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ORIGINAL RESEARCH
Carbonic Anhydrase IX Targeted Polyaspartamide
uorescent Probes for Tumor imaging
Yu Zhang
1,2,
*, Fan Liu
2,3,
*, Chuntao Shao
2,
*, Jun Huang
3
, Guoping Yan
1
1
College of Chemical and Material Engineering, Quzhou University, Quzhou, Zhejiang Province, 324000, People’s Republic of China;
2
School of
Materials Science and Engineering, Wuhan Institute of Technology, Wuhan, 430205, People’s Republic of China;
3
School of Chemical and Biomolecular
Engineering, University of Sydney, NSW, 2006, Australia
*These authors contributed equally to this work
Correspondence: Guoping Yan, Tel +86 570 8028560, Email guopingyan@qzc.edu.cn
Background: Precise intraoperative tumor delineation is essential for successful surgical outcomes. However, conventional methods
are often incompetent to provide intraoperative guidance due to lack specicity and sensitivity. Recently uorescence-guided surgery
for tumors to delineate between cancerous and healthy tissues has attracted widespread attention. The contrast-enhanced uorescent
imaging has been applied for non-invasive diagnosis of cancers using tumor-targeting uorescent probes.
Methods: The carbonic anhydrase IX targeted polyaspartamide uorescent compounds (SD-PHEA-NI) were synthesized by incor-
porating a tumor-targeting group of sulfadiazine (SD) and N-butyl-4-ethyldiamino-1,8-naphthalimide (NI) into water-soluble carriersof
poly-α,β-[N-(2-hydroxyethyl)-L-aspartamide] (PHEA). These derivatives were also characterized by Fourier transform infrared
spectroscopy, gel permeation chromatography, ultraviolet-visible spectroscopy, nuclear magnetic resonance spectroscopy and uores-
cence assays. The cellular uptake, cytotoxicity, and uorescence imaging ability were evaluated.
Results: Experiment results indicated that SD-PHEA-NI has low cytotoxic to Henrietta Lacks (HeLa) cells. Moreover, B16F10
melanoma cells can take up SD-PHEA-NI and show good green uorescent images. However, SD-PHEA-NI displayed a low-intensity
green uorescence signal in healthy human embryonic kidney (293T) cells.
Conclusion: SD-PHEA-NI can be considered a potential uorescent probe for the detection of tumors. This study has the potential to
enhance tumor diagnosis and image-guided surgical interventions by providing real-time information and robust decision support,
thereby reducing recurrence and complication rates and ultimately improving patient outcomes.
Keywords: uorescent imaging, polyaspartamide, sulfadiazine, naphthalimide, uorescent probes
Introduction
Renal cell carcinoma (RCC) originates from renal tubular epithelial cells and is responsible for approximately 90% of all
renal malignancies.
1–3
Due to the deep location of the kidney, the early clinical manifestations of RCC are insidious, and
the patients exhibit no obvious symptoms. More than 16% of patients develop distant metastasis at the time of the initial
diagnosis.
4,5
Surgical resection is currently the main treatment method, but the surgeon mainly relies on visual
inspection, palpation and subjective experience to distinguish between malignant lesions and adjacent (healthy) tissues.
The unclear boundary of the lesion leads to incomplete tumor resection, increases the risk of recurrence, and ultimately
affects the patient’s postoperative prognosis and survival rate.
6,7
Therefore, accurate assessment of the resection margin
during surgery is crucial for diagnosis, staging and ultimate patient prognosis.
Fluorescence imaging (FI) has emerged as a promising technique for intraoperative management of tumor margins
owing to its advantages, including real-time feedback, high specicity, high sensitivity, and ionizing radiation-free, as
well as low cost.
8–10
Currently, uorescent probes have been used for in vivo imaging and differentiation of normal
tissue and tumor tissue, including small-molecule probes, nanoparticle-based probes, and quantum dots.
