Nanoscale Coordination Polymers Codeliver Carboplatin and Gemcitabine for Highly Effective Treatment of Platinum-Resistant Ovarian Cancer

Article abstract of DOI:10.1021/acs.molpharmaceut.6b00466

Due to the ability of ovarian cancer (OCa) to acquire drug resistance, it has been difficult to develop efficient and safe chemotherapy for OCa. Here, we examined the therapeutic use of a new self-assembled core-shell nanoscale coordination polymer nanoparticle (NCP-Carbo/GMP) that delivers high loadings of carboplatin (28.0 ± 2.6 wt %) and gemcitabine monophosphate (8.6 ± 1.5 wt %). A strong synergistic effect was observed between carboplatin and gemcitabine against platinum-resistant OCa cells, SKOV-3 and A2780/CDPP, in vitro. The coadministration of carboplatin and gemcitabine in the NCP led to prolonged blood circulation half-life (11.8 ± 4.8 h) and improved tumor uptake of the drugs (10.2 ± 4.4% ID/g at 24 h), resulting in 71% regression and 80% growth inhibition of SKOV-3 and A2780/CDDP tumors, respectively. Our findings demonstrate that NCP particles provide great potential for the codelivery of multiple chemotherapeutics for treating drug-resistant cancer.

Full text of DOI:10.1021/acs.molpharmaceut.6b00466

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Article
Nanoscale coordination polymers co-deliver carboplatin and gemcitabine
for highly effective treatment of platinum-resistant ovarian cancer
Christopher Poon, Xiaopin Duan, Christina Chan, Wenbo Han, and Wenbin Lin
Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.6b00466 • Publication Date (Web): 06 Oct 2016
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Nanoscale coordination polymers co-deliver carboplatin and gemcitabine for highly
effective treatment of platinum-resistant ovarian cancer
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Authors: Christopher Poon,† Xiaopin Duan,† Christina Chan, Wenbo Han, and Wenbin Lin*
Department of Chemistry, University of Chicago, 929 E 57th Street, Chicago, IL 60637, USA; Eꢀ
mail: wenbinlin@uchicago.edu; Tel: 1 773 834 7163
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Abstract:
Due to the ability of ovarian cancer (OCa) to acquire drug resistance, it has been difficult to
develop efficient and safe chemotherapy for OCa. Here, we examined the therapeutic use of a
new selfꢀassembled coreꢀshell nanoscale coordination polymer nanoparticle (NCPꢀCarbo/GMP)
that delivers high loadings of carboplatin (28.0±2.6 wt.%) and gemcitabine monophosphate
(8.6±1.5 wt.%). A strong synergistic effect was observed between carboplatin and gemcitabine
against platinumꢀresistant OCa cells, SKOVꢀ3, and A2780/CDDP in vitro. The coꢀadministration
of carboplatin and gemcitabine in the NCP led to prolonged blood circulation halfꢀlife (11.8 ±
4.8 h) and improved tumor uptake of the drugs (10.2 ± 4.4 %ID/g at 24 h), resulting in 71%
regression and 80% growth inhibition of SKOVꢀ3 and A2780/CDDP tumors, respectively,
compared to the control group. Our findings demonstrate that NCP particles provide great
potential for the coꢀdelivery of multiple chemotherapeutics for treating drugꢀresistant cancer.
Keywords: Nanoscale coordination polymers, Carboplatin, Gemcitabine, Synergistic Effect,
Platinumꢀresistant ovarian Cancer
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Introduction
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Ovarian cancer (OCa) is the deadliest of all gynecological cancers, accounting for 5% of all
cancer deaths among women.1 Despite progress made with the introduction of a platinumꢀtaxane
combination as firstꢀline treatment for OCa, the development of platinum (Pt) resistance often
leads to relapse, and patients’ eventual succumb to tumors.2ꢀ4 The ability of OCa cells to resist
multiple chemotherapeutics hinders successful treatment of OCa, with 85% of OCa patients
relapsing following their successful firstꢀline treatment.5, 6 Ptꢀresistant disease is often seen when
patients develop recurrence within six months of the initial Pt treatment.7, 8 Treating the disease
with Pt/taxane or other Pt based therapies again following first relapse has shown to be
ineffectual.9 The mechanisms of drug resistance include the decreased metabolism of drugs to
the active chemotherapeutics,10 cell uptakes and efflux of the drugs,11, 12 cellular tolerance to the
drug,13 or increased DNA repair.14ꢀ16 Combination therapy can counter these mechanisms of
resistance without increasing dosages to the point of toxicity.
The combination of carboplatin (carbo) and gemcitabine was approved by the FDA in 2006
for the treatment of advanced recurrent ovarian cancer occurring at least 6 months after
completion of Ptꢀbased (e.g., carbo or cisplatin) therapy. Gemcitabine, a nucleotide analog,
induces apoptosis by replacing cytidine during DNA replication.17 Together with carbo, this
combination therapy has been shown to work effectively against drugꢀresistant tumors without
the added toxicity of other Pt cytotoxins (e.g., cisplatin).18ꢀ21 However, gemcitabine is often
limited by its poor pharmacokinetics and rapid metabolic deactivation.22ꢀ24 Furthermore, the
combination with Pt often enhances gemcitabine’s hematological toxicity.25, 26 Therefore, there is
an urgent need to develop a delivery system for combination drugs that can overcome the
limitations imposed by free platinum drugs and gemcitabine.
Nanoparticleꢀbased drug delivery systems can more easily control the many relevant
properties of drugs than traditional small molecule agents. These include physical properties
such as size and surface charge that are dictated by nanoparticle synthesis or assembly, and
important pharmacokinetic parameters such as circulation halfꢀlife and drug release kinetics.27ꢀ33
In particular, nanoparticle drug delivery systems that are able to deliver multiple therapeutics
agents have shown to enhance antitumor efficacy, overcome multidrug resistance, and reduce
addictive toxicity.34ꢀ42 We have developed a novel delivery vehicle, nanoscale coordination
polymers (NCPs), for various cancer therapies,31, 43ꢀ45 including a combination therapy delivery
system carrying oxaliplatin and gemcitabine for the synergistic treatment of pancreatic cancer.46
These nanoparticles combine the organic and inorganic properties of nanoparticles, as well as
their intrinsic biodegradability, to prolong blood circulation halfꢀlives and effectively treat
tumors with minimal side effects.27, 28 Herein, we report that NCPs can coꢀdeliver carbo and
gemcitabine for synergistic therapy of Ptꢀresistant OCa. More importantly, a superior synergistic
effect was found in vitro against two Ptꢀresistant OCa cell lines, SKOVꢀ3 and A2780/CDDP.
