Specific modification and self-transport of porphyrins and their multi-mechanism cooperative antitumor studies

Article abstract of DOI:10.1039/d0tb02847a

In order to reduce the toxicity and side effects of anti-tumor drugs and improve their therapeutic effect against cancer, photodynamic and chemical combination therapy has been exploited. However, the complicated preparation and metabolic toxicity of photosensitizer-loaded materials remain major obstacles for bioapplications. In this study, we designed and prepared a specific photosensitizer self-transporting drug-delivery system. First, 5,10,15,20-tetrakis(4-aminophenyl)-21H,23H-porphine (TAPP) was modified using specific molecules ofd-α-tocopheryl polyethylene glycol 1000 succinate (TPGS) with a certain antitumor effect, to prepare a specific fluorescent amphiphilic system (TAPP-TPGS). Then, the drug-loaded fluorescence nanomicelle (TAPP-TPGS/PTX) was formedviaself-assembly using the amphiphilic system and the anticancer drug paclitaxel (PTX). The carrier material could be used as a drug tracer and cancer therapy reagent to synergistically trace the chemotherapy drug and treat cancers. The biocompatibility and the enhanced antitumor effect of TAPP-TPGS/PTX were confirmed byin vitroandin vivoexperiments. To detect the synergistic anticancer effect enhanced by TPGS, TAPP-mPEG synthesized with a similar method as TAPP-TPGS was used for a comparative analysis. The results showed that the excellent synergistic anticancer effect of the TAPP-TPGS/PTX was enhanced due to the introduction of TPGS. Thus, the specific porphyrin self-transporting nanomicelle is a very promising carrier material for applications in biomedicine.

Full text of DOI:10.1039/d0tb02847a

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PAPER
Specific modification and self-transport of
porphyrins and their multi-mechanism
cooperative antitumor studies†
Cite this: J. Mater. Chem. B, 2021,
9, 3180
Shuxin Jia,a Shaochen Wang,a Shanshan Li,a Peng Hu,a Shuling Yu, *a Jiahua Shi*a
b
and Jintao Yuan
*
In order to reduce the toxicity and side effects of anti-tumor drugs and improve their therapeutic effect
against cancer, photodynamic and chemical combination therapy has been exploited. However, the
complicated preparation and metabolic toxicity of photosensitizer-loaded materials remain major
obstacles for bioapplications. In this study, we designed and prepared a specific photosensitizer self-
transporting drug-delivery system. First, 5,10,15,20-tetrakis(4-aminophenyl)-21H,23H-porphine (TAPP)
was modified using specific molecules of D-a-tocopheryl polyethylene glycol 1000 succinate (TPGS)
with a certain antitumor effect, to prepare a specific fluorescent amphiphilic system (TAPP-TPGS). Then,
the drug-loaded fluorescence nanomicelle (TAPP-TPGS/PTX) was formed via self-assembly using the
amphiphilic system and the anticancer drug paclitaxel (PTX). The carrier material could be used as a
drug tracer and cancer therapy reagent to synergistically trace the chemotherapy drug and treat
cancers. The biocompatibility and the enhanced antitumor effect of TAPP-TPGS/PTX were confirmed by
in vitro and in vivo experiments. To detect the synergistic anticancer effect enhanced by TPGS, TAPP-
mPEG synthesized with a similar method as TAPP-TPGS was used for a comparative analysis. The results
showed that the excellent synergistic anticancer effect of the TAPP-TPGS/PTX was enhanced due to the
introduction of TPGS. Thus, the specific porphyrin self-transporting nanomicelle is a very promising
carrier material for applications in biomedicine.
Received 7th December 2020,
Accepted 16th March 2021
DOI: 10.1039/d0tb02847a
rsc.li/materials-b
attracted attention as feasible strategies to effectively treat
cancer.14–16
1. Introduction
Cancer remains one of the deadliest diseases and causes millions
In particular, PDT—a promising noninvasive and effective
of deaths annually.1,2 Traditional chemotherapy usually has serious treatment modality—has been clinically approved for cancer
side effects. Also, its clinical application efficacy is restricted by therapy. PDT utilizes light of specific wavelengths to activate
multidrug resistance (MDR).3 To overcome these problems, various photosensitizers (PSs) and generate reactive oxygen species
nanomedicine strategies have been developed for sustained, (ROS) that can destroy biomacromolecules and organelles,
controlled, and targeted delivery of anticancer drugs.4,5 Because and induce cancer cell death. Furthermore, the ROS can inhibit
of their synergistic treatment advantages,6–9 combination therapies, the drug-efflux P-glycoprotein (P-gp) pumps of MDR cells,
such as the use of chemotherapy drugs with photothermal therapy, which increases the drug concentration in the tumor area.17
immunotherapy, photodynamic therapy (PDT), and so on,10–13 have Previous studies have shown that the synergistic effects of PDT
and chemotherapy result in significantly better therapeutic
effects in comparison to either PDT or chemotherapy alone.
Therefore, the combination of PDT and chemotherapy has
a Key Laboratory of Natural Medicine and Immune-Engineering of Henan Province,
Henan University, Kaifeng, Henan, 475004, People’s Republic of China.
