Programmed drug delivery system based on optimized “size decrease and hydrophilicity/hydrophobicity transformation” for enhanced hepatocellular carcinoma therapy of doxorubicin

Article abstract of DOI:10.1016/j.nano.2018.02.006

Requirements on drug delivery systems to surmount a complex series of pathophysiological barriers bear “cascading contradictions”, especially size and hydrophilicity/hydrophobicity contradiction. Herein, a programmed drug delivery system (GNPs-Dox-Lac) based on optimized “size decrease and hydrophilicity/hydrophobicity transformation” was developed by combination the gelatin nanoparticle (GNPs) and prodrug Doxorubicin-Lactose (Dox-Lac). The results showed that GNPs-Dox-Lac (133.3 nm) were kinetically stable in blood circulation and inclined to accumulate at the tumor site. Then the degradation of the GNPs triggered by tumor extracellular matrix metalloproteinase-2 (MMP2) led to the release of prodrug Dox-Lac (Mw 898 Da) to facilitate the tumor tissue penetration and cellular uptake. Last, pH-responsive disassociation of Dox-Lac in tumor cells resulted in the free Dox (Mw 543 Da) release to induce toxicity. As expected, GNPs-Dox-Lac achieved superior tumor inhibition rate of 90.8% with low toxicity in vivo, suggesting its potential for enhanced hepatocellular carcinoma (HCC) therapy of doxorubicin in future.

Full text of DOI:10.1016/j.nano.2018.02.006

Accepted Manuscript
Programmed drug delivery system based on optimized “size
decrease and hydrophilicity/hydrophobicity transformation” for
enhanced hepatocellular carcinoma therapy of doxorubicin
Yuanyuan Liu, Lian Li, Lijia Li, Zhou Zhou, Fengling Wang,
Xiaofeng Xiong, Rui Zhou, Yuan Huang
PII:
S1549-9634(18)30038-8
doi:10.1016/j.nano.2018.02.006
NANO 1761
DOI:
Reference:
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Revised date:
Accepted date:
16 September 2017
3 January 2018
8 February 2018
Please cite this article as: Yuanyuan Liu, Lian Li, Lijia Li, Zhou Zhou, Fengling Wang,
Xiaofeng Xiong, Rui Zhou, Yuan Huang , Programmed drug delivery system based on
optimized “size decrease and hydrophilicity/hydrophobicity transformation” for enhanced
hepatocellular carcinoma therapy of doxorubicin. The address for the corresponding
author was captured as affiliation for all authors. Please check if appropriate. Nano(2018),
doi:10.1016/j.nano.2018.02.006
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ACCEPTED MANUSCRIPT
Programmed drug delivery system based on optimized “size decrease and
hydrophilicity/hydrophobicity transformation” for enhanced hepatocellular carcinoma
therapy of doxorubicin
Yuanyuan Liu, MSc, Lian Li, PhD, Lijia Li, MSc, Zhou Zhou, PhD, Fengling Wang, MSc,
Xiaofeng Xiong, MSc, Rui Zhou, PhD and Yuan Huang, PhD *
Key Laboratory of Drug Targeting and Drug Delivery System (Ministry of Education), West
China School of Pharmacy, Sichuan University. No. 17, Block 3, South Renmin Road,
Chengdu610041, P.R. China
*
Corresponding author
Prof. Yuan Huang; Tel/Fax: +86-028-85501617; E-mail: huangyuan0@163.com
Funding: This work was supported by the National Natural Science Foundation for
Distinguished Young Scholars (81625023) and the National Natural Science Foundation of
China (81473167).
Conflict of interest: The authors declare no conflict of interest.
Word count for Abstract: 149
Number of References: 35
Number of tables: 1
Word count for manuscript: 4993
Number of figures: 8
Number of Supplementary online-only files: 1
1

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Abstract
Requirements on drug delivery systems to surmount a complex series of pathophysiological
barriers bear “cascading contradictions”, especially size and hydrophilicity/hydrophobicity
contradiction. Herein, a programmed drug delivery system (GNPs-Dox-Lac) based on
optimized “size decrease and hydrophilicity/hydrophobicity transformation” was developed by
combination the gelatin nanoparticle (GNPs) and prodrug Doxorubicin-Lactose (Dox-Lac).
The results showed that GNPs-Dox-Lac (133.3 nm) were kinetically stable in blood circulation
and inclined to accumulate at the tumor site. Then the degradation of the GNPs triggered by
tumor extracelluer matrix metalloproteinase-2 (MMP2) led to the release of prodrug Dox-Lac
(Mw 898 Da) to facilitate the tumor tissue penetration and cellular uptake. Last, pH-responsive
disassociation of Dox-Lac in tumor cells resulted in the free Dox (Mw 543 Da) release to
induce toxicity. As expected, GNPs-Dox-Lac achieved superior tumor inhibition rate of 90.8%
with low toxicity in vivo. Suggesting its potential for enhanced hepatocellular carcinoma
(HCC) therapy of doxorubicin in future.