NCPꢀCarbo/GMP shows enhanced cellular uptake, prolonged blood circulation, and elevated
tumor uptake, resulting in improved antitumor efficacy in Ptꢀresistant OCa tumor xenograft
models.
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Results
Synthesis and characterization of NCP-Carbo/GMP. The carbo prodrug, carboꢀ
bis(phosphonic acid), was synthesized and characterized by NMR spectroscopy and mass
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spectrometry (Figures S1ꢀS6). DOPAꢀcapped NCPꢀCarbo/GMP nanoparticles containing the
carbo prodrug and gemcitabine monophosphate (GMP) were initially synthesized by reverse
microemulsion (Figure S7). The drugꢀcontaining core was capped by DOPA via Znꢀphosphate
interactions. DOPA further selfꢀassembled into a monolayer via hydrophobicꢀhydrophobic
interactions, making the particles dispersible in organic solvents. The surface was further coated
with DSPC, cholesterol, and DSPEꢀPEG2k at a 1:1:0.75 molar ratio. The Zꢀaverage, numberꢀ
average, PDI, and zetaꢀpotential for NCPꢀCarbo/GMP were 85.3±0.7 nm, 64.9±1.7 nm,
0.069±0.013, and ꢀ6.02±0.55 mV, respectively, by dynamic light scattering (DLS) measurement
(Figure 1B and Table 1). The nearꢀneutral surface charge indicated that PEG chains were
successfully coated on the particle surface. Transmission electronic microscopy (TEM) showed
uniform spherical nanoparticles with a diameter of ~20 nm (Figure 1C), implying that the lipid
coating did not disrupt the NCP core. DOPAꢀNCPꢀCarbo/GMP gave carbo loadings of 28.0±2.6
wt.% (54.4±5.1 wt.% prodrug loading) by ICPꢀMS. Gemcitabine monophosphate (GMP)
loadings were found to be 8.6±1.5 wt.% by UVꢀVis (Figure S12). No change was seen in the
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particle size upon exposure in 30 nM bovine serum albumin in phosphate buffer saline at 37 C
after 24 h (Figure S13). The in vitro study showed that NCPꢀCarbo/GMP exhibited excellent
stability.
A
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NCP-Carbo/GMP
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Figure 1. (A) Schematic of NCPꢀCarbo/GMP (B) Numberꢀaverage size distribution of NCPꢀ
Carbo/GMP particles. Bare and lipid coated particles were measured in THF and PBS,
respectively. (C) TEM micrographs of NCPꢀCarbo/GMP. Scale bar = 100 nm.
Table 1. Sizes, polydispersities, and zeta potentials of NCPꢀCarbo/GMP particles.
ZꢀAve diameter NumberꢀAve
PDI
Zeta Potential
(mV)
NCPs
(nm)
diameter (nm)
37.1±0.4#
DOPAꢀNCPꢀ 56.9±0.2#
Carbo/GMP
0.112±0.003 NA
NCPꢀ
Carbo/GMP
85.3±0.7$
64.9±1.7$
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0.069±0.013$ ꢀ6.02±0.55$
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#Measured in THF. $Measured in PBS. Data are expressed as means±SD.
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In vitro drug release. The release profiles of carbo and GMP from DOPAꢀNCPꢀCarbo/GMP
and NCPꢀCarbo/GMP were investigated in PBS with or without 5 mM cysteine at 37 C at pH
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7.4 (Figure S14). In the absence of cysteine, DOPAꢀNCPꢀCarbo/GMP revealed rapid burst
release due to the dissociation of phosphate groups with Zn2+, with 57% and 33% cumulative
release of Pt and GMP before 2 h, respectively. In contrast, only 19% Pt and 21% GMP release
were observed for NCPꢀCarbo/GMP after 24 h. 5 mM cysteine was added to PBS in order to
investigate drug release from DOPAꢀNCPꢀCarbo/GMP in a simulated intracellular environment.
These conditions accelerated the drug release of DOPAꢀNCPꢀCarbo/GMP with 82% of Pt and
57% of GMP release after 2 h, indicating that cysteine can penetrate into the NCP core and
reduce the carbo prodrug into active carbo. Both the reduction of PtꢀO bonds and the dissociation
of phosphate groups with Zn2+ accelerated the drug release. However, NCPꢀCarbo/GMP
exhibited similarly slow drug release patterns in PBS and PBS supplemented with 5 mM
cysteine. The lipid coating on the particle surface prevented the penetration of cysteine into the
NCP core, thereby rendering the particle more stable. The external lipid layer may be
incorporated into the cell or plasma membrane during and after endocytosis, disrupting the lipid
coatings, allowing cysteine to penetrate the particle core, and triggering the reductive
degradation of NCPs to release the therapeutic payload.
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In vitro cytotoxicity and the synergistic effect. To evaluate the potency of NCPꢀ
Carbo/GMP, in vitro cytotoxicity was performed on SKOVꢀ3 (Figure 2A, B) or A2780/CDDP
(Figure 2D, E) ovarian cancer cells treated with nanoparticles or free drugs at different carbo or
gemcitabine concentrations for 72 h. The cell viability was measured by MTS assay. As seen in
Figure 2 and Table 2, the IC50 of carbo and GMP against SKOVꢀ3 were 24.21 ± 0.96 ꢀM and
1.89 ± 0.26 ꢀM, respectively. When free carbo and GMP were combined (Carbo&GMP), the
carbo and GMP IC50 values were dramatically decreased by 12ꢀfold and 2.5ꢀfold, respectively
(carbo IC50= 2.01 ± 0.62 ꢀM or GMP IC50=0.74 ± 0.23 ꢀM). NCPꢀCarbo/GMP showed
comparable cytotoxicity as their corresponding free drug counterparts, with carbo IC50=1.91 ±
0.55 ꢀM and GMP IC50=0.70 ± 0.20 ꢀM. In contrast, monotherapeutic NCPs IC50 values of
carbo and GMP were 22.82 ± 2.49 ꢀM and 1.98 ± 0.34 ꢀM, respectively. Similar results were
shown with A2780/CDDP cells with NCPꢀCarbo/GMP, with IC50 values that are about 7.6ꢀfold,
14.4ꢀfold, 2.3ꢀfold, and 2.4ꢀfold lower than free carbo, NCPꢀCarbo, free GMP, and NCPꢀGMP,
respectively.