11–13
Small-
molecule probes, such as indocyanine green (ICG) and 1.8-naphthalimide, are widely applied in FI to improve
International Journal of Nanomedicine 2025:20 639–651 639
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International Journal of Nanomedicine
Open Access Full Text Article
Received: 25 October 2024
Accepted: 3 January 2025
Published: 15 January 2025
contrast and sensitivity.
11,14
In particular, polymeric nanoparticles (PNs) have demonstrated signicant potential in
drug delivery, improved biocompatibility and the ability to be easily functionalized. At present, some polymeric
nanoparticles labeled with tumor-targeting molecules have been reported to effectively deliver uorescent probes to
the tumor regions. These systems can increase the imaging efcacy for tumors and improve the half-life, solubility,
and stability, which dramatically reduces potential side effects in the body.
15,16
In general, a uorescent probe is
chemically attached to polymer biomaterials,
17
such as polylysine and poly(acrylic acid). The encapsulating materials
including liposomes, polymersomes, polymeric nanoparticles, and micelles.
18,19
Glycol chitosan-based polymeric
nanoparticles have been investigated to develop therapeutic drugs and uorescent agents for tumor accumulation and
treatment.
20
As a water-soluble synthetic protein-like polymer, polyaspartamide possesses nontoxic, nonantigenic, good
biocompatibility and degradability, and then can be easily modied through side chain groups. The degradation
products of poly(amino acid)-based biomaterials are nontoxic small-molecule nutrients, which can be used or excreted
by physiological processes in the body. So it is widely applied as a plasma extender and drug delivery carrier.
21,22
Some polymer pro-drugs were produced by a covalent link of anti-inammatory agents and antiviral drugs with poly-
α,β-[N-(2-hy droxyethyl)-D,L-aspartamide] (PHEA). They indicated increased drug stability, solubility, and bioavail-
ability. Tumor-targeting polymer MRI contrast agents were made by incorporating gadolinium diethylenetriamine-
pentaacetic acid (Gd-DTPA) into PHEA. Therefore, PHEA can be used as an ideal polymer carrier for uorescent
probes.
23,24
Currently, contrast-enhanced FI has attracted wide interest in non-invasive detection of cancers. However, some
probes are restricted to tumor imaging due to no tumor-targeting biodistribution. Moreover, tumor-targeting uorescent
probes can be used to effectively monitor targeted molecular imaging by the conjugation of uorescent probes with
tumor-targeting molecules, such as peptides,
15,25
folate,
26,27
antigens,
28
and cell surface receptors.
29
Carbonic anhydrase (CA IX) is notably over-expressed in clear cell RCC and is often used as a biomarker for this
type of cancer due to its high specicity and correlation with tumor aggressiveness.
30,31
Therefore, the design and
construction of ultrasonic molecular probes targeting CA IX can realize targeted molecular imaging of RCC. Sulfadiazine
(SD) derivatives are known for their high specicity for transmembrane carbonic anhydrase (CA) isoforms, cost-
effectiveness, and broad availability, and then are developed as tumor-targeting moieties in anticancer drug formulations.
Some SD derivatives can accumulate into Walker carcinoma or Yoshida sarcoma to treat cancers and suppress
metastasis.
32,33
SD-conjugated Gd-DTPA derivatives demonstrated high uptake by Hepatoma and Ehrlich ascites
carcinoma cells in mice. SD-containing PHEA gadolinium complexes showed high uptake and enhanced MRI in
hepatomas.
24,34
Naphthalimide derivatives are usually applied as organic dyes, luminophores, and anticancer agents due to their good
photophysical and chemical property.
35,36
To enhance the accuracy of intraoperative uorescent probes using water-
soluble carriers, this study introduces a carbonic anhydrase IX targeted polyaspartamide uorescent probes (NI-PHEA-
SD). This probe incorporates a carbonic anhydrase IX-targeting group of sulfadiazine (SD) and N-butyl-4-ethyldiamino-
1,8-naphthalimide (NI) into water-soluble carriers of poly-α,β-[N-(2-hydroxyethyl)-L-aspartamide] (PHEA). The probe
responds specically to over-expressed carbonic anhydrase IX, enabling precise localization of renal cell carcinoma
during surgery. Tumor lesions smaller than 1 mm in diameter can be accurately identied using PHEA-NI-SD and
completely excised under uorescence imaging guidance.