attracted significant attention in the field of nanomedicine
E-mail: yushuling@henu.edu.cn, sjiahua@henu.edu.cn, jtyuan@zzu.edu.cn
b College of Public Health, Zhengzhou University, Zhengzhou, 450001, People’s
because of its excellent antitumor effect.18–20
Republic of China
The PS plays a very important role in PDT.21 Phytochlorin
(Ce6), indocyanine green (ICG), and protoporphyrin IX are
usually used as PSs due to their high rate of reactive oxygen
species (ROS) production. However, the application of these PSs
is limited because of their instability and high cost. Porphyrin,
however, is a readily available and cheap PS.22,23 However, the
† Electronic supplementary information (ESI) available: Materials; the detailed
processes of TAPP preparation; micelles stability test; 1H NMR spectrum of TAPP
(Fig. S1); mass spectrometry of TAPP (Fig. S2); 1H NMR spectrum of TAPP-TPGS
(Fig. S3); 1H NMR spectrum of TAPP-mPEG (Fig. S4); PTX standard curve, release
profile of TAPP-mPEG (B) and TAPP-TPGS (C) nanomicelles loaded with PTX at
different pH values (Fig. S5). See DOI: 10.1039/d0tb02847a
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complicated preparation and metabolic toxicity of porphyrin- similar method. The two prepared nanomicelle types were char-
loaded materials limit their bioapplication. acterized by nuclear magnetic resonance spectroscopy (NMR)
Carriers loading PSs have a pivotal impact on the therapeutic and mass spectroscopy (MS). The morphologies of the nano-
effect of PDT.23 However carriers synthesized using complex micelles were characterized with transmission electron micro-
methods or unapproved excipients could result in undesired scopy (TEM). The ROS produced by nanomicelles was detected
side effects, and metabolic and biocompatibility problems by using the ROS probes 1,3-diphenylisobenzofuran (DPBF) and
in vivo.24 D-a-Tocopheryl polyethylene glycol 1000 succinate 2,7-dichlorodihydrofluorescein (DCFH-DA). Finally, the synergistic
(TPGS), formed by esterification of PEG 1000 and vitamin E anticancer effect of TAPP-TPGS/PTX (TPGS, chemotherapy, and
succinate, possesses an amphiphilic structure and exhibits an PDT) and that of TAPP-mPEG/PTX were studied by performing
extraordinary permeation effect toward hydrophobic and hydro- in vitro and in vivo experiments.
philic drug molecules.25,26 Furthermore, TPGS can improve
cellular uptake, enhance the solubility of various drugs, target
the mitochondria of cancer cells, and induce apoptosis of cancer
2. Experimental section
2.1. Synthesis of 5,10,15,20-tetrakis(4-aminophenyl)-
cells; however, TPGS exhibited nontoxicity toward normal
tissues.27–29 TPGS was recognized as a safe pharmaceutical
21H,23H-porphine (TAPP)
adjuvant and was approved by the US FDA; it has been widely p-Nitrobenzaldehyde (50 mmol, 7.561 g), acetic anhydride (86 mmol,
used as a solubilizer, emulsifier, additive, and stabilizer in 8.2 mL), and propionic acid (200 mL) were added to a two-necked
various drug-delivery systems.30–32 Therefore, the aim of this flask. The reaction system was heated to 80 1C in an oil bath, and
present work is to realize the best possible therapeutic effect then the mixture solution of freshly distilled pyrrole (50 mmol,
with the approved excipients and a simple synthesis routine.
3.5 mL) and propionic acid (10 mL) were slowly added dropwise into
We prepared a specific porphyrin self-transporting nano- the two-necked flask by using a constant-pressure dropping funnel
scale drug-delivery system for cancer synergistic treatment with condensation. After the dropwise addition, the temperature
(Scheme 1). The porphyrin was synthesized via a simple method, was raised to 135 1C and the mixture was refluxed for 30 min. The
and then the arms of porphyrin were modified with TPGS to reaction solution was then cooled to room temperature and main-
prepare a specific amphiphilic system (TAPP-TPGS). Next, the tained overnight at 4 1C; then, the obtained precipitate was filtered,
amphiphilic system and the anticancer drug paclitaxel (PTX) and the filter cake was washed with distilled water (3 ꢀ 80 mL) and
underwent self-assembly to form the nanomicelle loaded with dried under vacuum. The crude product was dissolved in 80 mL of
the drug (TAPP-TPGS/PTX). We expected TAPP-TPGS/PTX to pyridine, and the solution was refluxed for 1 h, cooled to the room
result in enhanced cellular uptake and a long blood-circulation temperature, and then maintained at 0 1C for 24 h. The solid was
time owing to the introduced TPGS and the combination therapy collected and washed with acetone until the washing liquid became
(i.e., drug, TPGS, and PDT) to effectively inhibit the growth of colorless. Finally, the solid was dried under vacuum and collected
tumors and prolong the survival time of tumor-bearing mice. again: a purple solid (1.093 g) was obtained in a yield of about 11%.
In order to confirm the increasing cellular uptake and inducing
The prepared product (1.3 mmol, 1.0069 g) was dissolved in
of apoptosis by TPGS, TAPP-mPEG/PTX was also prepared by a 50 mL of concentrated hydrochloric acid, and the solution was
Scheme 1 (A) Synthetic scheme for TAPP-mPEG and TAPP-TPGS and Schematic diagram of TAPP-mPEG and TAPP-TPGS self-assembled nanomi-
celles. (B) Schematic diagram of the synergistic therapy of TAPP-TPGS/PTX in tumor.
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bubbled with nitrogen for 20 min. At the same time, SnCl2ꢁ water to remove residual acetone, the dialysate was freeze-
2H2O (19.1 mmol, 4.3127 g) was dissolved in 70 mL of con- dried, and the product was stored at 4 1C.
centrated hydrochloric acid, and the solution was also bubbled
2.5. Morphological characterization
with nitrogen for 20 min. Then, these two solutions were mixed,
and reacted at 80 1C for 30 min. Thereafter, the heating of the
reaction system was stopped, and it was cooled to the room
temperature. Then, the reaction was continued in an ice bath.
Next, 62.5 mL of NH2ꢁH2O was added to the reaction system
for neutralizing the concentrated hydrochloric acid. A solid
appeared and was filtered with a sand core funnel, and finally,
the filtrate was collected. Next, 100 mL of a 5% NaOH solution
was added to the filtrate, and the solution was vigorously
stirred. The resultant solid was collected by filtering, and the
filter cake was washed three times with distilled water and
dried under vacuum. The crude product was further purified in
acetone by using a Soxhlet extractor, and a purple solid (TAPP)
was obtained in a yield of 15.1%; it was analyzed using 1H NMR
and MS.