Keywords: Drug delivery system; Size decrease; Hydrophilicity/hydrophobicity
transformation; Environment-responsive; hepatocellular carcinoma therapy
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Background
A desired drug delivery system needs to flexibly overcome a complex series of
pathophysiological barriers, including insufficient tumor accumulation, restricted intratumoral
1, 2
diffusion and low efficiency of cellular uptake in tumor tissues . Unfortunately, requirements
on the delivery systems to surmount all those barriers bear “cascading contradictions”. So
increasing the therapeutic efficiency of anti-cancer drug delivery remains an enormous
challenge. What's worse, mishandling any one of the contradictions would profoundly
decrease the drug delivery efficiency, resulting in insufficient final drug concentration in tumor
3
, 4
cells and weakening treatment effect of solid tumors .
As reported, active targeting nanoparticles with a size range of 50–200 nm are suitable for
5
, 6
accumulation at the tumor site both by passively and actively manner . However, the dense
extracellular matrix (ECM) and elevated interstitial fluid pressure (IFP) in the tumor
microenvironment could severely impede the nanoparticles to the tumor peripheral region
7
because of their large size . Thus, there is a “size contradiction” in the requirements for tumor
accumulation and tumor penetration. It has been found that releasing the small molecule drug
8
after arriving at tumor sites could significantly facilitate deep tumor penetration . Therefore, an
ideal drug delivery system should be relatively large in its initial size to enhance tumor
accumulation by EPR effect and able to switch into small molecular weight therapeutics to
facilitate tumor penetration once “docked” at tumor sites. What’s more, the penetration of
small therapeutics through tumor tissue is determined by diverse physicochemical properties,
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especially by aqueous solubility. Water-soluble drugs could distribute readily in the
extracellular matrix (ECM) and diffuse efficiently through intercellular spaces; whereas,
9
lipid-soluble drugs could penetrate lipid cell membranes and be transported through cells .
Unluckily, lipid-soluble drugs, such as doxorubicin (Dox), could not penetrate deeply into
tumor tissue through cells, due to the intercalating ability and avidity of Dox to bind to DNA,
which make them trapped and consumed inside tumor cells located in the margin of the tumor
1
0
tissue, leaving a reduced amount available for diffusion to distal cells . Thus, it is easy to find
another “hydrophilicity/hydrophobicity contradiction” in the requirements for tumor
penetration and cellular uptake of Dox. Therefore, endowing hydrophilic property to
doxorubicin (Dox) in the form of prodrug to relatively increase the extracellular matrix (ECM)
distribution might be considered as a simple and effective method to achieve deep penetration
through intercellular spaces.
Lactose (Lac), a hydrophilic glucosyl-galactose disaccharide, has been widely used as a
hepatocyte targeting ligand, which can specifically interact with the asialoglycoprotein-1
receptor (ASGPR1) over-expressed on mammalian hepatocytes and actively internalized via
1
1
receptor-mediated endocytosis . In order to balance the “hydrophilicity/hydrophobicity
contradiction”, water-soluble lactose acid was covalently conjugated with Dox via a
pH-responsive hydrazone bond to form a hydrophilic active targeting prodrug Dox-Lac.
Although prodrug Doxorubicin-Lactose (Dox-Lac) could achieve deep tumor penetration and
deliver Dox to ASGPR1 over-expressed tumor cells, the delivery efficiency of this small
molecule (Mw 898 Da) needed further optimization while administered systemically in vivo,
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considering the above mentioned “size contradiction”. To improve the delivery efficiency of
Dox in vivo, gelatin nanoparticles (GNPs) were prepared by simple self-assembly and
crosslinking of gelatin, which is the substrate of metal matrix protease-2 (MMP2)
1
2, 13
over-expressed in tumors
. Then the prodrug Dox-Lac molecules were conjugated onto the
surface of GNPs to form a programmed drug delivery system (GNPs-Dox-Lac).
Inspired by those principles discussed above, this programmed drug delivery system
(GNPs-Dox-Lac) could be considered as a promising and reliable approach to deliver Dox
specifically to the site of action with high efficiency and optimized therapeutic effectiveness
towards hepatocellular carcinoma (HCC).
Methods
Synthesis and characterization of Dox-Lac
The synthetic details are described in supplementary materials. Briefly, lactobionolactone was
first synthesized via the lactonization of lactose acid, and then converted to
lactobionohydrazide (Lac-NHNH ) by hydrazinolysis. Lac-NHNH further reacted with Dox
2
2
1
to produce Dox-Lac. The structures of Dox-Lac were confirmed by H NMR spectra (400 MHz,
DMSO-d6) and mass spectra.
