotin-conjugate in this study is via SMVT, a
secondary pump whose driving force is regulated by the
intracellular ATP hydrolysis.
Fig. 3 (a) Effect on the uptake of 5 in in HeLa cells in the presence of vitamins biotin,
pantothenic acid, ascorbic acid and folic acid. The cells were incubated with 2 μM of
the probe in presence of the vitamin under high glucose serum free DMEM media at 37
℃, λex = 458 nm, bandpath filter (505-550 nm). (b) Fluorescence intensity (F.I.) per cell
in the images of the panel (a). The images were analysed by Image J software. The data
are presented as mean ± SD (n =5).
results clearly indicate that all vitamins used in this study fail to
induce any inhibitory effect on the uptake of probe 2 (Fig. S4).
These results indicate that 5 enters the cells selectively via SMVT,
whereas the transport of 2 is independent of SMVT, confirming the
differential uptake processes.
To check whether or not the physiological regulation of the SMVT
activity for biotin uptake into cells works for the biotin-conjugates,
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In summary, we have designed and developed biotinylated probes
with varying hydrophilicity to investigate their cellular uptake
mechanism and behavior. Compared with the non-biotinylated
fluorescent probes, probe 5 exhibits preferential cellular uptake
among other biotinylated probes through SMVT-protein receptors
under sodium-ion dependent manner. In addition, the cellular
uptake behavior is regulated under PKC-mediation by utilizing
intracellular ATPs. Taken together, these data collectively highlight
the critical role of hydrophilicity on the cellular uptake processes of
biotin-based cancer targeting imaging agents. The use of these
guidelines will expand the current cancer cell labelling/targeting
toolbox and also offer the potential to improve their cellular uptake,
which is crucial for the development of in vivo imaging systems and
facilitate rational screening and allow for efficient diagnosis and
monitoring of treatment response and more importantly patient
satisfaction allowing for the implementation of precision medicine
as part of standard patient care.
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