The morphology of the micelles was observed through transmis-
sion electron microscopy (TEM, JEM-2100, Japan). Micelles were
dissolved in distilled water to form a solution (1 mg mLꢂ1), and
the solution was dripped onto a copper mesh, which was dried at
room temperature. Then, the morphology of the micelles was
observed.
2.6. Hemolysis experiment
The eyeball blood of healthy Kunming mice was collected and
centrifuged to obtain the red blood cells. The red blood cells
were dispersed into normal saline to prepare the 4% red blood
cell suspension. Then, the 4% red blood cell suspension was
placed in 18 centrifuge tubes, respectively, and the blood
volume of every tube was 2 mL. Solutions of TAPP-mPEG,
TAPP-mPEG/PTX, TAPP-TPGS, and TAPP-TPGS/PTX at different
concentrations (300, 150, 75, and 37.5 mg mLꢂ1) were prepared,
respectively, and 2 mL of each solution was separately added
into the above centrifuge tubes. Next, 2 mL of physiological
saline and deionized water were also added, followed by the 4%
red blood cell suspension. The red blood cells with normal
saline were used as the negative control group, and the red
blood cells with deionized water were used as the positive
control group. The samples were incubated at 37 1C for 4 h
and then centrifuged and photographed. Finally, the super-
natant was taken to measure the UV absorption at 540 nm. The
following formula was used to calculate the hemolysis rate of
different groups:
2.2. Synthesis of TAPP-mPEG and TAPP-TPGS
First, carboxyl mPEG and carboxyl TPGS were prepared with the
method reported in ref. 33. The synthesis method of TAPP-
mPEG is as follows: carboxyl mPEG (400 mg, 0.2 mmol), DMAP
(24.5 mg, 0.2 mmol), and PyBOP (104.08 mg, 0.2 mmol) were
added to a round-bottomed flask, and then, DMF (15 mL) was
added into the system to dissolve the samples. Next, TAPP
(33.74 mg, 0.05 mmol) was added into the solution, which was
magnetically stirred at room temperature for 24 h, and purified
by dialysis against deionized water for 48 h. The dialysis solution
was freeze-dried, and finally, a dark green powder was obtained
with the yield of 72.1%. The synthesis and purification method
for TAPP-TPGS was the same as that for TAPP-mPEG; a dark
green solid was obtained in a yield of 71.9%. The products were
characterized by 1H NMR.
Hemolysis ð%Þ
(1)
Abssample ꢂ Absnegative control
¼
ꢀ 100%
Abspositive control ꢂ Absnegative control
2.3. Preparation of TAPP-mPEG and TAPP-TPGS micelles
Blank micelles of TAPP-mPEG and TAPP-TPGS were prepared
by the solvent evaporation method as follows: TAPP-mPEG or
TAPP-TPGS (5 mg) was dissolved in 2 mL of an acetone solution,
and then this solution was slowly added dropwise to 5 mL of
deionized water with vigorously stirring. Then, the acetone was
volatilized under a nitrogen atmosphere, and the residual liquid
was dialyzed against deionized water. Finally, the dialysate was
lyophilized, and the product was stored at 4 1C.
2.7. Determination of PTX loading in micelles and release
profiles in vitro
The PTX loading in the micelles was quantified by measuring the
UV absorption intensity of PTX. The nanomicelles were dissolved
in ethanol, and then the concentration of PTX in the ethanol
solution was calculated according to formula (2), and the drug
loading content was calculated to be 8.7% by using eqn (3).
The standard curve equation: Y = 0.039 + 0.0375X (R2 = 0.99873)
2.4. Preparation of PTX-loaded TAPP-mPEG and TAPP-TPGS
micelles
(2)
Drug loading ðDLÞ
The drug loading of TAPP-mPEG and TAPP-TPGS micelles was
completed by a solvent evaporation method as follows: TAPP-
mPEG or TAPP-TPGS (20 mg) and PTX (10 mg) were dissolved in
4 mL of acetone, and the solution was stirred at room tem-
perature for 4 h. Then, the solution was slowly added dropwise
(3)
Mass of drug loaded in micelles
¼
ꢀ 100%
Mass of micelles
TAPP-mPEG/PTX and TAPP-TPGS/PTX (260 mg PTX/mL) were
to deionized water with vigorously stirring, and thereafter, the each dissolved separately in 1 mL saline, and the solutions were
acetone was removed by volatilizing under a nitrogen atmo- dialyzed against 10 mL of phosphate buffer solution with
sphere. The resultant solution was dialyzed against deionized different pH values (pH = 7.4, 6.5, 5.5) in a 1 kDa membrane
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at 37 1C with gentle agitation for 72 h. Then, 3 mL of the and resuspended in the PBS (1 mL) to detect the fluorescence of
dialysis medium was taken out at predetermined time points DCF by using an inverted fluorescence microscope.
(0, 2, 4, 8, 12, 24, 48, 72 h), and the same volume of fresh
2.11. Cytotoxicity assay
medium was added immediately. The content of PTX was
measured by using a UV spectrophotometer, and the cumula- To investigate the cytotoxicity of TAPP-mPEG, TAPP-mPEG/PTX,
tive drug release was calculated by using equation (4).
TAPP-TPGS, and TAPP-TPGS/PTX, 4T1, MCF-7, HepG2, and
HL-7702 cells were seeded in a 96-well plate with a density of
5 ꢀ 103 cells per well and cultivated in the DMEM medium at
37 1C in a humidified atmosphere of 5% CO2. Various concen-
trations of TAPP-mPEG, TAPP-TPGS, PTX, TAPP-mPEG/PTX,
and TAPP-TPGS/PTX were added into the 96-well plate with
4T1, MCF-7, and HepG2 cells (two groups for each concen-
tration: no-laser-irradiation group and laser-irradiation group).