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Preparation and Characterization of GNPs, GNPs-Dox-Lac and GNPs-Dox
The gelatin nanoparticle (GNPs) was firstly prepared from the self-assembly and crosslinking
of gelatin, then the Dox-Lac molecules were conjugated onto the surface of GNPs. The details
are described in supplementary materials. The dynamic light scanning (DLS) size and zeta
potential of the GNPs, GNPs-Dox, GNPs-Dox-Lac were measured on a Zetasizer Nano ZS90
(Malvern Instruments, UK). The morphology and structure were observed via transmission
electron microscopy (TEM, H-600,Hitachi, Japan). The fluorescence emission spectra and
UV–Vis absorption of GNPs-Dox-Lac, GNPs-Dox, Dox-Lac and Dox were recorded by
UV-Vis spectrophotometer (Varian, USA) and spectrofluorophotometer (RF-5301 PC,
Shimadzu, Japan), respectively. The Dox-loading content, defined as the weight percentage of
Dox in nanoparticles (w/w), was quantified by UV-Vis analysis at 487nm using a UV-Vis
spectrophotometer. A calibration curve was prepared with the standard Dox solution under the
same condition.
Stability and MMP 2-triggered degradation of GNPs
For stability study, particle size changes were measured in 100% fetal bovine serum (FBS) or
pH 7.4 Dulbecco's modified Eagle's medium (DMEM, containing 10％FBS) medium by
dynamic light scanning (DLS) at different time points (1h,4h,8h,12h, 24h). To study the
degradation performance of GNPs triggered by MMP2, the morphology of GNPs-Dox-Lac
(with/without MMP2 for 4h) were captured by TEM after dyed by uranyl acetate, and particle
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size changes were also determined by DLS at 0h,1h,2h,4h.
In vitro drug release
The Dox-Lac release experiment was performed using a dialysis method. The release of
Dox-Lac from GNPs were studied at 37 ºC under different pH conditions (pH7.4 or 6.5),
5
ug/mL MMP2 in TNCB buffer, HepG2 cells and Lo2 cells conditioned media. 2 mL of the
prepared GNPs-Dox-Lac (equivalent of 2 mg/mL Dox) was transferred to a dialysis bag
MWCO 35 KDa), which was then immersed into 38 mL corresponding buffer. At desired time
(
points, 200ul of the release medium was taken out and replaced with 200ul of fresh medium.
Then the concentration of Dox-Lac was quantified by a Varioskan Flash (Thermo Fisher
Scientific, MA, USA) with excitation at 487 nm and emission at 553 nm. All the release
experiments were conducted in triplicate.
The Dox release experiment was determined by High Performance Liquid Chromatography
(HPLC) using a Diamonsil ODS column (250 × 4.6 mm, 5 mm). 2mg/mL Dox-Lac were
studied under different pH conditions (pH 5.0 or 6.5). At desired time points, 10 ul of
supernatant was analyzed by HPLC, the mobile phase consisted of deionized water and
acetonitrile (gradient 25 to 62 percent acetonitrile in water, v/v) at a flow rate of 1 mL/min. All
the release experiments were conducted in triplicate.
Animals
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Male Kunming mice (18−20g, 7-8weeks old) were purchased from Jiang’an Animal Center of
Sichuan University (Chengdu, China). All animal-handling procedures were performed
according to the Guide for the Care and Use of Laboratory Animals of the National Institutes of
Health, and all of the animal procedures in this study were approved by the Laboratory Animal
Management Committee of Sichuan University.
Ex vivo imaging and tissue distribution
The details establishment of H22 tumor-bearing mouse was described in supplementary
materials. H22-bearing Kunming mice were randomly divided into 4 groups (n=12 each group).
Different formulations (200 µL) were administered via tail vein injection at a dose of 2.5
mg/kg Dox equivalents when the tumor volume reached around ~150 mm. The mice were
sacrificed at 1, 8, 16,24h respectively, and various tissues such as the heart, liver, spleen, lung,
kidney and tumor were collected and imaged. The bio-distribution at 8h was also qualitatively
analyzed using liquid chromatography/mass spectrometry (LC-MS/MS) (Agilent
Technologies Co.Ltd. USA). The collected tissues were suspended in 4-foldsaline (w/v) and
homogenate thoroughly. 3-fold chloroform/methanol（4:1，v/v）was added, the mixture was
vortexed for 10min, followed by centrifuging at 13500 rpm (10 min), repeated 3 times. Then
5
μL of supernatant was analyzed by LC-MS/MS. The amount of Dox was calculated according
to standard curves. Data were represented as mean ±SD (n = 5).
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Cell culture
Human hepatoma cell line (HepG2) and human normal hepatocyte cell line (Lo2) were
maintained in Dulbecco’s modified Eagle’s medium (DMEM; Hyclone, USA), including 10%
fetal bovine serum(10％FBS ) and 1% penicillin (100 IU/mL) /streptomycin (100 μg/mL),
cultured in incubator containing 5% CO at 37 °C. All experiments were performed with cells
2
in the logarithmic growth phase.
