Bioresponsive cancer-targeted polysaccharide nanosystem to inhibit angiogenesis
Received 11 April 2017
Accepted for publication 8 August 2017
Published 10 October 2017 Volume 2017:12 Pages 7419—7431
Checked for plagiarism Yes
Review by Single anonymous peer review
Peer reviewer comments 3
Editor who approved publication: Dr Linlin Sun
Fang Yang, Xueyang Fang, Wenting Jiang, Tianfeng Chen
Department of Chemistry, Jinan University, Guangzhou, China
Abstract: With many desirable features, such as being more effective and having multiple effects, antiangiogenesis has become one of the promising cancer treatments. The aim of this study was to design and synthesize a new targeted bioresponsive nanosystem with antiangiogenesis properties. The [email protected](POP) nanosystem was constructed by the polymerization of Ulva lactuca polysaccharide and N-isopropyl acrylamide, decorated with methoxy polyethylene glycol and Arg–Gly–Asp peptide, and encapsulated with anticancer complex [Ru(phen)2p-MOPIP](PF6)2·2H2O. The nanosystem was both pH responsive and targeted. Therefore, the cellular uptake of the drug was greatly improved. Moreover, the [email protected](POP) had strong suppressive effects against vascular endothelial growth factor (VEGF)-induced angiogenesis through apoptosis. The [email protected](POP) significantly inhibited VEGF-induced human umbilical vein endothelial cell migration, invasion, and tube formation. These findings have presented new insights into the development of antiangiogenesis drugs.
Keywords: polysaccharide nanosystem, antiangiogenesis, cancer-targeted, bioresponsive
It was reported that the overall cancer death rate dropped 25% from 1991 to 2014 in the USA.1 But lots of cancers are hardly cured, and some are still very difficult to treat. Chemotherapy, a common cancer treatment to attack cancer cells,2 can induce severe side effects that may influence a person’s quality of life.3 Targeting angiogenesis represents a novel strategy for the development of anticancer therapies.4,5 Both chemotherapy and antiangiogenesis are systemic treatments, but antiangiogenesis drugs work on where new blood vessels are forming.6,7 Therefore, they do not induce severe side effects that chemotherapy does.8 Antiangiogenesis was proposed in the early 1970s, but until 2004 the first antiangiogenesis drug, Avastin (bevacizumab), was approved by the US Food and Drug Administration (FDA) to treat colon cancer. Bevacizumab is a monoclonal antibody which targets vascular endothelial growth factor (VEGF). It has been established that VEGF is a key driver of sprouting angiogenesis and is overexpressed in most solid cancers. Several other antiangiogenesis drugs have since been approved by the FDA, such as Erbitux (cetuximab), Vectibix (panitumumab), Herceptin (trastuzumab), Tarceva (erlotinib), Nexavar (sorafenib), and Sutent (sunitinib).9 Some of them are orally active drugs. Antiangiogenesis drugs have become an important part of treatment for many types of cancer.
Ulva lactuca, also known as sea lettuce, is an edible green alga in the division Chlorophyta. Sea lettuce, a food for humans, is eaten raw in salads and cooked in soups. It is a soluble dietary fiber and has high content of protein and high concentrations of iron as well as a variety of vitamins and minerals. It was reported that U. lactuca polysaccharides contained glucose, glucuronic acid, xylose, rhamnose, and sulfate groups. Ulva lactuca (UL) polysaccharide exhibited antiviral, anti-peroxidative and anti-hyperlipidemic properties.10,11 Polysaccharides, as functionalizing agents, have been widely used in cancer nanotechnology because of their excellent biocompatibility, stability, nontoxicity, etc.5 The nanomaterials decorated by polysaccharides exhibited enhanced cell-permeating and cancer-targeting abilities.12 Therefore, in this study, the nanosystem was originally synthesized by the polymerization of N-isopropyl acrylamide (NIPAM) and UL polysaccharide.
It was reported that the newer antiangiogenesis drugs also attacked tumor cells themselves.13,14 The use of antiangiogenesis drugs in conjunction with irradiation and other chemotherapeutic drugs has led to improved treatment outcomes.15,16 It is the goal of scientists to design new drugs that are more effective and have multiple effects. In the years to come, such drugs may blur the line between antiangiogenesis drugs and other cancer treatments. In this study, our goal was to design a targeted antiangiogenic nanosystem, which is also pH responsive.17 Drug delivery systems with a pH response have been widely used, because they can swell and shrink in different pH condition so as to achieve targeted drug delivery and efficient drug release in cancer cells.18,19 The Arg–Gly–Asp (RGD) peptide, an excellent targeting reagent, can recognize the integrin receptor which is overexpressed in a lot of human tumor cells and is able to bind to both tumor cells and endothelial cells.20 Therefore, this pH-responsive nanosystem was also decorated with RGD peptide to achieve targeted drug delivery.