After 24 h, one group was irradiated with a laser (660 nm) with
the optical power density being 18 mW cmꢂ2, and the irradiation
time per well was 10 min. Then, the cells were incubated in the
incubator. TPGS and TAPP-TPGS with various concentrations
nꢂ1
P
V e
Ci þ V0Cn
1
Er ¼
(4)
mdrug
Er: Cumulative drug release (%), Ve: Sample volume (mL), V0:
Total volume of the released medium (mL), Ci: Concentration
of the drug in the ith sample (mg mLꢂ1), Cn: Concentration of
the drug in the nth sample (mg mLꢂ1), m: Total amount of drug
in the drug-loaded micelle (mg).
2.8. Singlet oxygen detection
To evaluate the singlet oxygen generation capability of TAPP- were added into the 96-well plate with HL-7702 cells. After 24 h,
50 mL of MTT was added to each well, and the cells were
incubated for 4 h at 37 1C in an incubator with 5% CO2. Then,
100 mL of DMSO was added to each well and shaken for 10 min
mPEG and TAPP-TPGS nanomicelles, 1,3-diphenylisobenzofuran
(DPBF), a sensitive ROS probe, was employed to detect the
singlet oxygen produced by the two micelles. The singlet oxygen
was identified by the emission spectra of the fluorescence probe on a shaker to fully dissolve the crystals. The absorbance of each
DPBF. DPBF (300 mL, 100 mg mLꢂ1) was added to the TAPP-mPEG well at 570 nm was measured by using a microplate reader.
or TAPP-TPGS (2.7 mL, 111 mg mLꢂ1) methanol solution. The
2.12. In vivo fluorescence imaging
power density of 18 mW cmꢂ2. The absorption spectrum of the All the animal experiments were performed under a protocol
solution was irradiated with a 660 nm laser with the optical
approved by the Institutional of Animal Care and Use Committee,
Henan University. Tumor-bearing BALB/c mice models (four mice)
were established by subcutaneous injection of cancer cells at the
right armpit of mice. After one week, the four mice were divided
DPBF at 480 nm was recorded by using an ultraviolet-visible
spectrometer at 2, 4, 6, and 8 min, respectively.
2.9. In vitro cellular internalization
4T1 cells were incubated in the DMEM medium at a density of into two groups with two mice in each group. TAPP-mPEG/PTX
5.0 ꢀ 103 cells per well at 37 1C under a 5% CO2 atmosphere for and TAPP-TPGS/PTX (200 mL, 1 mg mLꢂ1) were each injected into
24 h. After 24 h, the medium was replaced with a medium contain- one of the two groups of tumor-bearing mice through the tail vein.
ing one of the following: TAPP-mPEG or TAPP-TPGS (20 mg mLꢂ1). Then, in vivo fluorescence imaging of the tumor-bearing mice was
The cells were cultured for another 4 h at 37 1C in a CO2 atmosphere. conducted at predetermined time points (2, 4, 6, 8, 12, 24, 48, 72,
After 4 h, the medium was discarded, and the cells were washed 96, 120, 144 h) with isoflurane-induced anesthesia via an IVIS
three times with PBS (pH = 7.4) and fixed with paraformaldehyde Lumina XRMS Series (USA, PerkinElmer) in vivo imaging system.
(4%) for 10 min. Then, the paraformaldehyde was poured off and Finally, the main organs (heart, liver, spleen, lung, and kidney)
the cells were washed three times with PBS, stained with DAPI for and tumor of each group of mice were collected for fluorescence
10 min, and washed again with PBS. Laser confocal scanning imaging to detect the accumulation of drug-loaded materials in
microscopy was employed to perform the fluorescence imaging of tumors and different organs.
the cells, and the excitation wavelength was 488 nm.
2.13. Evaluation of antitumor activity in vivo
2.10. Intracellular ROS generation
The 4T1 tumor-bearing mice were randomly divided into ten
The generated ROS was detected with 2,7-dichlorodihydrofluorescein groups with six mice per group. One group was injected with
(DCFH-DA), which could react with the ROS to generate fluor- saline and the other was injected with PTX via the tail. TAPP-
escent 2,7-dichlorofluorescein (DCF) as a probe to detect the mPEG, TAPP-TPGS, TAPP-mPEG/PTX, and TAPP-TPGS/PTX
generated ROS. 4T1 cells were seeded in six-well plates (105), and (PTX dose = 5 mg kgꢂ1) were each injected into two (out of
the cells were co-cultured with 300 mg mLꢂ1 TAPP-TPGS/PTX and eight) groups of mice via the tail vein; one of these two groups
TAPP-PEG/PTX nanomicelles at 37 1C for 6 h. Then, the medium was subjected to laser irradiation, and the other group was not.
was removed and the DCFH-DA working solution (1 mM) was The tail vein injections were given on the first, fourth, and
added into six-well plates. The control group was not irradiated seventh days. Then, laser irradiation of one group each treated
with the laser, whereas the experimental group was irradiated with TAPP-mPEG, TAPP-TPGS, TAPP-mPEG/PTX, and TAPP-
with a 660 nm laser for 10 min with the optical power density TPGS/PTX was performed 24 h after each injection, by using a
being 18 mW cmꢂ2. Then, the samples were cultured for another 660 nm laser at a power density of 18 mW cmꢂ2 for 5 min. The
30 min in darkness. The cells were washed with PBS three times other four groups that were not subjected to irradiation were
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used as the control. The tumor size and body weight per group mPEG. The results confirm that TAPP-mPEG was successfully
were monitored every other day up to 14 days, and the tumor prepared. Further, Fig. S4 (ESI†) clearly shows the characteristic
volume was calculated according to the following formula: peaks of TAPP and the characteristic peak of the polyethylene-
width2 ꢀ length/2 (where width was the tumor width at the glycol repeat unit (–CH2CH2O–) in TPGS at 3.64 ppm.
shortest dimension, and length was the longest dimension).