Materials and Methods
Materials
Polysuccinimide (PSI),
24,34
sodium sulfadiazine (SDNa),
24
NI,
35,36
and PHEA
24,34
were synthesized using previously
reported methods cited in the literatures. Human embryonic kidney (293T) cells, B16F10 mouse melanoma cells, and
Henrietta Lacks (HeLa) cells were provided by the China Center for Type Culture Collection of Wuhan University,
China, and cultured according to a previously described method.
37
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Synthesis of Polyaspartamide Containing SD and NI Groups (SD-PHEA-NI)
Polysuccinimide (PSI, 0.97g, 10 mmol) was dissolved in N,N-dimethylformamide (DMF, 10mL) and N-butyl-4-ethyl-
diamino-1,8-naphthalene imide (NI,1.24g, 0.2mmol, 0.4 equiv. to the repeat structure units of PSI) was added to the
solution with rapid stirring at room temperature. The reaction solution was continued with stirring for 48h at 80°C and
further cooled to room temperature. Subsequently, the solution of ethanolamine (1 mL, 18 mmol, 1.8 equiv. to the
repeating structural units of PSI) in DMF (3mL) was added to the reaction solution with rapid stirring at 0°C. The
reaction mixture was stirred continuously for 4h at 0°C and more for 12h at room temperature. The resulting mixture
was ltered and precipitated using a solution of dichloromethane and n-hexane (V/V, 2/1). The precipitate was
collected from ltration and dried under vacuum. The residual solid was dissolved with distilled water in a dialysis
bag and dialyzed against distilled water for 48 h. After ltration, the dialyzed solution was evaporated, and the solid
residue was dried under vacuum to producepolyaspartamide containing naphthalene imide groups (PHEA-NI)
(1.59g, 71%).
PHEA-NI (0.97g, 10 mmol) and triethylamine (200 μL) were dissolved in DMF (10 mL) and bromoacetyl bromide
(130.5μL, 0.87 mmol, 0.51 equiv. for the repeat structural units of PSI or 76.8 μL, 0.51 mmol, 0.3 equiv. to the repeat
structure units of PSI) was added, respectively, to the solution with rapid stirring at 0°C. The reaction was continued with
stirring for 3 h at 0°C and more for 1 h at room temperature. Most of the solvent was removed under vacuum, and the
resultant mixture was precipitated with a solution of dichloromethane and n-hexane (V/V, 2/1). The solid residue was
reprecipitated from DMF against dichloromethane and n-hexane, ltered and dried under vacuum to obtain a solid (Br-
PHEA-NI (I): 0.72 g, 73%, or Br-PHEA-NI (II): 0.8 g, 83%).
Br-PHEA-NI (0.66g, 2 mmol) was dissolved in N,N-dimethylformamide (DMF, 10 mL). SDNa (I:, 0.56 g, 2mmol, or
II: 0.41 g, 1.5 mmol) and tetrabutyl ammonium hydroxide (1 mL) were added, respectively, to the solution with rapid
stirring at room temperature. The reaction was continued with stirring for 48h.The resulting mixture was ltered and
precipitated using a solution of dichloromethane and n-hexane (V/V, 2/1). After ltration, the precipitate was collected
and dried under vacuum to produce a solid. The solid was dissolved with distilled water in a dialysis bag and dialyzed
against distilled water for 48 h. After ltration, the dialyzed solution was evaporated and the solid residue was dried
under vacuum to give polyaspartamide containing sulfadiazine and naphthalene imide groups (SD-PHEA-NI) (L
1
:0.79g,
84% or L
2
:0.65 g, 83%). The average molecular weight (Mn) was measured using Gel Permeation Chromatography
(GPC). SD-PHEA-NI (L
1
):
1
H NMR (DMSO-d
6
, δ ppm): 8.25, 6.55 (CH, sulfadiazine), 7.47 (CH, naphthalene ring),
4.63 (OCCH
2
N), 3.89, 3.36, 3.15, 2.68, 2.51 (CH
2
, CH), 1.55, 1.29, 0.89 (CH
3
); Mn: 1.59 × 10
4
, Polydispersity: 1.15;
FT-IR (KBr, ν
max
, cm
−1
): 3419 (-OH), 2930 (C-H), 1669, 1510 (CO-NH), 1385 (C-N), 1120 (SO
3
), 910, 770, 669 (C
6
H
4
);
UV (H
2
O,λ
max
, nm): 266, 286, 439.