In this study, we designed a novel [email protected](POP) antiangiogenic nanosystem. The polyelectrolyte core of the nanosystem was constructed by the polymerization of U. lactuca polysaccharide and NIPAM and decorated with RGD peptide and methoxy polyethylene glycol (mPEG). The anticancer complex Ru(POP) ([Ru(phen)2p-MOPIP](PF6)2·2H2O), an anticancer complex with significant anticancer activity and strong auto-fluorescence, was encapsulated in this nanosystem.21,22 The nanosystem overcame the poor stability and low solubility of Ru(POP). It was also pH responsive and targeted, and the cellular uptake of the drug was greatly improved. This [email protected](POP) had strong suppressive effects against VEGF-induced angiogenesis through apoptosis. It also significantly inhibited VEGF-induced human umbilical vein endothelial cell (HUVEC) migration, invasion, and tube formation both in vitro and in vivo. These findings have provided meaningful insights into the development of antiangiogenesis drugs.
Materials and methods
RGD was obtained from China Gier Biochemistry Company (Shanghai, China). NIPAM, mPEG, N-hydroxysuccinimide (NHS), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), ammonium persulfate (APS), 2-mercapto benzoic acid (MBA), methyl thiazolyl tetrazolium (MTT), propidium iodide (PI), 4′,6-diamidino-2-phenylindole (DAPI), and dihydroethidium (DHE) were purchased from Sigma-Aldrich Co., St Louis (MO, USA). Tetraethylorthosilicate (TEOS), polyethyleneimine (PEI, 99%), triethanolamine, and hexadecyltrimethylammonium bromide (CTAB, 99%) were obtained from Aladdin Chemistry Company (Shanghai, China). The HUVECs were purchased from Guangzhou Jennio Biotech Co., Ltd. (Guangzhou, China).
Preparation of U. lactuca polysaccharide
U. lactuca powder was purchased from Research Center of Hydrobiology of Jinan University (Guangzhou, China). To extract the polysaccharide, the sample powder was refluxed with ethanol in a water bath for 2 h. Then, the precipitate was heated at 100°C in water (pH =4) for another 2 h. The filtrate was lyophilized, and the polysaccharide was obtained.
Synthesis of [email protected](POP)
First, NIPAM (0.5 g) and UL polysaccharide (0.25 g) were dissolved in deionized water, and then stirred overnight. Afterward, 0.025 g of MBA was added, and the mixture was heated at 75°C in a three-necked flask with nitrogen gas. Heating of the mixture was continued after the addition of 0.025 g of APS at 60°C for 4 h. After centrifuging and lyophilizing, UL polysaccharide-NIPAM (UP) was obtained. Second, mPEG was decorated to the surface of UP by dissolving UP (500 mg) in water, adding NHS (100 mg), EDC (100 mg), and mPEG (100 mg), and reacting at room temperature for 24 h. mPEG-UL polysaccharide-NIPAM (mUP) was obtained after dialysis for 24 h. Furthermore, NHS, EDC, and RGD were added and stirred for 24 h. mPEG-UL polysaccharide-NIPAM-RGD (mUPR) was obtained after 24 h of dialysis. Finally, Ru(POP) was encapsulated in mUPR to form [email protected](POP) by the reaction of mUPR and Ru(POP) for 24 h.
The morphology, particle size, and zeta potential of [email protected](POP) were characterized by transmission electron microscopy (TEM; H-7650; Hitachi Ltd., Tokyo, Japan) and Nano-ZS instrument (Malvern Instruments, Malvern, UK). The Fourier transform infrared (FTIR; Equinox 55; Bruker, Ettlingen, Germany) spectroscopy was conducted in the range of 4,000-500 cm−1.
Cell culture and MTT assay
The HUVECs were cultured in F12 medium including 10% fetal bovine serum (FBS), 0.01% streptomycin, and 1% penicillin in 5% CO2 atmosphere at 37°C. To perform MTT assay, 2,000 cells/well of HUVECs were seeded in a 96-well plate for 24 h, and different concentrations of [email protected](POP) were added and incubated for 72 h. Next, 30 μL of MTT was added to each well for 3 h, and then the medium was replaced with dimethyl sulfoxide (DMSO). The cell viability of HUVECs was measured by the absorbance at 570 nm with a microplate spectrophotometer.