The CI provides a quantitative measure of synergism (CI < 1), additivity (CI = 1), or
antagonism (CI > 1) for the drug combinations. The CI was around 0.5 for NCPꢀCarbo/GMP
against the monotherapeutic NCP and free drugs over a large range of drug effect level for both
SKOVꢀ3 (Figure 2C) and A2780/CDDP (Figure 2F). Synergy was thus seen between carbo and
GMP in both Ptꢀresistant ovarian cancer cells. These results suggest that coꢀdelivery of carbo and
gemcitabine could overcome drug resistance, leading to much enhanced anticancer efficacy
against ovarian tumor models.
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Figure 2. In vitro cytotoxicity plots and combination indices (CI) of carbo/GMP combinations
on SKOVꢀ3 (AꢀC) and A2780/CDDP (DꢀF) cells. The cell viabilities on SKOVꢀ3 and
A2780/CDDP cells were measured after a 72 h exposure to NCPꢀCarbo, NCPꢀGMP, NCPꢀ
Carbo/GMP, or free drugs (carbo, or GMP). Data are mean ± S.D. (n=6).
Table 2. Carbo IC50 values of carbo, GMP, NCPꢀCarbo, NCPꢀGMP, NCPꢀCarbo/GMP against
SKOVꢀ3, and A2780/CDDP cells (the numbers in parenthesis refer to GMP concentrations).
Carbo(ꢁM)
GMP
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Carbo&GMP
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NCPꢀ
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GMP
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NCPꢀ
Carbo/GMP
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(1.1±0.2)
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(0.7±0.2)
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1.9±0.6
(0.7±0.2)
1.3±0. 2
(0.5±0.1)
A2780/CDDP
18.7±1.0 (1.2±0.2)
(0.3±0.1)
Data are expressed as means±SD.
In vitro cell apoptosis. To investigate the synergistic effect of carbo and GMP on cell
apoptosis, Annexin V staining and flow cytometry analysis were performed to investigate cell
apoptosis induced by free drugs or nanoparticle formulations. After incubation for 24 h, SKOVꢀ3
and A2780/CDDP cells were stained with Alexa Fluor 488 conjugated Annexin V and observed
using CLMS. NCPꢀCarbo/GMP induced highest level of cell apoptosis, as evidenced by the
presence of the most and brightest green fluorescence. Carbo&GMP, NCPꢀGMP and GMP also
induced high level of cell apoptosis, while carbo and NCPꢀCarbo resulted in much less cell
apoptosis (Figures S16 and S17).
We further quantified cell apoptosis by flow cytometry (Figure S18). Similar to the
cytotoxicity assay and CLSM analysis, NCPꢀCarbo/GMP showed the highest ability to induce
cell apoptosis, resulting in 62.05% and 65.94% apoptosis for SKOVꢀ3 and A2780/CDDP cells,
respectively. Carbo&GMP also showed significantly increased cell apoptosis in both ovarian
cancer cell lines with 52.67% and 35.43% of cells undergoing apoptosis in SKOVꢀ3 and
A2780/CDDP, respectively. Free GMP and NCPꢀGMP showed comparable apoptotic cells,
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ranging from 47ꢀ49% in SKOVꢀ3 and 26ꢀ35% in A2780/CDDP. Little or no apoptotic cells were
seen in PBS, carbo, and NCPꢀcarbo for both ovarian cancer cell lines.
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In vitro cellular uptakes. RhodamineꢀBꢀdoped NCPꢀCarbo/GMP (RhBꢀNCPꢀCarbo/GMP)
nanoparticles were synthesized for confocal microscopy studies. As seen in the DLS (Table S3)
and TEM images (Figures S19 and S20), no difference was observed in the size distribution and
morphology between dyeꢀdoped particles and NCPꢀCarbo/GMP. To directly observe the NCPꢀ
Carbo/GMP uptake, Rhodamine Bꢀdyed nanoparticles were incubated with either SKOVꢀ3
(Figure 3A) or A2780/CDDP (Figure 3B) human ovarian cancer cells and then observed under
CLSM. After a 1 h incubation, the nanoparticles were readily taken up by the cells, as evidenced
by the coꢀlocalization of green fluorescence (Lysotracker Greenꢀstained endosome) and red
fluorescence (Rhodamine Bꢀdyed nanoparticle) seen around the exterior of the nucleus. The red
fluorescence signal was greatly enhanced after 4 h and 24 h, indicating an increased uptake of
this nanoparticle over time.
This result was further supported by timeꢀdependent cellular uptake of NCPꢀCarbo/GMP in
SKOVꢀ3 (Figure 3C, D) and A2780/CDDP (Figure 3E, F) as determined by ICPꢀMS. Free carbo
showed highest cellular uptake at 1 h, but it decreased by ~50% at 24 h, indicating that the
uptake of free carbo is rapid. In contrast, the cellular uptake of NCPꢀCarbo/GMP increased
gradually over time and was significantly higher compared to the free combination drugs. At 24
h, the Pt uptake was 4 times and 5 times higher than the free combination treatments in SKOVꢀ3
and A2780/CDDP, respectively. The cellular uptake of free GMP remained constant throughout
the 24ꢀh experiment in A2780/CDDP cells, but decreased by ~50% at 24 h in SKOVꢀ3 cells. The
uptake of NCPꢀCarbo/GMP, in term of GMP, also showed a slight gradual increase over time,
but no significant difference was observed.
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Figure 3. CLSM images of coꢀlocalization of RhB (red) from carbo/GMP particles with a late
endosome and lysosome marker, LysoTracker (green), in SKOVꢀ3 (A) and A2780/CDDP (B)
cells after incubation for different times. Scale bars: 50 ꢀm. Cellular uptake of carbo/GMP and
NCPꢀCarbo/GMP in SKOVꢀ3 (C,D) and A2780/CDDP (E,F) cells determined by ICPꢀMS (Pt
uptake) and UVꢀVis (GMP uptake). Data expressed as means±SD (N=3).