SD-PHEA-NI (L
2
):
1
H NMR (DMSO-d
6
, δ ppm): 8.25, 6.57 (CH, sulfadiazine), 7.47 (CH, naphthalene ring), 4.61
(OCCH
2
N), 3.89, 3.42, 3.14, 2.66, 2.51 (CH
2
, CH), 1.53, 1.29, 0.89 (CH
3
); Mn: 1.82 × 10
4
, Polydispersity: 1.19; FT-IR
(KBr, ν
max
, cm
−1
): 3448 (-OH), 2928 (C-H), 1664, 1507 (CO-NH), 1385 (C-N), 1121 (SO
3
), 910, 771, 668 (C
6
H
4
); UV
(H
2
O, λ
max
, nm): 266, 286, 439.
Vitro Cytotoxicity Assay
HeLa cells (2 × 10
5
/mL) were plated in 96 wells plates in the RPMI-1640 medium supplemented with 10% fetal
bovine serum (Gibco). Co., USA, 100 µg/mL streptomycin and 100 units/mL penicillin) and the number of cells in
each well was 2 × 10
4
. The cells were incubated for 24 h in an incubator (37°C, 5% CO
2
), and residual liquid was then
removed and the solution of polyaspartamide containing sulfadiazine and naphthalene imide groups (SD-PHEA-NI:
L
1
, L
2
, 100 µL)in growth medium was added. After a 48h incubation, thiazolyl blue (3-[4,5-dimethylthiazol-2-yl]-
2,5-diphenyltetrazolium bromide (MTT, 20µL, 5.0 mg/mL) solution in phosphate-buffered saline (PBS) was added to
each well. The cells were incubated again for 4h. After removal of growth medium, dimethyl sulfoxide (DMSO,
100µL) was added and the solution shaken for 30 min. The absorbance (optical density: OD
492
) was measured at 492
nm using a DG-3022A ELISA-Reader (Hercules, CA, United States) and expressed as a percentage relative to control
cells (no SD-PHEA-NI).
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Fluorescent Imaging Assay in Healthy Cells
Human embryonic kidney cells (293T; 2 × 10
5
/mL) were implanted in 6-well plates (1 × 10
5
/mL) in DMEM (10% fetal
bovine serum, 100 units/mL penicillin and 100 µg/mL streptomycin). The cells were incubated for 24 h in an incubator
(37°C, 5% CO
2
). After separation of growth medium, pure growth medium or the solution of SD-PHEA-NI (L
1
, L
2
or
PHEA-NI, 1 mL, 100 µg/mL) in growth medium was added, respectively. After 2h of incubation, the residual liquid was
moved again, and the cells were rinsed with PBS three times. Subsequently, the cells were xed with paraformaldehyde
(0.5 mL, 4%) for 10 min and stained with benzophenone imine (Hoechst, 5 μg/mL) for more 10 min. The cell
morphology and density were observed using a confocal laser scanning microscope (CLSM) (TCS SP8, Leica,
Germany).