3.3. Preparation and characterization of TAPP-mPEG and
TAPP-TPGS micelles
The surviving mice were observed until the 45th day.
2.14. Histology study
Both TAPP-mPEG and TAPP-TPGS micelles were prepared by
the solvent evaporation method. The morphologies of TAPP-mPEG
and TAPP-TPGS micelles were characterized by transmission
electron microscopy (TEM). As shown in the Fig. 1A, TAPP-
mPEG existed as almost-spherical nanoparticles, and the particle
size was about 18 nm. The morphology and the particle size
distribution of TAPP-TPGS are shown in Fig. 1B; TAPP-TPGS also
existed as spherical nanoparticles, and the particle size was
about 20 nm. In addition, Fig. 1C and D show that the UV
absorption, fluorescence intensity, and the Tyndall phenomenon
of two nanomicelles on first day and fourteenth day, respectively.
The result displayed that the UV absorption, fluorescence inten-
sity, and the Tyndall phenomenon of two nanomicelles were
almost the same on the first day and the fourteenth day, which
proved that both micelles had good stability.
TAPP-mPEG, TAPP-TPGS, TAPP-mPEG/PTX, and TAPP-TPGS/
PTX (PTX dose = 5 mg kgꢂ1) were each injected into two (out
of eight) groups of 4T1 tumor-bearing mice (two mice per
group) via the tail vein; one of these two groups was subjected
to laser irradiation, and the other group was not. The tail vein
injections were given on the first, fourth, and seventh days.
Then, laser irradiation of one group each treated with TAPP-
mPEG, TAPP-TPGS, TAPP-mPEG/PTX, and TAPP-TPGS/PTX was
performed 24 h after each injection, by using a 660 nm laser at
a power density of 18 mW cmꢂ2 for 5 min. The other four
groups of mice treated with TAPP-mPEG, TAPP-TPGS, TAPP-
mPEG/PTX, and TAPP-TPGS/PTX without irradiation were used
as the control. Two days after the last PDT treatments, the main
organs (heart, liver, spleen, lung, and kidney) and tumor of the
eight groups were collected for hematoxylin and eosin (H&E)
staining, TUNEL staining, and PCNA.
3.4. Hemolysis experiment
The problem of hemolysis due to non-specific interactions of drug
carriers with hemoglobin must be solved for the practical applica-
tion of drug carriers in the field of nanomedicine. Therefore, the
blood compatibility of nanocarriers needs to be investigated
before biological application. As shown in Fig. 2A and B, at a
certain concentration range (from 37.5 mg mLꢂ1 to 300 mg mLꢂ1),
the hemolysis rate increases with the concentration of the nano-
micelles, but for both nanomicelle types, it is much less than 5%.
Further, the images show that no obvious hemolysis occurred in
all samples. All results prove that the prepared nanomicelles had
excellent biocompatibility and met the standard medical require-
ments. Therefore, the prepared nanomicelles can be used as
medical materials for cancer treatment.
2.15. Statistical analysis
The results of all experiments are presented as the mean +
standard deviation (SD). The data were analyzed using the SPSS
software (version 22.0; SPSS Inc., Chicago, IL, USA). The statistical
significance of differences was evaluated by two-way ANOVA
analysis: p o 0.05 and p o 0.01 were considered to be statisti-
cally significant and highly statistically significant, respectively.
3. Results and discussion
3.1. Synthesis and characterization of TAPP
1
TAPP was characterized by H NMR (300 MHz, chloroform-d)
3.5. Detection of reactive oxygen species (ROS)
and MS, and the results are shown in Fig. S1 and S2 (ESI†),
respectively. The characteristic peaks at 8.90 ppm, 7.99 ppm,
7.06 ppm, and ꢂ2.72 ppm all correspond to TAPP: those at
7.99 ppm and 7.06 ppm were the characteristic peaks of the
hydrogen of the phenyl group, and those at 8.90 ppm and ꢂ2.72 ppm
were the characteristic peaks of the pyrrolyl group. The NMR
spectrum of TAPP is shown in Fig. S1 (ESI†), and the results
prove that TAPP was synthesized successfully. The characteristic
peak of TAPP in Fig. S2 (ESI†) is at m/z 675.45, which further
demonstrates the successful synthesis of TAPP.
ROS is an important indicator for determining whether nano-
micelles can be used in PDT. DPBF was used as a ROS trapping
agent to detect the ROS produced by nanomicelles under laser
irradiation. As DPBF only reacts with singlet oxygen and the
superoxide anion in active oxygen species, the experimental
results can objectively reflect the ROS yield. DPBF absorbance
decreases when ROS is generated; that is, with an increasing
amount of ROS, the absorption value of DPBF decreases.
Obviously, the absorbance of the DPBF solution containing
nanomicelles is significantly smaller than that of the DPBF
solution, indicating that both nanomicelles can produce ROS at
660 nm laser irradiation (Fig. 2C). These results further confirm
3.2. Synthesis and characterization of TAPP-mPEG and TAPP-
TPGS
TAPP-mPEG and TAPP-TPGS were characterized by 1H NMR that porphyrin, as a PS, is suitable for PDT.