In vitro intracellular fluorescence localization
In brief, 20,000 cells/mL of HUVECs were seeded in 2 cm dishes and cultured for 24 h. Next, the [email protected](POP) was added at different time points. The DAPI and Lyso-Tracker were added preceding the observation time of 0.5 h and 2 h. Finally, the cells were washed with PBS three times and observed by fluorescence microscopy (IX51; Olympus Corporation, Tokyo, Japan).
In vitro cell migration
HUVECs were seeded and incubated in six-well plates for 24 h. Monolayer cells were wounded by 100 μL pipette tips and washed with PBS three times. Fresh medium and [email protected](POP) were added and incubated for 24 h. Then, the migrated cells were photographed and quantified by manual counting.
In vitro cell invasion
The Boyden chamber is a useful tool to study the effects of [email protected](POP) on HUVEC invasion. Briefly, the Boyden chamber was precoated with matrigel for 6 h at 37°C. HUVECs (5×104 cells/mL) with serum-free medium were placed in the upper compartment and were allowed to migrate into the lower compartment, in which [email protected](POP) was present with F12 medium. Nonmigratory cells were scraped off 24 h later, and migratory cells were fixed in methanol, washed, and stained with Giemsa solution. Experiments were performed in triplicate transwells, and the number of cells that migrated to the lower side of the membrane was determined.
In vitro tube formation assay and chorioallantoic membrane assay
The effects of [email protected](POP) on tube formation and chorioallantoic membrane were evaluated as reported.17 Briefly, Matrigel was first thawed at 4°C overnight. Then, 100 μL Matrigel per well was added into prechilled 48-well plate and incubated for 30 min at 37°C to allow the gel to solidify. HUVECs (5×104 cells/mL) were added into the matrigel layer, as well as [email protected](POP). After 8 h, the tube formation was visualized under an Olympus microscope (Olympus Corporation, Tokyo, Japan). The chorioallantoic membrane (CAM) assay was used to analyze the effect of [email protected](POP) on angiogenesis in vivo. First, the fertilized eggs were cleaned with 75% ethanol and incubated at 37°C in an egg incubator for 5 days. Then, the eggs were cracked open, and different concentrations of [email protected](POP), with or without VEGF, were gently injected on the chorioallantoic membrane. After the embryos were incubated for another 2 days, the CAM was observed under an Olympus microscope.
Flow cytometric analysis
As described in our previous paper,18 the Beckman flow cytometry was used to analyze the cell cycle distribution.
Experiments were repeated at least three times. The differences between the treatment groups were analyzed by one-way ANOVA multiple comparisons using SPSS 13.0 statistical package (SPSS Inc., Chicago, IL, USA).
Results and discussion
Characterization of [email protected](POP)
In this study, a novel cancer-targeting nanosystem, [email protected](POP), was designed and synthesized (Scheme 1). The polyelectrolyte core was constructed by utilizing the polymerization of NIPAM and UL polysaccharide with APS as an initiator and MBA as a crosslinker, followed by surface modification with mPEG and RGD peptide. According to the previous research,23 the decoration of mPEG increased the solubility, stability, and biocompatibility of the nanosystem. Moreover, RGD was a cancer-targeting ligand. Then, this drug delivery system (mUPR) was loaded with anticancer drug Ru(POP) to form [email protected](POP) (Figure 1A and B). The average particle size of [email protected](POP) was shown in Figure 1C and D. As shown in Figure 1D, the average particle size of [email protected](POP) was 250 nm, which was approximately the TEM result as shown in Figure 1C. The TEM showed the image of homogeneous particles with a diameter of about 200 nm, and the nanosystems were stable in 40 days (Figure S1). To determine the stability of [email protected](POP), the particle sizes in DMEM and DMEM with 10% FBS were observed (Figure 1E). The results showed that the particle sizes had little changes within 24 h in both media. Moreover, the nanoparticles were stable with a diameter of about 300 nm after 30 days. FTIR spectra (Figure 1F) were used to further characterize the [email protected](POP) nanosystem. As shown in Figure 1F(a), the peaks at 1,243 cm−1 and 1,180 cm−1 were contributed to the symmetrical stretching vibration of C–O–C and the stretching vibration of C–N from Ru(POP). These two peaks were also presented in the spectrum of [email protected](POP) (Figure 1F[d]). The peaks at 1,540 cm−1 and 1,380 cm−1 were the NH bending for secondary amides and isopropyl group bending vibrations of NIPAM (Figure 1F[b–d]). The peak at 1,600 cm−1 corresponded to the stretching vibration of amide from RGD (Figure 1F[c and d]). The difference and appearance of the abovementioned peaks indicated the successful synthesis of [email protected](POP).
Scheme 1 Scheme of synthesis of [email protected](POP).