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Pharmacokinetics and biodistribution. The pharmacokinetics and biodistribution of NCPꢀ
Carbo/GMP were investigated to determine the extent of MPS clearance and the relative uptake
of major MPS organs versus tumor on CT26ꢀtumor bearing mice (Figure 4A). The Pt distribution
was quantified by ICPꢀMS and the GMP amount in the blood was quantified by HPLCꢀMS/MS.
By i.p. injection, the blood circulation halfꢀlives were fitted with a oneꢀcompartment model using
PK solver. NCPꢀCarbo/GMP resulted in Pt and GMP blood circulation halfꢀlives of 11.8 ± 4.8 h
(Figure 4B) and 9.4 ± 1.4 h (Figure 4C), respectively. There were low percentages of injected
dose per gram tissue (% ID/g) in organs associated with MPS activity such as the liver (3.8 ± 2.8
% ID/g), spleen (3.7 ± 3.6 % ID/g), and kidney (4.8 ± 2.2 % ID/g), supporting the ability of
NCPꢀCarbo/GMP to evade MPS clearance. The slow blood clearance and low MPS uptake led to
the high tumor accumulation of the drug. The Pt distribution in tumor tissue increased over time,
reaching a maximum at 24 h (10.2 ± 4.4 %ID/g), which indicated the high accumulation and
long retention of NCPꢀCarbo/GMP in tumor tissue.
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Figure 4. Percentage injected dose per gram (% ID/g) (A) of Pt in tissues after intraperitoneal
administration of NCPꢀCarbo/GMP in CT26 tumorꢀbearing mice at time points 5 min, 1 h, 3 h, 5
h, 8 h, 24 h, and 48 h. Data are mean ± S.D. (n=3). Average observed and predicted timeꢀ
dependent Pt (B) and GMP (C) distributions in blood after administration of NCPꢀCarbo/GMP
(n=3). The oneꢀcompartment model was used in determining the Pt and GMP distribution in
blood.
Anti-tumor activity in vivo. The antiꢀtumor activity of NCPꢀCarbo/GMP was evaluated in
SKOVꢀ3 and A2780/CDDP subcutaneous xenograft murine models. Mice bearing SKOVꢀ3 or
A2780/CDDP tumors were treated once every three days for a total of three i.p. injections at a
dose of 10 mg carbo/kg and 2.4 mg GMP/kg. All of the mice were sacrificed at day 19 for
SKOVꢀ3 and day 8 for A2780/CDDP, when the control tumors reached above 2000 mm3. As
shown in Figure 5A, NCPꢀCarbo/GMP showed significant tumor regression in SKOVꢀ3 tumors,
with a reduction of 71% compared to the initial tumor volume. For the more aggressive
A2780/CDDP tumor, NCPꢀCarbo/GMP also demonstrated dramatic tumor inhibition, with a
tumor inhibitory rate (TIR) of 80% (Figure 5C). The tumor weight of NCPꢀCarbo/GMPꢀtreated
group was 90ꢀfold and 12ꢀfold smaller than that of control in SKOVꢀ3 (Figure 5B) and
A2780/CDDP (Figure 5E) tumors, respectively. On the contrary, mice treated with Carbo&GMP
showed a similar tumor growth pattern as PBS, suggesting that free combination drugs caused no
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antitumor efficacy, possibly because free carbo and GMP do not significantly accumulate in
tumor tissue.
TUNEL assay was performed on the resected tumors to further substantiate and quantify the
in vivo apoptosis. Apoptotic cells showing high fluorescence intensity indicative of DNA
fragmentation were seen at a higher proportion of total cells in tumors treated with NCPꢀ
Carbo/GMP (Figure 6B and 6D). NCPꢀCarbo/GMP induced 92.7±2.4% and 89.3±2.8% tumor
cell apoptosis for SKOVꢀ3 and A2780/CDDP, respectively, whereas Carbo&GMP caused
4.0±0.4% and 3.6±0.5% apoptosis for SKOVꢀ3 and A2780/CDDP, respectively. NCPꢀ
Carbo/GMP thus exhibits superior anticancer efficacy as compared to free drugs. Further
analysis of the tumor tissue by H&E staining supported the TUNEL results of NCPꢀCarbo/GMP
inducing the most apoptosis and necrosis compared to low or no apoptosis and necrosis in
Carbo&GMP and PBSꢀtreated tumors (Figure 6A and 6C).
No obvious signs of toxicity were observed in vivo after treatment with NCPꢀCarbo/GMP, as
evaluated by body weight evolution, immunogenic response, and histology of major organs. No
significant weight loss was observed in NCPꢀCarbo/GMP treated group, indicating the absence
of severe systemic toxicity (Figures S24 and S25). Negligible differences were observed across
treatment groups in the levels of proꢀinflammatory cytokines assessing acute immunogenic
response (Figure S26) and histological assessments (Figure S27). No statistically significant
differences were observed between the control and NCPꢀCarbo/GMP group in terms of the proꢀ
inflammatory cytokine production. From the H&Eꢀstained sectioned tissues of the heart, liver,
lung, spleen, and kidney, no acute pathological change was detected in NCPꢀCarbo/GMPꢀtreated
groups.
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Figure 5. Subcutaneous SKOVꢀ3 xenografts: (A) In vivo tumor growth inhibition. Carbo (dose,
10 mg/kg) and gemcitabine (4.6 mg/kg) and NCPꢀCarbo/GMP (doses, 10 mg/kg and 4.6 mg/kg)
were administered on day 0, 3, and 6. Data are expressed as means±S.D. (n=5). (B) Endꢀpoint
tumor weights. Data are expressed as means±S.D. (n=5). Subcutaneous A2780/CDDP
xenografts: (C) In vivo tumor growth inhibition. Carbo (dose, 10 mg/kg) and gemcitabine (4.6
mg/kg) and NCPꢀCarbo/GMP (doses, 10 mg/kg and 4.6 mg/kg) were administered on days 0, 3,
and 6. Data are expressed as means±S.D. (n=5). (D) Endꢀpoint tumor weights. Data are
expressed as means±S.D. (n=5), ***p<0.001.