(300 MHz, Chloroform-d), and the results are displayed in Fig. S3
3.6. Release of drug from two nanomicelle types
and S4 (ESI†). In Fig. S3 (ESI†), the peaks at 8.91 ppm, 7.98 ppm,
and ꢂ2.74 ppm are the characteristic peaks of TAPP, and the peak In order to test the sustained release behavior of PTX loaded in
at 3.38 ppm is the characteristic peak of the methoxy hydrogen of TAPP-mPEG and TAPP-TPGS nanomicelles in vitro, the drug-
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Fig. 1 TEM images and particle size distribution of TAPP-mPEG (A) and TAPP-TPGS (B). The Tyndall phenomenon, fluorescence image and UV-Vis
absorption spectra of TAPP-mPEG (a) and TAPP-TPGS (b) on the 1st day (C). The Tyndall phenomenon, fluorescence image and UV-Vis absorption
spectra of TAPP-mPEG (a) and TAPP-TPGS (b) on 14 th day (D).
loaded nanoparticles were dissolved in physiological saline and fluorescent dyes. 4T1 cells were used to analyze the cellular
added into the dialysis membrane; then, the dialysis uptake of the two nanomicelle types. 4T1 cells were incubated
membrane was immersed in 10 mL of PBS with pH values of with TAPP-mPEG and TAPP-TPGS for 4 h at 37 1C, and then, the
7.4, 6.5, and 5.5 with gentle shaking. At predetermined time cellular nuclei were dyed with 4,6-diamidino-2-phenylindole
points, the samples were collected to determine PTX release by (DAPI). The results were obtained via confocal laser scanning
using the standard curve method. The standard curve of PTX microscopy (CLSM). As shown in Fig. 3, homogeneous red
was first established and is shown in Fig. S5A (ESI†). Fig. S5B fluorescence was observed in the cytoplasm, indicating nano-
and C (ESI†) show the results of the in vitro release experiments micelle uptake by cells through endocytosis. The fluorescence
for the two drug-loaded nanomicelles. The two nanomicelle intensity in the TAPP-TPGS treatment group was stronger than
types showed similar results: 72% and 70.2% of PTX were that in the TAPP-mPEG treatment group, confirming that TPGS
released from TAPP-mPEG/PTX and TAPP-TPGS/PTX, respec- can improve cellular uptake.
tively, after 72 h of incubation at pH 5.5. The released drug
amount reached 48% and 47.1%, respectively, after 72 h of
incubation at pH 6.5. However, the amounts were 30.2% and
30.9%, respectively, after 72 h of incubation at pH 7.4. These
as an indicator to trace intracellular singlet oxygen generation by
results demonstrate that the two nanomicelles were pH-sensitive.
The pH in the microenvironment of the tumor tissue is lower than
results are shown in Fig. 4. No obvious green fluorescence was
that in normal cell tissue. The amount of the drug released from
nanomicelles under acidic conditions is significantly higher than
laser irradiation, while strong green fluorescence could be detected
that in a neutral environment, which is conducive to cancer
3.8. Intracellular ROS detection
In order to further investigate ROS generation, DCFH-DA was used
TAPP-TPGS/PTX and TAPP-4PEG/PTX nanomicelles, and the
observed in the samples treated with the two nanomicelles without
in the samples treated with the two nanomicelles with laser
targeted therapy.
irradiation; this implies that the treatment with the two nano-
micelles and laser irradiation could generate large amounts of
singlet oxygen. Further, the fluorescence intensity of the group
3.7. Cellular uptake
TAPP-mPEG and TAPP-TPGS exhibit red fluorescence; thus, treated with TAPP-TPGS/PTX with laser irradiation is stronger
the samples are free from fluorescent labeling with organic than that of the group treated with TAPP-PEG with laser irradiation;
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Fig. 2 Blank nanomicelles and drug-loaded nanomicelles hemolysis tests in vitro (A) and (B). Singlet oxygen probe DPBF detection of TAPP-mPEG and
TAPP-TPGS singlet oxygen production at 660 nm illumination (C).
and the fluorescence quantitative results displayed that the mean are shown in Fig. 5: Fig. 5A–C show that the TAPP-mPEG blank
fluorescence intensity the group treated with TAPP-TPGS/PTX with micelles were non-toxic to 4T1, MCF-7, and HepG2 within a
laser irradiation is 1.70 times that of the group treated with TAPP- certain concentration range without laser irradiation, which
PEG with laser irradiation. This is because TPGS improves the demonstrated that TAPP-mPEG blank micelles have excellent
cellular uptake, which is consistent with the results of the cellular biocompatibility. The experimental results also showed that the
uptake experiment.
toxicity of TAPP-TPGS without irradiation to 4T1, MCF-7, and
HepG2 cells gradually increased with the concentration of
samples; the reason for this is that TPGS has a certain anti-
tumor effect. A significant anti-tumor effect could be detected
in groups subjected to laser irradiation. The results prove that
the two materials had an excellent photodynamic effect. Under
laser irradiation, the cytotoxicity of TAPP-TPGS to 4T1, MCF-7,
and HepG2 cells was stronger than that of TAPP-mPEG. These
results prove that porphyrin has a good photodynamic ther-
apeutic effect, and TPGS has an auxiliary anti-tumor effect. The
toxicities of TPGS and TAPP-TPGS toward HL-7702 cells were
evaluated, as shown in Fig. 5D. The results show that TPGS and
TAPP-TPGS did not have significant toxicity to HL-7702, which
further confirmed that the TAPP-TPGS nanomicelle showed
good biocompatibility. Therefore, TAPP-TPGS is superior to
TAPP-mPEG as an anticancer drug-loaded material.
3.9. Cytotoxicity
The cytotoxicity of the materials before and after drug loading
was evaluated using various cells by the 3-(4,5-dimethyl-2-
thiazolyl)-2,5-diphenyl-2-H-tetrazolium (MTT) assay. The results
In Fig. 6A–C, the cell viabilities of 4T1, MCF-7, and HepG2
cells treated with PTX and PTX with laser irradiation were
almost the same, which proves that PTX did not have photo-
dynamic effect and that the cytotoxicity was enhanced with
increasing drug concentration. Further, the TAPP-mPEG/PTX
treatment cells showed obvious toxicity, which confirmed that
Fig. 3 Confocal fluorescence micrographs of 4T1 cells after incubation
with TAPP-mPEG and TAPP-TPGS for 4 h at 37 1C.