Fluorescent Imaging in Tumor Cells
B16F10 cells (1 × 10
5
/mL) were implanted in 6 wells plates in DMEM (100 units/mL penicillin,10% fetal bovine serum
and 100 µg/mL streptomycin). The cells were incubated for 48 h in an incubator (37°C, 5% CO
2
). After separation of
growth medium, pure growth medium or the solution of SD-PHEA-NI (L
1
or L
2
, 1 mL, 100 µg/mL) in the growth
medium was added, respectively. After 2h of incubation, residual liquid was moved again and the cells were rinsed with
PBS three times. Subsequently, the cells were xed with paraformaldehyde (0.5 mL, 4%) for 10 min and stained with
4’,6-diamidino-2-phenylindole (DAPI, 5 μg/mL) for more 10 min. The cell morphology and density were observed using
a confocal laser scanning microscope (CLSM) (TCS SP8, Leica, Germany).
Cellular Uptake Test
B16F10 melanoma cells (1 × 10
5
/mL) were implanted in 24 wells plates in RPMI-1640 media (100 units/mL
penicillin, 100 µg/mL streptomycin and 10% fetal bovine serum (Gibco). Co., USA)). The cells were incubated for
24 h in an incubator (37°C, 5% CO
2
). After removal of growth medium, 200 µL of pure growth medium or growth
Scheme 1 Synthetic route to polyaspartamide uorescent probes containing naphthalimide and sulfadiazine groups (SD-PHEA-NI).
https://doi.org/10.2147/IJN.S500614
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medium containing SD-PHEA-NI (0.25 μmol/L) was added, respectively. After a 2-h incubation, the growth medium
was removed again, and the cells were washed three times with PBS and subsequently xed with paraformaldehyde
(0.5mL, 4%) for 10 min. The cell morphology and density were observed using an IX-70 inverted uorescence
microscope (Olympus Co., Ltd., Japan).
Figure 1
1
H NMR spectra of SD-PHEA-NI (L
1
: a and L
2
: b).
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An inhibited cell uptake assay was performed according to the method described above. B16F10 melanoma cells (1 ×
10
5
/mL) were implanted in 24 wells plates in RPMI-1640 media. The cells were incubated for 24 h in an incubator
(37°C, 5% CO
2
). After removal of growth medium, growth medium containing SD (25 μmol/L, 200 µL) was added and
then incubated for 1 h in an incubator (37°C, 5% CO
2
). After removal of growth medium, a solution of SD-PHEA-NI
(0.25 μmol/L, 200 µL) in growth medium was added and incubated for 2 h. The growth medium was removed again, and
then the cells were washed three times with PBS and subsequently xed with paraformaldehyde (0.5mL, 4%) for 10 min.
The cell morphology and density were observed using an IX-70 inverted uorescence microscope.
Results and Discussion
Synthesis and Characterization
Two polyaspartamides containing sulfadiazine and naphthalimide groups (SD-PHEA-NI: L
1
, L
2
) were synthesized by the
incorporation of uorescence molecule NI and tumor-targeting group sulfadiazine into a polyaspartamide carrier
(Scheme 1). The experimental data from UV, FT-IR,
1
H NMR, GPC and uorescenctspectra provided evidence for the
formation of SD-PHEA-NI (Figures 1–5).
1
H NMR spectra of SD-PHEA-NI (L
1
, L
2
) displayed typical peaks of sulfadiazine and naphthalimide groups, which
appeared at 8.25, 6.55, 1.55–0.89 ppm, and 7.47 ppm, respectively (Figure 1a and b), which indicated that SD and NI
were covalently bound to polyaspartamide, respectively. The average grafted mole ratios of SD and NI groups
Figure 2 IR spectra of SD-PHEA-NI (L
1
: a and L
2
: b).
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Figure 3 GPC spectra of SD-PHEA-NI (L
1
: a and L
2
: b).
Figure 4 UV of polyaspartamide uorescent probes (SD-PHEA-NI).