Cellular uptake
4
HepG2 cells and Lo2 cells were seeded in 6-well plates (1×10 cells/well) and cultured for 24 h.
Then, each sample was added and the cells were further incubated for another 2h or 4 h, and the
untreated cells were used as negative control. For cellular uptake mechanism assays, cells were
pre-treated with free lactose (1mg/ml) for 2h. Next, each well was washed twice with
phosphate-buffered saline (PBS, pH 7.4), and the Dox-Lac, GNPs-Dox, GNPs-Dox-Lac
solution with equivalent Dox concentration (10 µg/mL) in the presence of free Lactose
(1mg/ml) was added and further incubated for another 4 h. The amount of Dox inside cells was
analyzed by flow cytometry (BD FACS Calibur).
4
HepG2 cells were seeded to 12-well plates at a density of 1×10 cells per well and incubated for
2
4 h. The cells were then washed twice with PBS, and incubated with each sample with
equivalent Dox concentration (10 µg/mL) for 4 h, then each well were washed three times with
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PBS, and the nucleus labeled with DAPI. The fluorescence intensity of Dox and DAPI was
recorded with a confocal laser scanning microscope (CLSM; FluoView 253 TM FV1000,
Olympus) at an excitation wavelength of 543 nm.
In vitro cytotoxicity study
The cytotoxicity of various samples were studied by MTT assay. Briefly, the HepG2 cells and
3
Lo2 cells were seeded at the density of 7×10 cells per well in 96-well plates and further
incubated until suitable density. After incubated with serial dilutions of each sample at same
Dox concentration range for 48 h, the cultured medium were removed and washed with PBS,
and then 20uL MTT (5mg/mL) was added and incubated for 4 h. The residual blue formazan
crystals were then dissolved in 150 µL DMSO per well and agitated for 15 min, the absorbance
was measured at 570 nm via a Varioskan Flash Multimode Reader.
Tumor penetration in HepG2 Multicellular Spheroids
The details establishment of three-dimensional (3D) HepG2 multicellular spheroids (MCSs)
was described in supplementary materials. When the diameter of MCSs reached about
4
00-500μm, they were treated with the samples at equivalent Dox concentration (10 µg/mL)
for 4 h, rinsed three times with ice-cold PBS, followed by fixed with 4% paraformaldehyde for
5 min. To analyze the fluorescent intensity in spheroids by a laser scanning confocal
microscopy, the spheroids were transferred to glass bottom petri-dish. Tumor sections (10 µm
1
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thick) were harvested by confocal laser scanning microscopy (CLSM) with Z-stack images,
and semi-quantitative intensity of inner region at different sections were calculated by image-J.
In vivo pharmacodynamics
H22-bearing Kunming mice were randomly divided into 5 groups (n=8 each group). When the
3
tumor volume reached around 150 mm , the mice were intravenously injected with free Dox,
Dox-Lac, GNPs-Dox and GNPs-Dox-Lac at a dose of 2.5 mg/kg Dox equivalents for 4 times.
Meanwhile, mice injected with saline were used as a control group. The tumor length and
width were measured and the body weights were weighed every other day. Finally, mice were
sacrificed at 21 day, the excised tumors were weighed and taken an image. The tumor volume
2
and tumor growth inhibitory (TGI) were calculated using these formulas: volume = 1/2 × LW
（
L and W represented the maximum and minimum diameter of the tumor）; TGI = [(WC-
We)/W ] × 100% （W and We are the tumor weight of the control group and experimental
C
C
group）, respectively. The excised major organs, including the livers, hearts, spleens, lungs
and kidneys were fixed in 4% paraformaldehyde and embedded in paraffin blocks. Then each
section was cut into 4 mm slices and stained with hematoxylin and eosin (H&E), and
visualized with an optical microscope (Axiovert 40 CFL, Carl Zeiss, Shanghai, China).
Maximum tolerated dose Studies
Maximum tolerated dose (MTD) was performed on healthy male kunming mice (18−20g).
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Four groups of mice received a single dose of 10, 15, 20 and 25 mg/kg Dox equivalents (n= 3)
by intravenous injection, respectively. Body weight and survival of the mice were monitored
for 14 days after injection. The MTD was estimated based on below 15% loss of body weight
and no death within 14 days.
Statistical analysis
Data were shown as the mean± standard deviation (SD). The significance of the experiential
data was evaluated by the Statistical Program for Social Sciences (SPSS) software 17.0 (SPSS
Inc., Chicago, IL), p value<0.05 and <0.001 were considered indications of statistical
diff erence and statistically significant diff erence, respectively.