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Figure 6. (A) Representative CLSM images of TUNEL assays and H&E staining of SKOVꢀ3
tumor tissues. DNA fragment in apoptotic cells was stained with fluoresceinꢀconjugated
deoxynucleotides (green) and the nuclei were stained with DAPI (blue). Top bar = 50 µm.
Bottom bare = 100 ꢀm. (B) The percentages of TUNELꢀpositive cells in SKOVꢀ3 tumor tissues.
(C) Representative CLSM images of TUNEL assays and H&E staining of A2780/CDDP tumor
tissues. DNA fragment in apoptotic cells was stained with fluoresceinꢀconjugated
deoxynucleotides (green) and the nuclei were stained with DAPI (blue). Top bar = 50 µm.
Bottom bar = 100 ꢀm. (D) The percentages of TUNELꢀpositive cells in A2780/CDDP tumor
tissues. Data are expressed as means±S.D. (n=3), ***p<0.001.
Discussion
Combinations of two or more chemotherapeutic agents are often employed as firstꢀline
therapy in the clinical treatment of a variety of cancers. The different drugs can either affect
distinct molecular targets in order to delay molecular and genetic mutations by cancer cells to
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resist the chemotherapy, or target similar cellular mechanisms for synergistically enhanced
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therapy.47,
However, significant differences in physical and chemical properties of
chemotherapeutic drugs cause difficulties in optimizing treatment regimens. The
pharmacokinetics and pharmacodynamics of each combination therapy component is unique,
which results in inconsistent drug concentrations in tumor sites and suboptimal efficacy.49, 50 Coꢀ
delivering multiple chemotherapeutics within a single nanocarrier provides distinct advantages
over administering multiple singleꢀagent nanoparticles or free drugs. Physical and chemical
differences in solubility and charge of individual drugs are nullified when incorporated together
onto a nanoparticle, allowing for a consistent ratio of drug at the tumor site. 51ꢀ53 Specifically, the
modular synthesis of NCP allows for highly reproducible drug loadings and the coreꢀshell
structure endows nanoparticle stability and controlled release kinetics for both drugs.
Here, we utilized the NCPꢀplatform that combines the high drug loadings of
chemotherapeutics carboplatin and gemcitabine at a precise ratio to elicit therapeutic effect in a
single nanocarrier for Ptꢀresistant OCa treatment. A major benefit of using nanoparticles of about
10ꢀ100 nm in size for drug delivery is their intrinsic ability to target tumors by the enhanced
permeability and retention (EPR) effect.54, 55 NCPꢀCarbo/GMP are an optimal size that takes
advantage of the leaky vasculature to accumulate in the tumor, while sufficiently avoiding both
renal filtration which removes particles less than 40 nm and mononuclear phagocytic system
(MPS)ꢀmediated clearance of particles over 200 nm. In addition, the nearꢀneutral surface charge
of NCPꢀCarbo/GMP provides the possibility for the particles to resist opsonization. NCPꢀ
Carbo/GMP also showed very high stability under physiological conditions, such that drug
leakage during systemic circulation was minimized and drug effects were primarily focused at
the tumor site to reduce side effects.56ꢀ58 All of these characteristics contribute to the particles’
prolonged circulation time and improved tumor accumulation.
Once the particles enter the tumor cells, the lipid bilayer on the surface likely incorporates
into the lipid assemblies of cell and plasma membranes, exposing the core of the particle to
endogenous reducing agents, such as cysteine, for degradation of the particle to release the
therapeutic payloads. The incorporation of lipid bilayer into the cell membranes might be able to
prevent the efflux of the released drugs, as shown by gradual increased cellular uptake over time.
While the cellular uptake rapidly decreased for free drugs over time due to the efflux effect of
cells. As a result, NCPꢀCarbo/GMP has 5ꢀtimes higher cellular uptake as compared to the free
drugs. The enhanced tumor uptake led to effectively regression of the Pt resistant SKOVꢀ3
tumors and dramatically inhibition of the aggressive A2780/CDDP tumors in vivo. The tumor
weights were ~90ꢀfold and ~12ꢀfold lower as compared to the control and free combination
drugs, respectively.
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Conclusion
We developed an NCP platform that combines carbo and gemcitabine for successful
treatment of Ptꢀresistant OCa. We show here that carbo and gemcitabine produce a synergistic
effect in SKOVꢀ3 and A2780/CDDP, and this synergy is retained when this combination is coꢀ
delivered in an NCP. NCPꢀCarbo/GMP showed a long blood circulation halfꢀlife and high drug
accumulation in the tumor, resulting in superior tumor regression in vivo when compared to free
combination drugs. Equipped with high antitumor therapeutic potential and low systemic
toxicity, NCPs possess the ideal characteristics to coꢀdeliver multiple chemotherapies to tumors
for enhanced cancer therapy.
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Experimental Procedures
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Materials, cell lines, and animals. All starting materials were purchased from Sigmaꢀ
Aldrich (Louis, MO) and Fisher Scientific (Pittsburgh, PA) unless otherwise noted and used
without further purification. 1,2ꢀdioleoylꢀsnꢀglyceroꢀ3ꢀphosphate (DOPA), 1,2ꢀdistearoylꢀsnꢀ
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glyceroꢀ3ꢀphosphocholine
(DSPC),
cholesterol,
and
1,2ꢀdistearoylꢀsnꢀglyceroꢀ3ꢀ
phosphoethanolamineꢀNꢀ[amino(polyethylene glycol)2000] (DSPEꢀPEG2k) were purchased
from Avanti Polar Lipids (Alabaster, AL). Gemcitabine monophosphate (GMP) was synthesized
from gemcitabine following the literature59.
1H NMR spectra were recorded on a Bruker NMR 400 DRX Spectrometer at 400 MHz. The
mass spectra of Carbo prodrug, Carboꢀbis(phosphonic acid), and its intermediates were
determined on electrospray ionization mass spectrometry (ESIꢀMS, Agilent 6130) by dissolving
the prodrug in water, deprotonated with excess 3 M NaOH, and then filtered through 0.2 ꢀm
syringe filter. ESIꢀMS was taken in positive mode, with a fragmentation voltage of 100 V from
620ꢀ720 MW/z.