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topolyaspartamide (mol%) in SD-PHEA-NI (L
1
, L
2
) molecules were: L
1
,7.6% and 16%, and L
2
,5.4% and 18.2%,
respectively.
IR spectra of SD-PHEA-NI (L
1
, L
2
) showed characteristic absorption peaks of amide bonds (CONH) at
1669–1507 cm
−1
, amino groups (NH) at 3448–3419 cm
−1
, and benzyl groups varying from 910 to 668 cm
−1
, respectively
(Figure 2a and b), which demonstrated that both SD and NI were covalently bound to PHEA. SD-PHEA-NI (L
1
, L
2
) had
number-average molecular weights (Mn) of 1.59×10
4
and 1.82 × 10
4
, respectively (Figure 3a and b).
UV spectra indicated that SD-PHEA-NI had the same characteristic absorption peaks (266, 286 nm: SD, and 439 nm:
NI) as those of SD and NI (Figure 4). The uorescent spectra showed that SD-PHEA-NI (L
1
, L
2
) had a maximum
excitation wavelength of 437 nm and maximum emission wavelength of 525 nm (Figure 5). It appears that SD-PHEA-NI
possesses the similar typical uorescence emission wavelength to NI. Therefore, SD-PHEA-NI (L
1
, L
2
) retains the same
photophysical property as NI for uorescent imaging. Moreover, they should possess good tumor-targeting property to
achieve highly sensitive enhanced imaging of tumor cells.
Vitro Cytotoxicity
The growth and metabolism of HeLa cells incubated with SD-PHEA-NI (L
1
, L
2
) are shown in Figure 6. At
a concentration (1 µg/mL) of the solution of SD-PHEA-NI (L
1
, L
2
), the viability of HeLa cells incubated with SD-
Figure 6 In vitro cytotoxicity of SD-PHEA-NI (L
1
, L
2
) to HeLa cells.
Figure 5 Fluorescent spectra of polyaspartamide uorescent probes (SD-PHEA-NI).
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PHEA-NI (L
1
, L
2
) was 66.3% and 86.5%, respectively. At a concentration (100 µg/mL) of the solution of SD-PHEA-NI,
the viability of HeLa cells incubated with SD-PHEA-NI (L
1
, L
2
) was 56% and 83.9%, respectively. This illustrates that
SD-PHEA-NI (L
1
, L
2
) exhibited low cytotoxicity in HeLa cells.
Sulfadiazine (SD) is an antibacterial and anti-inammatory drug used clinically. SD-PHEA-NI (L
1
, L
2
) possessed
high uptake and good tumor-targeting properties for tumor cells. SD-PHEA-NI (L
1
, L
2
) may selectively accumulate in
HeLa cells and exhibit high anticancer efciency to HeLa cells during increasing of incubation concentration. Moreover,
SD-PHEA-NI (L
1
) had slightly higher cytotoxicity to HeLa cells than that of SD-PHEA-NI (L
2
) because the content of
the SD groups attached to SD-PHEA-NI (L
1
) was larger than that of SD-PHEA-NI (L
2
).
Fluorescent Imaging
Fluorescent imaging of healthy 293T cells and B16F10 melanoma cells cultured with SD-PHEA-NI (L
1
, L
2
) is shown in
Figures 7 and 8, respectively, when excited by white and blue light (excitation wavelength: 450 nm). Healthy 293T cells
cultured with SD-PHEA-NI (L
1
, L
2
) and PHEA-NI displayed a low-intensity green uorescent image when excited by
blue light (Figure 7) because 293T cells present a rather low expression of transmembrane CA isoforms and cannot show
high uptake of SD-PHEA-NI (L
1
, L
2
). Meanwhile, the cells cultured in growth medium did not show uorescence when
excited by blue light.