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Fig. 4 DCF fluorescence intensity of 4T1 cells after treatment of TAPP-mPEG/PTX and TAPP-TPGS/PTX without and with NIR laser irradiation detected
by fluorescent inverted microscopy. Fluorescence imaging (A) and mean fluorescence intensity (B).
Fig. 5 In vitro inhibition effects of TAPP-mPEG and TAPP-TPGS on 4T1 cells (A), MCF-7 cells (B), HepG2 cells (C), Cytotoxicity of TPGS and TAPP-TPGS (D).
Fig. 6 In vitro inhibition effects of PTX, TAPP-mPEG/PTX and TAPP-TPGS/PTX on 4T1 cells (A), MCF-7 cells (B), HepG2 cells (C).
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the drug loaded in the nanomaterials could be released success- PTX could enter into the tumor region and the fluorescence signal
fully while maintaining the efficacy of PTX. The toxicities of 4T1, became strong 4 h following intravenous injection. This implies
MCF-7, and HepG2 cells treated with TAPP-TPGS/PTX were that TAPP-TPGS/PTX can enter the tumor faster. To further
higher than those of the cells treated with TAPP-mPEG/PTX; this compare the distribution of the two drug-loaded micelles in
confirms that TPGS could assist the drug in treating cancer. mice, the ROI function of IVIS Lumina XRMS Series was also
When various cells treated with TAPP-TPGS/PTX were irradiated, used to quantify the fluorescence signal of the tumor region, and
the maximum inhibition rate of 4T1, MCF-7, and HepG2 cells the results are shown in Fig. 7B. The photon counts in the tumor
reached 97%, 93.6%, and 94%, respectively. The in vitro results region gradually increased from 2 h after the injection of TAPP-
demonstrate that the loaded drug, TPGS, and photodynamic mPEG/PTX and TAPP-TPGS/PTX, and a maximum was observed
effect result in the effective synergistic cancer treatment.
24 h post-injection in the group treated with TAPP-TPGS/PTX; on
the other hand, the photon counts reached a maximum 48 h
following injection of TAPP-mPEG/PTX. Thus, the two nanomicelles
3.10. In vivo fluorescence imaging
The fluorescence imaging of porphyrin could enable the tracking accumulated in the tumor areas via the enhanced permeability and
of the drug delivery and biodistribution of drug-loaded materials. retention (EPR) effect, and TAPP-TPGS/PTX accumulated in the
In order to study the biodistribution of the drug and the materials, tumor area more quickly than did TAPP-mPEG/PTX. Thereafter,
the in vivo fluorescence imaging of the mice treated with TAPP- with time, the fluorescence intensity in the tumor regions of the two
mPEG/PTX and TAPP-TPGS/PTX was carried out, and the results groups both gradually decreased. However, the fluorescence signal
are shown in Fig. 7. Fig. 7A shows that both drug-loaded micelles in the tumor region could still be detected 144 h post-injection,
entered and accumulated in the tumor areas, and remained there indicating that the two nanomicelles remained for a long time in
for a long time. The two drug-loaded micelles could be excreted the tumor region. In addition, TAPP-TPGS/PTX accumulated quickly
through the hepatoenteric metabolism. Further, the tumor area in the tumor area, and the fluorescence intensity in the tumor
showed a weak fluorescence signal 4 h after injection in the group region of the group treated with TAPP-TPGS/PTX was stronger than
treated with TAPP-mPEG/PTX, while 2 h after injection, TAPP-TPGS/ that in the tumor region of the group treated with TAPP-mPEG/PTX
Fig. 7 (A) In vivo fluorescence imaging of tumor-bearing mice after intravenous injection of TAPP-mPEG/PTX and TAPP-TPGS/PTX. (B) The average
fluorescence intensity in the tumor region of the two groups of mice treated with TAPP-mPEG/PTX and TAPP-TPGS/PTX was recorded as total photon
counts per tumor corresponding to the fluorescence images in the mice bearing tumors. (C) The fluorescence image of various organs 168 h after
intravenous injection. (D) The average fluorescence intensity in the dissected tumor from the two groups of mice treated with TAPP-mPEG/PTX and
TAPP-TPGS/PTX at 144 h was recorded as total photon counts per tumor corresponding to the fluorescence images in the mice bearing tumor.
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at every time point; this indicates that TAPP-TPGS/PTX could TAPP-TPGS and TAPP-TPGS/PTX to the treated tumor-bearing mice
accumulate at a faster rate and reach a maximum concentration (Fig. 8B). Because the weight of the mice steadily increased in all
value. Further, the fluorescence intensity of the tumor region in the treatment groups, the toxic effects of TAPP-TPGS and TAPP-TPGS/
group treated with TAPP-TPGS/PTX decreased more slowly than that PTX could be considered to be negligible. The median survival time
of the tumor region in the group treated with TAPP-mPEG/PTX. of mice treated with TAPP-TPGS/PTX and laser irradiation was
Thus, TAPP-TPGS/PTX is more promising than TAPP-mPEG/PTX as longer than 45 d, while that of the mice treated with PTX and
a medical nanomaterial for cancer combination therapy.