Results
Synthesis and characterization of Dox-Lac
The synthetic route of Doxorubicin-Lactose (Dox-Lac) was shown in Figure 1, A. The
1
structures of Dox-Lac were confirmed by H NMR spectra (400 MHz, DMSO-d ) and mass
6
spectrum. As shown in Figure 1, B, the proton peak from the secondary amine of the
acylhydrazone group at 11.6 ppm, the aroma protons (7.6-8.2 ppm) and the methyl groups
(3.98 and 1.19 ppm) from the doxorubicin, and the disappearance of the proton peak from
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+
diazanyl-group indicated the purity of the prodrug. Besides, 898.2, calculated for [M+H] ,
could be found on the mass spectrum (Figure 1, C). All the results were in agreement with
1
4
previous study , demonstrating that Dox-Lac had been synthesized successfully.
Preparation and characterization of GNPs-Dox-Lac
The preparation process of GNPs-Dox-Lac was shown in Figure 2, A. TEM showed that GNPs
had a spherical structure (Figure 2, B). The hydrate diameter size was about 130.2 nm and the
zeta potential was about -11.11 mV determined by DLS (Figure 2, C and Table 1). The
hydrate diameter size of GNPs-Dox and GNPs-Dox-Lac were about 132.7 nm and 133.3 nm
respectively, indicating that the small molecule modifications hardly changed the size
1
5
compared to GNPs . Nevertheless, the zeta potential decreased from -11.11 mV to -5.96 mV
and -5.38 mV respectively, due to the carboxyl groups of GNPs decreased after amidation with
Dox or Dox-Lac. The UV–Vis absorbance showed an intense absorption band at 487 nm, and
the fluorescence showed a strong emission band at 553 nm, which are the characteristic peaks
of Dox (Figure 2, D and E). All the results suggested both GNPs-Dox and GNPs-Dox-Lac
were successfully constructed.
Stability and MMP2-triggered degradation of GNPs-Dox-Lac
For stability analysis, 100％ fetal bovine serum (100％FBS) was used to mimic the blood
circulation environment in vivo, and Dulbecco’s modified Eagle’s medium (DMEM)
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containing 10％ fetal bovine serum(10％FBS) was used to simulate the cell culture medium in
vitro. As a result, the hydrated particle size of GNPs-Dox-Lac hardly changed for 24h in both
medium (Figure 3, A), indicating that the GNPs-Dox-Lac were kinetically stable, and
appropriate for achieving a long circulating time and tumor accumulation in vivo.
Gelatin is the substrate of matrix metalloproteinase-2 (MMP2), which is over-expressed in
1
6
hepatocellular carcinoma . To evaluate MMP2 triggered degradation performance of
GNPs-Dox-Lac, the particle size changes were recorded by dynamic light scanning (DLS)
after incubation with MMP2 for 0, 1, 2, 4h, and the morphology study was conducted by using
transmission electron microscope (TEM ) after 4h incubation. DLS showed that the particle
size decreased gradually with the expanding of incubated time (Figure 3, C). After incubation
with MMP2 for 4h, irregular shapes and smaller size were observed in TEM image (Figure 3,
B), indicating that the GNPs-Dox-Lac could be degraded by MMP2 effectively.
In vitro drug release
The secretions of MMP2 from the HepG2 cell line was firstly studied by gelatin zymography.
As presented in Figure 4, A, a high level of MMP2 expression could be observed in the
conditioned medium of HepG2 cells. It was reported that almost no MMP2 secreted from
1
7
human normal hepatocyte cell line Lo2 . Then, whether the Dox-Lac could be released from
GNPs following the degradation of GNPs-Dox-Lac triggered by MMP2 were further
investigated, also the Dox-Lac release profiles at pH 7.4 or 6.5 were determined. Only a very
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low percentage of Dox-Lac release could be observed either at pH7.4 or 6.5, indicating that pH
had no influence on prodrug release. Obviously, the prodrug release was significantly
accelerated, with 77.1% and 63.2% of Dox-Lac released after 24h incubation in the presence of
MMP2 and in HepG2 cells culture media, respectively. However, only less than 20 % of
Dox-Lac released in Lo2 cells culture media. These results indicated that MMP2 could
facilitate the release of Dox-Lac from GNPs-Dox-Lac.
The hydrazone bond has been widely used as a pH-sensitive spacer between anticancer drug
and carrier, which was relatively stable at physiological conditions, but low pH value could
1
8, 19
accelerate its hydrolysis
. As shown in Figure 4, B, about 83.1% of Dox released from
Dox-Lac at pH5.0 after 24h incubation, indicating the pH-responsive disassociation of
Dox-Lac.