Human ovarian cancer cells SKOVꢀ3 and murine colon cancer cells CT26 were purchased
from the American Type Culture Collection (Rockville, MD) and grown in McCoy’s 5A and
RPMI 1640, respectively, supplemented with 10% fetal bovine serum (FBS, Gibco, Grand
Island, NY). Cisplatinꢀresistant human ovarian cancer cells A2780/CDDP were obtained from
Developmental Therapeutics Core, Northwestern University, and cultured in RPMI 1640
containing 10% FBS. All cells were cultured in a humidified atmosphere containing 5% CO2 at
37 oC.
BALB/c female mice (6 weeks, 18ꢀ22 g) and athymic female nude mice (6 weeks, 18ꢀ22 g)
were provided by Harlan Laboratories, Inc (USA). The study protocol was reviewed and
approved by the Institutional Animal Care and Use Committee (IACUC) at the University of
Chicago.
Carbo prodrug synthesis. Carbo synthesis was based on previous literatures.60ꢀ62 Carbo was
synthesized by adding equivalent amounts of Ag salt of cyclobutanedicarboxylic acid (Agꢀcarbo,
1.51 g) to Pt(NH3)2I2 (2.05 g) in minimal H2O and stirred at room temperature and in darkness
for 48 h. The solution was filtered, and the filtrate was evaporated, resulting in a white solid. The
product cisꢀdiammine(1, 1ꢀcyclobutanedicarboxylato)ꢀplatinum(II) (Carbo) was recrystallized in
1:1 H2O : EtOH. Yield: 80%. 1H NMR in D2O: δ 2.8 (t, 4H); δ 1.8 (quintet, 2H).
Carbo (1.04 g) was reacted with 30% H2O2 (8 mL) in H2O (16 mL) at 70 °C for 5 h in
darkness. Addition of EtOH precipitated a white solid, Carboꢀ(OH)2, which was washed twice
1
with EtOH. Yield: 80%. H NMR in D2O: δ 2.67 (t, 4H); δ 2.0 (quintet, 2H). The M/Z of
[M+H]+ was determined to be 406.1 (expected M/Z= 406.1).
Carboꢀ(OH)2 (0.8 g) was dissolved in minimal DMF, to which was added 4 equivalents of
diethoxyphosphinyl isocyanate (1.2 mL). The mixture was allowed to react overnight in
darkness. The solution was filtered, and the resulting lightly yellow Carboꢀbis(phosphoester)
product was precipitated by addition of Et2O. The product was then washed twice with Et2O.
1
Yield: 78%. H NMR in D2O: δ 4.05 (m, 8H); δ 2.60 (m, 3H); δ 2.39 (m, 1H); δ 1.93 (m, 2H).
The M/Z of [M+H]+ was determined to be 764.2 (expected M/Z= 764.1).
Trimethylsilyl bromide (TMSBr, 800 ꢀL) was added slowly to the Carboꢀbis(phosphoester)
(0.8 g) dissolved in minimal DMF at 4 °C, and the solution was stirred for 18 h at r.t. in darkness
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under N2 protection. A lightly yellow solid was collected after addition of DCM and then washed
twice with additional DCM. The solid was dissolved in MeOH and stirred overnight to hydrolyze
the
acid.
The
final
product,
cis,transꢀ[Pt(1,1ꢀ
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cyclobutanedicarboxylato)(NH3)2(OCONHP(O)(OH)2)2], [Carboꢀbis(phosphonic acid) prodrug],
was collected by precipitation by DCM and then washed twice with additional DCM. Yield:
1
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73%. H NMR in D2O: δ 2.57 (t, 3H), 2.43 (m, 1H), and 1.91 (m, 2H). C NMR in D2O:
δ
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179.9; 179.5; 55.7; 35.1; 28.0; and 15.4. 195Pt in D2O: δ 174 using K2PtCl4 as reference. The M/Z
of [M+MeOH]+ was determined to be 684.2 (expected M/Z= 684.0).
Particle synthesis and characterization. DOPAꢀNCPꢀCarbo/GMP was synthesized in
reverse microemulsions. To synthesize DOPAꢀNCPꢀCarbo/GMP, carbo prodrug (331.3 ꢀL, 11.5
ꢀmol), GMP sodium salt solution (68.7 ꢀL, 5.0 ꢀmol), and DOPA (44 ꢀmol) were added to a 10
mL aliquot of TritonꢀXꢀ100 (0.3 M in 1.5 M hexanol/cyclohexane) solution to form a W=7.4
microemulsion. Another microemulsion of 10 mL TritonꢀXꢀ100 (0.3 M, 1.5 M
hexanol/cyclohexane) containing Zn(NO3)2·6H2O (262 ꢀmol) was also prepared. The two
microemulsions were stirred vigorously at room temperature for 15 min. The microemulsion
containing the Zn complex was added to the other microemulsion dropwise. The combined
emulsion was stirred for an additional 30 min at room temperature. After the addition of 20 mL
ethanol, the particles were washed once with ethanol and twice with 50% (v/v)
ethanol/tetrahydrofuran (THF) before being redispersed in THF.
Carboꢀloaded nanoparticles were dried, weighed, digested in concentrated nitric acid
overnight, and diluted with water to determine the carbo loading by inductively coupled plasmaꢀ
mass spectrometry (ICPꢀMS, Agilent 7700x ICPꢀMS). GMP loading was determined by UVꢀVis
spectroscopy and thermogravimetric analyses (TGA). Particles were digested overnight in 6 M
hydrochloric acid, and the concentration of GMP in the solution was determined by the
absorbance at 275 nm using a Shimadzu UVꢀ2401PC UVꢀVis Spectrophotometer. A baseline
spectrum was recorded using 6 M hydrochloric acid. Using the Ptꢀdrug loading from ICPꢀMS
and corresponding to the standards of carboplatin, the absorbance from Pt was subtracted from
the total absorbance to determine the GMPꢀdrug loading.
DOPAꢀcapped NCPꢀCarbo/GMP nanoparticles were further coated with DSPC, cholesterol,
and DSPEꢀPEG2k to increase their stability and allow them to circulate longer in the blood. A
THF solution of DSPC, cholesterol, DSPEꢀPEG2k (molar ratio 1:1:0.75), and DOPAꢀcapped
NCP nanoparticles was added dropwise to 500 ꢀL of 30% (v/v) ethanol/H2O at 60 °C under
strong stirring. THF was evaporated, and the dispersion was allowed to cool to room temperature
before use. NCPꢀCarbo nanoparticle carrying only carbo, and NCPꢀGMP particles carrying
gemcitabine monotherapy were prepared similarly as NCPꢀCarbo/GMP.