TAPP-mPEG/PTX and laser irradiation was 28 d and 38 d, respec-
Further, 144 h after injection, mice in the two groups were tively. These results prove that PDT combined with TPGS and
sacrificed, and the tumors as well as major organs were chemotherapy can effectively prolong the survival time of tumor-
collected and subjected to fluorescence imaging. Fig. 7C shows bearing mice (Fig. 8C). Thus, the in vitro and in vivo results confirm
that no fluorescence signal was detected in any major organ, that the prepared specific nanomaterial is promising for cancer
while a strong fluorescence signal could be detected in the treatment.
tumor areas. The fluorescence intensity of the tumor areas was
quantified by calculating the photon count, and the results
3.12. Histological analysis
showed that the fluorescence intensity of the tumor region in
the group treated with TAPP-TPGS/PTX was stronger than that
in the group treated with TAPP-mPEG/PTX (Fig. 7D), which
further proved that TAPP-TPGS/PTX could more easily accumu-
late and remain in the tumor area than could TAPP-mPEG/PTX.
In order to further evaluate the toxicity of TAPP-TPGS/PTX and
TAPP-TPGS, the major organs of tumor-bearing mice including
heart, liver, spleen, lung and kidney were collected and stained
using the hematoxylin and eosin (H&E) for pathology analysis,
and the results are shown in Fig. 9A. All materials and drug-
loaded materials treatment groups with laser and without laser
irradiation have no obvious toxicity to normal tissues, which
3.11. In vivo antitumor effect
The in vivo anti-tumor effect of TAPP-TPGS/PTX with laser indicates that the material and drug-loaded materials had
irradiation was evaluated with 4T1 tumor-bearing mice as the excellent biocompatibility and was promising for cancer therapy.
model animals, and the results are shown in Fig. 8. As illustrated
Further, TUNEL staining and PCNA assays were performed
in Fig. 8A, the tumor inhibition rate when TAPP-mPEG/PTX was to evaluate the anti-tumor effect of TAPP-TPGS/PTX with and
used was higher than that when PTX was used, which indicates without laser irradiation (Fig. 9B). TUNEL assay results display
that TAPP-mPEG/PTX was effective in drug delivery and success- that a few apoptosis cells were detected in PTX, TAPP-mPEG/
ful drug release in the tumor area, while maintaining the efficacy PTX and TAPP-TPGS treatment groups and TAPP-TPGS/PTX.
of PTX. Further, the tumor inhibition effect was similar for TAPP-mPEG(L+), TAPP-TPGS(L+), TAPP-TPGS/PTX and TAPP-
TAPP-TPGS and TAPP-mPEG/PTX, indicating that TPGS could mPEG/PTX(L+) treatment groups showed a significantly higher
inhibit tumor growth similar to antitumor drugs. Further, the rate of apoptosis. The TAPP-TPGS/PTX(L+) treatment group has
group treated with TAPP-TPGS/PTX showed a stronger suppres- the most apoptosis cells among treatment groups. These results
sion effect than the groups treated with TAPP-TPGS and TAPP- prove TAPP-mPEG and TAPP-TPGS have excellent photo-
mPEG/PTX, which proves that TPGS and the anti-tumor drug dynamic action, the introduction of TPGS can effectively induce
effectively treat cancer in combination. In addition, all groups cancer cells apoptosis, and the synergistic therapy of TPGS,
subjected to laser irradiation displayed excellent tumor suppression drug and photodynamic therapy exhibits excellent anti-tumor
effect, while the group treated with TAPP-TPGS/PTX and laser effect. Furthermore, in the case of PCNA assay that determines
irradiation showed the most significant effect, further confirming the proliferation active cells, high proliferation levels are
the effective synergistic treatment with TPGS, the anti-tumor drug, observed in saline and TAPP-mPEG groups. Comparing to the
and PDT. The experimental results are consistent with the cytotoxi- PTX, TAPP-mPEG/PTX, TAPP-TPGS, TAPP-mPEG(L+), TAPP-
city test results. We also examined the potential toxic side effects of TPGS(L+), TAPP-TPGS/PTX and TAPP-mPEG/PTX(L+) treatment
Fig. 8 (A) The tumor volume in tumor-bearing mice from different treatment groups. *P o 0.05, **P o 0.01. (B) The body weight of tumor-bearing
mice from different treatment groups. (C) The survival rates of tumor-bearing mice from different treatment groups. Data in A and B are presented as
mean ꢃ SD (n = 6).
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Fig. 9 (A) Representative mouse tissue sections stained with hematoxylin and eosin (H&E). (B) PCNA (proliferation assay) and TUNEL (apoptosis assay)
stained 4T1 tumor tissue sections from mice that received different treatments.
groups, TAPP-TPGS/PTX with laser irradiation (TAPP-TPGS)/ involving the use of materials that assist in cancer treatment,
PTX (L+) could effectively inhibit the proliferation of the cancer chemotherapy, and PDT.
cells. The results further confirm the ideal synergistic treat-
ment effect of TPGS, chemotherapy, and PDT.
Conflicts of interest
There are no conflicts of interest to declare.
4. Conclusions
Acknowledgements
In conclusion, we prepared a specific drug-delivery nanomaterial
with photodynamic effect. The preparation method is simple,
This work was supported by the National Natural Science
and the materials used are biocompatible and approved by the
Foundation of China (Grant No.U1404508), the Key Scientific
FDA. TPGS with the anti-tumor effect was used to modify
Research Projects of Higher Education Institutions in Henan
porphyrin to form nanomicelles with a specific photodynamic
Province (Grant No. 20A430006 and 19A330006), the Young
effect, and the anti-tumor drug was loaded into the core of the
Talents Cultivation Program of the School of Medicine, Henan
nanomicelles. The TEM image shows that the size of the
University in 2019 (Grant No. 2019002), the Science and Tech-
nanomicelle is around 20 nm, which enables the utilization of
nology Development Program of Henan Province (Grant No.
the EPR effect of nanomaterials in the tumor environment.
212102310860).
Further, the results of hemolysis tests proved that the prepared
nanomicelle had excellent biocompatibility and met the stan-
dard medical requirements. In addition, a comparison of the
experimental results of using TAPP-mPEG/PTX and TAPP-TPGS/
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