Ex vivo imaging and tissue distribution
Whether the combination of nanovehicle and lactose mediated active targeting could improve
the tumor accumulation of GNPs-Dox-Lac, the ex vivo imaging was observed on H22 tumor
bearing mice after intravenously administration of free Dox, Dox-Lac, GNPs-Dox,
GNPs-Dox-Lac, respectively for 1h,8h, 16h, 24h. As displayed in Figure 5, A, the active
targeting prodrug Dox-Lac failed to achieve a higher tumor accumulation, which was similar
to the free Dox. As reported that the active targeting process cannot be separated from the
2
0
passive targeting because it occurs only after passive accumulation in tumors . Meanwhile,
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GNPs-Dox and GNPs-Dox-Lac led to much stronger fluorescence intensities in tumor, which
might benefit from the EPR effect triggered by nanovehicle. More exhilaratingly, the
GNPs-Dox-Lac displayed the highest fluorescent intensity in tumor than GNPs-Dox at all
times, and even retained a high concentration at 24h, indicating that lactose modification could
further increase the tumor-targeting ability of GNPs-Dox-Lac. In addition, previous study
reported that effective tumor penetration and ligand-targeting ability could achieve long-time
2
1
tumor retention . Quantitative analysis at 8h was studied using LC-MS/MS to further confirm
the in vivo fate (Figure 5, C), tumor accumulation of GNPs-Dox-Lac was about 1.51-fold,
2
.33-fold, 2.91-fold higher than GNPs-Dox, Dox-Lac and Dox, respectively. Tumor slices
harvested at 8h also showed a similar tendency (Figure 5, B). These results consistently
demonstrated that enhanced tumor accumulation and longer tumor persistence in vivo could be
achieved by the combination of enhanced passive targeting of nanovehicle and lactose
mediated active targeting during the intact stage of GNPs-Dox-Lac. In addition, the
distribution of GNPs-Dox-Lac in normal tissues indicated that they were mainly metabolized
2
2, 23
by liver and excreted by kidney, the results was consistent with previous studies
.
Cellular uptake and cytotoxicity in vitro
The cell uptake was analyzed using flow cytometry quantitatively and confocal laser scanning
microscope qualitatively. HepG2 cells and Lo2 cells were incubated with of different
formulations for 2h or 4 h. As shown in Figure 6, A and C, time-dependent cellular uptake in
each group could be seen on both cells. The fluorescence intensity of GNPs-Dox-Lac inside
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cells was higher than that of GNPs-Dox with a 1.25-fold increase for 4h in HepG2 cells, but
there were no significant difference on Lo2 cells. It has been reported that the lactose group is
a specific ligand binding to the asialoglycoprotein-1 receptor (ASGPR1) on the membrane of
2
4
25
HepG2 cells , but Lo2 cells displayed low levels of ASGPR1 expression . The results
indicating that lactose modification could improve the cellular uptake of Dox-Lac and
GNPs-Dox-Lac. Notably, both GNPs-Dox and GNPs-Dox-Lac exhibited stronger fluorescent
intensity in HepG2 cells than that on Lo2 cells (p＜0.01), which might be related to the
MMP2 triggered drug release by degradation of nanoparticles. The Dox-Lac showed
increased florescent intensity compared to GNPs-Dox-Lac (p＜0.01), which is similar to the
previous reports, free CPP-Dox molecule showed higher cellular uptake than CPP conjugated
2
6
nanoparticles . Free Dox showed remarkably increased cellular uptake compared to Dox-Lac
(p＜0.01), this might be attributed to the different cellular uptake pathways that Dox-Lac
2
7
through endocytosis and free Dox through passive diffusion . Confocal microscopy images
(Figure 6, C) further illustrated above results.
In addition, the lactose receptor-mediated endocytosis was directly examined by the
competition of excess free lactose acid (Figure 6, D). GNPs-Dox displayed no significant
decrease in cellular uptake expectedly, while apparent inhibition was observed in
GNPs-Dox-Lac and Dox-Lac (p＜0.05). The results further confirmed that the lactose
receptor-mediated endocytosis was involved and played an important role in the cellular
uptake of Dox-Lac and GNPs-Dox-Lac.
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The cytotoxicity was further verified by MTT assay. After 48 h of incubation, each group
showed a dose-dependent toxicity both towards HepG2 cells (Figure S2, A) and Lo2 cells
(Figure S2, B). As depicted in Figure 6, E, there were no significant difference in efficacy
between GNPs-Dox-Lac (IC50=1.01 ug/mL) and GNPs-Dox (IC50=0.92 ug/mL) from Lo2
cells, unlike the results obtained from HepG2 cells. The results strongly address the
the MMP2-responsive prodrug release and specific lactose-targeting to liver cancer cells,
which led to the enhanced antitumor activity. In accordance with the previous reported that
2
8
the higher cellular uptake led to the stronger cytotoxicity . In addition, the highest
cytotoxicity of free Dox compared to other groups may attribute to the incomplete drug
2
9
release of prodrug and Dox conjugated NPs over a short period .
Tumor penetration in HepG2 multicellular spheroids
The final Dox concentration in solid tumor cells would also be affected by efficient tumor
3
0
penetration due to the differences between cell mono-layer and solid tumor tissues .
Three-dimensional multicellular tumor spheroids (3-D MCSs) possess an established
extracellular matrix density as well as lower pH, hypoxia conditions and secrets such as MMPs
and proliferative gradients, which are analogous to the tumor microenvironment in
3
1
hypovascular and a vascular region of solid tumor . Therefore, HepG2 MCSs were
established to simulate the in vivo status of tumor penetration.