In vitro stability studies. Particle stability was evaluated in vitro in phosphate buffered
saline (PBS) buffer with bovine serum albumin (BSA)ꢀbinding and timeꢀdependent drug release.
BSA binding analysis was done by dispersing 0.45 mg of NCPꢀCarbo/GMP in 1 mL PBS
o
containing BSA (30 nM) at 37 C. DLS measurements were detected every hour for 24 hours to
determine the size of nanoparticles in suspension over time.
In vitro drug release. In vitro release profiles of Carbo and GMP from NCPꢀCarbo/GMP
o
were performed in 250 mL 1× PBS buffer with or without 5 mM cysteine at 37 C and pH 7.4.
DOPAꢀNCPꢀCarbo/GMP or NCPꢀCarbo/GMP (0.75 mg) were suspended in 4 mL of 1x PBS
buffer solution with or without 5 mM cysteine in a 10,000 MWCO pleated dialysis bag. The
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dialysis bag containing the nanoparticle suspension was added into the beaker containing 250
mL of 1x PBS buffer, incubated at 37 oC at pH 7.4, while stirring. Periodically, 1 mL aliquots of
solution were taken from the solution, and a fresh 1 mL of buffer solution with or without
cysteine was added to the beaker. The removed aliquot was digested in nitric acid or HCl and
analyzed by ICPꢀMS for Pt or UVꢀVis for GMP, respectively.
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In vitro cytotoxicity and synergistic effect. In vitro cytotoxicity assays were carried out on
SKOVꢀ3 and A2780/CDDP ovarian cancer cells. In 96ꢀwell plates, SKOVꢀ3 or A2780/CDDP
ovarian cancer cells were seeded at a density of 1000 cells/well and 750 cells/well, respectively,
in a total of 100 ꢀL McCoy or RPMIꢀ1640 containing 10% FBS. The cells were incubated at 37
oC for 24 h prior to drug treatment. The culture medium was replaced by fresh medium. Different
concentrations of carbo, GMP, free carbo plus GMP (Carbo&GMP) mixture (at the same NCPꢀ
Carbo and NCPꢀGMP drug dose), NCPꢀCarbo, NCPꢀGMP, and NCPꢀCarbo/GMP were added
and incubated at 37 C and 5% CO2 for 72 h, and cell viability was measured by MTS assay
(Promega, USA) based on the manufacturer’s manual. IC50 values were calculated from curves
constructed by plotting cell viability (%) versus drug concentration (ꢁM).
An additional in vitro cytotoxicity was performed on A2780/CDDP with similar conditions
above in which different concentrations of free drugs or particles were added for 4 h. The culture
medium was replaced with fresh medium, and the cells were incubated for an additional 44 h.
Cell viability was measured by MTS assay, and the IC50 values were determined.
The combination index (CI) was calculated using the following equation
o
ꢂꢃ
ꢂꢅ
ꢀꢁ =
+
ꢂꢄꢃ ꢂꢄꢅ
where D1 and D2 are concentrations of drug 1 and drug 2 in combination at a specific drug effect
level (eg. 50% inhibition concentration), while Dm1 and Dm2 are the concentrations of single
agent to reach the same drug effect level. CI values were plotted against drug effect level (ICx
values). CI values lower than 1 indicate synergism.
Cell apoptosis by confocal microscopy. SKOVꢀ3 and A2780/CDDP cells were seeded in 6ꢀ
well plates (5 × 104 cells/well), and the cells were treated with free drugs or particles at a
concentration of 1.9 ꢀM carbo or 0.7 ꢀM GMP for 24 h. Treated cells were harvested, washed
twice with iceꢀcold PBS, stained with Alexa Fluor 488 conjugated Annexin V and prodium
iodide (PI) for 15 min at room temperature in the dark, and then analyzed by flow cytometry.
For CLSM imaging, cells were seeded on 10 mm2 glass coverslips placed in 6ꢀwell plates at
a density of 5 × 104 cells per well and incubated with free drugs or particles at a concentration of
1.9 ꢁM carbo or 0.7 ꢀM GMP for 24 h. After fixing with 4% paraformaldehyde, cells were
stained with 10 ꢁg/mL of DAPI and Alexa Fluor 488 conjugated Annexin V and observed using
CLSM at excitation wavelengths of 405 and 488 nm to visualize nuclei (blue fluorescence) and
cell apoptosis (green fluorescence), respectively.
Cell apoptosis by flow cytometry. SKOVꢀ3 or A2780/CDDP were seeded at 500,000
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cells/well in 6ꢀwell plate containing 2 mL total volume of cell culture medium for 24 h at 37 C
and 5% CO2. The culture medium was replaced with fresh medium containing different drug
treatments at carbo concentration of 1.9 ꢀM for SKOVꢀ3 and 1.3 ꢀM for A2780/CDDP and/or a
GMP concentration of 0.7 ꢀM for SKOVꢀ3 and 0.5 ꢀM for A2780/CDDP. Following a 24 h
incubation, the floating and adherent cells were collected and stained with Annexin V/dead cell
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apoptosis kit with Alexa Fluor 488 annexin V and propidium iodide (PI, Invitrogen, USA) based
on manufacturer’s instructions. The apoptosis was analyzed on a flow cytometer (LSRII 3ꢀ8, BD,
USA).
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In vitro cellular uptakes and intercellular distribution. SKOVꢀ3 or A2780/CDDP cells
were seeded at a cell density of 5x105 cells per well and 2 mL total volume media containing
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10% FBS. Following 24 h of incubation at 37 C and 5% CO2, the culture medium was replaced
by 2 mL of fresh media containing aliquots of free carbo and GMP or NCPꢀCarbo/GMP at 0.96
ꢀM carbo (or 0.35 ꢀM GMP) for SKOVꢀ3 and 0.65 ꢀM carbo (or 0.24 ꢀM GMP) for
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A2780/CDDP. The cells were cultured for different time points (1 h, 2 h, 4 h, and 24 h) at 37 C
and 5% CO2. Media were removed and the cells were washed with 1× PBS three times. The
adherent cells were collected by trypsinization and washed further with 1× PBS three times. The
cell numbers were counted prior to the last wash. The cells were digested with concentrated
nitric acid or 6 M HCl and analyzed using ICPꢀMS for Pt content or UVꢀVis for GMP content.