B16F10 cells cultured with SD-PHEA-NI (L
1
, L
2
) exhibited good green uorescence images when excited with blue
light (Figure 8). However, the cells cultured with pure growth medium showed good growth conditions when excited by
white light, but no uorescent images when excited by blue light. Moreover, SD-PHEA-NI (L
1
, L
2
) exhibited better
uorescence properties than PHEA-NI without a tumor-targeting group in B16F10 cell imaging. It is likely that these
cells express high levels of transmembrane CA isoforms, which induce SD-PHEA-NI (L
1
, L
2
) to proactively accumulate
Figure 7 Fluorescent imaging of SD-PHEA-NI (L
1
, L
2
) to healthy 293T cells.
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in B16F10 cells mediated by the receptors. Therefore, SD-PHEA-NI (L
1
, L
2
) displayed high uptake by B16F10 cells due
to SD groups of tumor-targeting property.
Cellular Uptake
Cell uptake assays of SD-PHEA-NI (L
2
) were evaluated in B16F10 melanoma cells because SD-PHEA-NI (L
2
) was less
cytotoxic than SD-PHEA-NI (L
1
). Fluorescence imaging of B16F10 cells incubated with SD-PHEA-NI (L
2
) is shown in
Figure 9 when excited by white and blue light (excitation wavelength: 450 nm). B16F10 cells incubated with SD-PHEA-
NI (L
2
) displayed good green uorescence images when excited with blue light (Figure 9: B2). However, B16F10 cells
incubated with pure growth medium indicated no uorescence imaging when excited by blue light (Figure 9: B1).
In the inhibited cell uptake assay, B16F10 cells were incubated with SD solution for 1 h and subsequently SD-PHEA-
NI (L
2
, 0.25 μmol/L) solution later. B16F10 cells indicated the decreased intensity of green uorescence imaging when
excited by blue light (Figure 9: B3). The probable reason is that the transmembrane CA isoforms in tumor cells was
covered up early by SD. So the tumor cells cannot take up SD-PHEA-NI (L
2
)again and show an obvious decrease in
uorescence intensity after earlier inhibition of SD.
The comparable experimental values demonstrated that SD-PHEA-NI displayed good tumor-targeting property and
characteristic green uorescence imaging in B16F10 cells, whereas SD-PHEA-NI possessed a high specic selective
behavior to B16F10 cells. Moreover, SD-PHEA-NI was selectively taken up by receptor-binding afnity between SD
groups and the transmembrane CA isoforms.
Conclusions
Polyaspartamides containing sulfadiazine and naphthalene imide groups (SD-PHEA-NI) displayed similar UV and
uorescence property to NI. Moreover, SD-PHEA-NI exhibited low cytotoxicity to HeLa cells, high uptake and good
Figure 8 Fluorescent imaging of SD-PHEA-NI (L
1
, L
2
) to B16F10 cells.
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green uorescence imaging in B16F10 cells. Therefore, SD-PHEA-NI can be considered a potential probe for targeted
uorescent imaging in tumors.
Data Sharing Statement
Data will be made available on request.
Acknowledgments
The authors are grateful to the National Natural Science Foundation of China (Grant No.52303132), Key Project of
Zhejiang Province Natural Science Foundation (Grant No. LQZSZ25E030004), Key Science and Technology Projects of
Quzhou City (Grant No. 2024K132), Hubei Province Natural Science Foundation for Youths (Grant No.2022CFB710),
and Frontier Project of the Application Foundation of Wuhan Former Funded Science and Technology Program (Grant
No. 2020020601012252), Hubei Province, China.
Figure 9 Inhibited uorescence imaging of SD-PHEA-NI (L
2
) to B16F10 cells. (A1 and B1: Control B16F10 cells excited by white light and blue light, respectively; A2 and B2:
B16F10 cells incubated with SD-PHEA-NI (L
2
) excited by white light and blue light, respectively; A3 and B3: B16F10 cells that were previously incubated by SD (25μmol/L)
for 1h and subsequently incubated with SD-PHEA-NI (L2) (0.25μmol/L) later excited by white light and blue light, respectively.).
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Disclosure
The authors report no conicts of interest in this work.
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