The tumor penetration activity of different formulations in HepG2 MCSs was monitored by
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confocal laser scanning microscopy (CLSM) with Z-stack scanning (Figure 7), and the surface
of the MCSs was defined as 0 μm. At the scanning depth of 30 μm, the fluorescence intensity of
free Dox in the interior area of the HepG2 MCSs had dropped considerably. The relatively poor
penetration of doxorubicin might be due to its hydrophobicity and rapid uptake by perivascular
3
2
cells . Notably, the penetration capability of Dox-Lac was significantly increased (p＜0.01),
because the red fluorescence intensity inside the MCSs could be obviously observed at the
scanning depth of 70 μm and sporadic red fluorescence could still be seen even at the scanning
depth of 80 um, indicating the deep penetration and relatively uniform distribution of Dox-Lac.
Dox-Lac is a water-soluble molecule, which can distribute most readily in the extracellular
3
3
matrix (ECM), therefore efficiently diffuses around cells . Interestingly, GNPs-Dox-Lac
showed better tumor penetration capacity than Dox and GNPs-Dox, which might be caused by
the MMP2 triggered degradation of GNPs along with the release of Dox-Lac in tumor tissue.
However, GNPs-Dox-Lac still displayed a lower tumor penetration than Dox-Lac, which
might be due to the sustained Dox-Lac release from NPs responsive to MMP2.
In vivo pharmacodynamics and biosafety
Motivated by the enhanced tumor accumulation, balanced cellular uptake and tumor
penetration of GNPs-Dox-Lac, the in vivo pharmacodynamics of free Dox, Dox-Lac,
GNPs-Dox, and GNPs-Dox-Lac was evaluated on H22 tumor bearing mice. When the tumor
3
volume approximately 150 mm in average, the mice were treated with 2.5 mg/kg (equivalent
of Dox) of different formulations for each group at 1, 3, 5 and 7 day. From Figure 8, A, we
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could see that the tumor growth of saline treated mice was uncontrollable with the tumor size
increasing almost 25.59 times after 21 days. As shown in Figure 8, C, the tumor growth could
be suppressed by free Dox and Dox-conjugated formulations, especially the GNPs-Dox-Lac,
which significantly reduced the tumor progression and displayed a remarkable tumor growth
inhibition (TGI=90.8%). Photographs of tumors collected from each group at the last day
showed that GNPs-Dox-Lac treated group had the smallest tumor size (Figure 8, D). These
results confirmed the capability of the programmed delivery system to work smoothly as
expected in vivo.
To evaluate the biosafety of each formulation, the body weight and physical state were
simultaneously observed (Figure 8, B). Obviously, all groups of mice had no obvious body
weight decrease, which confirmed the well tolerance of this dose by mice. Interestingly, mice
treated with saline displayed emaciation and poor mental state, so the increase in body
weights may be owing to the remarkable increase in tumor volumes. Besides, the in vivo
biosafety was evaluated by examining histological changes of major organs by H&E. As
shown in Figure S3, the Dox and Dox-Lac groups showed some damages on heart, which
3
4
was caused exactly toxicity of Dox . Fortunately, the GNPs-Dox and GNPs-Dox-Lac groups
showed no noticeable structural changes in any organs, suggesting that gelatin NPs is a
promising delivery system for reducing therapeutic toxicity
Furthermore, the maximum tolerated dose (MTD) was estimated to determine tolerability and
the toxicity of the doxorubicin in gelatin nanoparticles by a single dose of 10, 15, 20 and 25
mg/kg Dox equivalents (n= 3) via intravenous injection, respectively. As shown in Figure 8,
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E and F, a stable increase of body weight and no death were appeared with the treatment of
GNPs-Dox-Lac in the doses of 10-20 mg/kg Dox equivalents, but body weight lose above 15%
with dose at 25 mg/kg Dox equivalent. Hui Zhang et al. have reported that the MTD of free
3
5
Dox in male Kunming mice was determined to be between 5 to 10 mg/kg . The MTD of
GNPs-Dox-Lac was between 20 to 25 mg/kg, which was approximately three fold higher
than that of free Dox. The high MTD of GNPs-Dox is consistent with its low adverse effects,
further indicating its biosafety.