Known cell numbers of SKOVꢀ3 or A2780/CDDP with no drug treatments were used as baseline
to subtract any interference from cellular components.
To directly observe the internalization and intracellular distribution of particles under CLSM,
Rhodamine B (RhB), a fluorescence marker, was doped into the particle by adding it to the
prodrug microemulsion during particle preparation. Cells were seeded on 10 mm2 glass
coverslips placed in 6ꢀwell plates and incubated with RhBꢀdoped NCPꢀCarbo/GMP for 1 h, 2 h,
4 h, and 24 h. Cells were washed with PBS three times, fixed with 4% paraformaldehyde, stained
with DAPI and LysoTracker Green, and observed under CLSM (FV1000, Olympus, Japan).
In vivo pharmacokinetic and biodistribution studies. To evaluate the pharmacokinetics
and biodistribution of NCPꢀCarbo/GMP, balb/c mice bearing CTꢀ26 tumors were
intraperitoneally injected with NCPꢀCarbo/GMP at 5 mg/kg carbo dose (or 1.5 mg/kg GMP
dose). Mice were sacrificed at 5 min, 1h, 3 h, 5h, 8 h, 24 h, and 48 h postꢀinjection. Their liver,
lung, spleen, kidney, heart, bladder, tumor, and blood were harvested and digested in
concentrated nitric acid for 24 h, and the Pt concentrations were analyzed by ICPꢀMS.
Pt concentration were analyzed using ICPꢀMS. GMP concentrations in plasma were further
analyzed using highꢀperformance liquid chromatographyꢀtandem mass spectrometry (HPLCꢀ
MS/MS, Agilent 6460 QQQ MSꢀMS). The extraction method followed a previous literature
procedure. To a 50 ꢀL plasma, 200 ꢀL iceꢀcold acetonitrile was added, vortexed, mixed, and
centrifuged. The resulting supernatant was evaporated and reconstituted in 100 ꢀL of water. An
injection volume of 20 ꢀL of sample was used. The autosampler and column temperatures were
kept at 4 and 30 oC, respectively. The samples were separated via a PGC Hypercarb column (100
x 2.1 ID, 5 ꢀm, Thermo Fisher Scientific) fitted with a guard column (Hypercarb 10 x 2.1, 5 ꢀm,
Thermo Fisher Scientific). A gradient mobile phase of (A) 10 mM ammonium acetate at pH 10
and (B) acetonitrile were used with the initial mobile phase of 95% solvent A and 5% solvent B
at a flow rate of 0.3 mL/min. After 2 min, solvent A was gradually decreased to 80% over 0.2
min and held at this condition for 5.6 min. The gradient was returned to 95% solvent A over 0.2
min, and this condition was held for an additional 7 min, for a total run time of 15 min. The mass
to charge transition was monitored from 342 to 231.
Anti-tumor activity in vivo. The antiꢀtumor activity was conducted on two subcutaneous
xenograft mouse models. SKOVꢀ3 cells (5 × 106 cells in 100 ꢀL medium/matrix gel (v/v 1:1)) or
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A2780/CDDP cells (5 × 106 cells in 100 ꢀL medium) were subcutaneously injected in the right
flank of mice. When the tumor volume reached around 100 mm3, mice were randomly divided
into 3 groups (n = 5), and i.p. injected with PBS, Carbo&GMP, and NCPꢀCarbo/GMP at a dose
of 10 mg carbo/kg and 2.4 mg Gem/kg every three days. Tumor sizes were measured every day
by a caliper and calculated as follows: (length × width2)/2. Body weight was also recorded every
day as an indicator of systemic toxicity. All mice were sacrificed when the tumor volume of PBS
group reached 2000 mm3.
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In vivo immunogenic response, hypersensitivity, and general toxicity evaluation of NCP.
At the endpoint of the in vivo efficacy, blood was collected, and the serum was separated for
immunogenic response analysis. TNFꢀα, IFNꢀγ, and ILꢀ6 production was determined by ELISA
(R&D Systems, USA). Blood from the control group was also analyzed under the same treatment
above for comparison. Organs (heart, liver, spleen, lung and kidney) were also harvested,
sectioned at 5ꢀꢀm thickness, stained with H&E, and observed for histological examination of
toxicity with light microscopy (Panoramic Scan Whole Slide Scanner, PerkinElmer, USA).
In vivo tumor cell apoptosis. SKOVꢀ3 and A2780/CDDP tumors were excised and
embedded in optimal cutting temperature (OCT) medium, sectioned at 5ꢀꢁm thickness. TdTꢀ
mediated dUTP nick end labeling (TUNEL) assay was performed using DNA Fragmentation
Detection Kit (Invitrogen, USA) and observed under CLSM to quantify in vivo apoptosis. Nuclei
were stained with DAPI (10 µg/mL), and DNA fragments in apoptotic cells were stained with
fluoresceinꢀconjugated deoxynucleotides (green). The number of TUNELꢀpositive cells was
divided by the total number of cells to calculate the percentage of apoptotic cells in the sample.
Statistical analysis. Results were expressed as means ± standard deviation (S.D.). ANOVA
was used to determine statistical significance. A P value < 0.05 was considered statistically
significant.
Acknowledgment
We acknowledge NIH (UO1ꢀCA198989) and the University of Chicago Medicine
Comprehensive Cancer Center (NIH CCSG: P30 CA014599) for funding support.
Author Contributions
†These authors contributed equally.
Abbreviations
OCa, ovarian cancer; Pt, platinum; Carbo, carboplatin; NCP, nanoscale coordination
polymer; DOPA, 1,2ꢀdioleoylꢀsnꢀglyceroꢀ3ꢀphosphate; GMP, gemcitabine monophosphate; THF,
tetrahydrofuran; PBS, phosphate buffered saline; CI, combination index; RhB, rhodamine B; TUNEL,
TdTꢀmediated dUTP nick end labeling
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