Discussion
In this study, a programmed drug delivery system (GNPs-Dox-Lac) was developed for
enhanced hepatocellular carcinoma therapy (HCC) by combination the gelatin nanoparticle
(GNPs) and small molecule prodrug Doxorubicin-Lactose (Dox-Lac). First, the
GNPs-Dox-Lac (133.3 nm, -5.68 mV) were kinetically stable in blood circulation, and inclined
to accumulate at the tumor site both benefitting from not only EPR effect triggered by GNPs
nanovehicle but also lactose mediated tumor targeting, as well as reduce the cardiotoxicity of
Dox. Second, after accumulation at the tumor site, the GNPs would be degraded by
extracellular matrix metalloproteinase-2 (MMP2), leading to the release of the small molecule
hydrophilic prodrug Dox-Lac (Mw 898 Da), which penetrate deeply into tumor and deliver
Dox to Asialoglycoprotein-1 receptor (ASGPR1) over-expressed tumor cells. Last, the free
Dox (Mw 543 Da) would be released specifically in lysosome via the hydrolysis of hydrazone
groups, and transferred to the nucleus to induce toxicity. Therefore, the system had the ability
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to surmount both the “size contradiction” and the “hydrophilicity/hydrophobicity
contradiction” in requirements for tumor accumulation, tumor tissue penetration and cellular
uptake. As expected, GNPs-Dox-Lac achieved superior tumor inhibition rate of 90.8% in
vivo, suggesting its potential for HCC in future.
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Figure legends
1
Figure 1. Synthesis and characterization of Dox-Lac. (A) Synthesis of Dox-Lac. (B) H
1
NMR spectra of Dox-Lac. H NMR (400 MHz, DMSO-d ) δ (ppm). (C) Mass spectra of
6
Dox-Lac.
Figure 2. Preparation and characterization of GNPs and GNPs-Dox-Lac. (A) Preparation
of GNPs-Dox-Lac. (B) TEM images of GNPs, Scale bar: 100 nm. (C) Hydrate size of GNPs
determined by DLS, the average intensity (Z- Average) and the polydispersity index (PDI).
(D) UV–Vis absorption and (E) fluorescence emission spectra of each simple.
Figure 3. MMP2-responsive degradation of GNPs-Dox-Lac. (A) Stability study of
GNPs-Dox-Lac in the medium of DMEM (pH7.4, 10％FBS) or 100％FBS. (B) TEM images
of GNPs-Dox-Lac incubated with or without MMP2（5 ug/ml）for 4h, Scale bar: 100 nm. (C)
The DLS size changes of GNPs-Dox-Lac treated with 5 ug/ml MMP2 for 4h, the size is
represented by the number intensity.
Figure 4. Drug release in vitro. (A) Gelatin zymography of secreted MMP2 in the conditioned
medium of HepG2 cells, and MMP2-responsive release profile of the Dox-Lac from
GNPs-Dox-Lac incubated with different pH values, MMP2 (5ug/ml), HepG2 cells and Lo2
cells culture media for 24h. (B) pH-responsive disassociation of Dox-Lac and Dox release
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profile incubated at pH 6.5 and pH 5.0 for 24 h.
Figure 5. Distribution of diff erent formulations in tumor and normal tissues. (A) Ex vivo
imaging of Dox in different organs of H22 tumor-bearing mice at various time points after i.v.
injection of different formulations. (B) Tumor tissue sections (10 μm thick) were harvested
from above 8h tumors by cryostat and imaged to show the Dox (red signal) accumulation in
tumors. (C) Quantitative analysis at 8h by LC-MS/MS. #p＜0.05 vs free Dox.
Figure 6. Cellular uptake and cytotoxicity in vitro. Flow cytometry analysis of cellular
uptake of different formulations at an equivalent Dox dose of 10 μg/mL incubated for 2h and 4
h from HepG2 cells (A) and Lo2 cells (C). (B) Confocal microscopy images of cellular uptake
of different formulations at an equivalent Dox dose of 10 μg/mL after 4h incubation from
HepG2 cells. (D) Cell-specific endocytosis inhibited by free lactose acid competition from
HepG2 cells. (E) The half maximal inhibitory concentration (IC ) from HepG2 cells and Lo2
5
0
&
cells. p＜0.01 vs. Dox-Lac, #p＜0.01 vs GNPs-Dox.
Figure 7. Penetration evaluation on HepG2 multicellular spheroids. (A) Representative
confocal microscopy images into HepG2 tumor spheroids to different depths after 4h
incubination. Scale bar: 100 µm. (B) Semi-quantitative fluorescent intensity of Dox at different
sections by image J.
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Figure 8. In vivo pharmacodynamics on H22 tumor bearing mice and biosafety. (A)
Tumor growth curves from H22 tumor-bearing Kunming mice of each group treated by saline,
free Dox, Dox-Lac, GNPs-Dox, and GNPs-Dox-Lac. (B) Body weight changes of mice during
treatment for 21 days. Mice were executed at the last day, and tumors were weighted (C) and
photographed (D). The tumor growth inhibition (TGI) was also calculated. Body weight
changes (E) and survival (F) of healthy Kunming mice 2 weeks after administration of
GNPs-Dox-Lac at different concentration of 10–25 mg/kg Dox equivalents (n= 3). Results
are means ± SD, n=8. &p＜0.01 vs saline, #p＜0.01 vs. free Dox. *p＜0.05, **p＜0.01.
$
(body weight lose above 15%) vs. saline.
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