Figure 1 (A, B) Design, delivery, and action mechanisms of bioresponsive cancer-targeted polysaccharide nanosystem to inhibit angiogenesis. (C) TEM images of [email protected](POP). (D) Size distribution of [email protected](POP). (E) Changes in the particle size of [email protected](POP) following incubation with DMEM or 10% FBS; (F) FTIR spectra of (a) Ru(POP), (b) UP, (c) mUPR, and (d) [email protected](POP).
pH response of [email protected](POP)
Four pH buffer solutions were used to demonstrate the pH-responsive swelling of the [email protected](POP) nanosystem (Figure 2A–F). The pH 7.4 was similar to the pH of normal tissues and organs; pH 6.8 was the pH of cytoplasm in cancer cells; pH 6.2 was the pH of tumor extracellular environment; pH 5.3 was the pH in lysosome.20 The zeta potential and particle size of [email protected](POP) in different buffer solutions are shown in Figure 2A and D. When the pH decreased from 7.4 to 5.3, the particle size increased from 250 nm to 950 nm, and the surface charge changed from −25 mV to +10 mV (Figure 2A). TEM (Figure 2B) and a scheme (Figure 1A) were used to further illustrate the pH response of the nanosystem. The TEM images showed that the diameter of the particles was about 200 nm at pH 7.4. However, the diameter of the nanoparticles expanded to about 800 nm when the pH decreased to 5.3 (Figure 2D). Under acidic conditions, there was a change in the ionization state due to the presence of carboxyl and amine group in the nanosystem. This produced an electrostatic repulsion between nanoparticles,24 resulting in the swelling of the polyelectrolyte core and the change of particle size. In vitro drug release of Ru(POP) was also performed in different pH values. As shown in Figure 2E, after 48 h, the drug release was about 85% at pH 5.3, which was almost three times more than that at pH 7.4. Therefore, this [email protected](POP) nanosystem had pH-responsive property which greatly increased the drug release in lysosome.
Figure 2 (A) Zeta potential of [email protected](POP) at different pH values. (B) TEM images of [email protected](POP) at pH 7.4 and 5.3. (C) Quantitative cellular uptake of Ru(POP), [email protected](POP), and [email protected](POP) in HUVECs (2×105 cells/mL). HUVECs were treated with Ru(POP) (10 μg/mL), [email protected](POP) (10 μg/mL), and [email protected](POP) (10 μg/mL) for different periods of time. The concentrations of Ru(POP) were determined by ICP-MS. (D) Change in the particle size of [email protected](POP) with pH value. (E) In vitro drug release of Ru(POP) from [email protected](POP) in different solution. (F) The cell viability of HUVECs treated with different concentrations of Ru(POP), [email protected](POP), and [email protected](POP) for 72 h. Each value represents mean ± SD (n=3).
Intracellular localization and cellular uptake of [email protected](POP)
It was reported that pharmaceutical activity of nanomedicine is mostly dependent on the efficiency of cellular uptake and drug delivery.22 The quantitative cellular uptake in HUVECs was originally determined (Figure 2C). HUVECs were treated with 10 μg/mL of Ru(POP), [email protected](POP), and [email protected](POP). The concentrations of Ru(POP) were detected by inductively coupled plasma mass spectrometry (ICP-MS). The results showed that the intracellular drugs were time dependent. The intracellular concentrations of [email protected](POP) were the highest, and the intracellular concentrations of Ru(POP) were the lowest. Moreover, Figure 2F also shows the significant differences of cell viability induced by Ru(POP), [email protected](POP), and [email protected](POP). The abovementioned results suggested that [email protected](POP) had a high cellular uptake, and the decoration with RGD as a target molecule greatly enhanced the antiangiogenic activity of [email protected](POP). Intracellular fluorescence localization was carried out to further investigate whether [email protected](POP) was in lysosome. As shown in Figure 3, the localization was studied by using Lyso-Tracker (red) to label lysosomes and DAPI to label the nucleus. The overlay results indicated that the drug successfully entered the cells through the lysosomes after treatment for 2 h.
Figure 3 Colocalization of [email protected](POP) (green fluorescence), Lyso-Tracker (red fluorescence), and DAPI (blue fluorescence) in HUVECs with an observation period of 8 h.
[email protected](POP) inhibited VEGF-induced cell migration, invasion, and tube formation in vitro
The early stages of angiogenesis have three steps: endothelial cell proliferation, migration, and tube formation.5 To determine whether [email protected](POP) could suppress the VEGF-induced endothelial cell migration and tube formation, wound healing, Transwell assay, and tube formation assay were performed. As shown in Figures 4A and B and S2, at the concentration of 1 μM, Ru(POP), [email protected](POP) and [email protected](POP) all strongly inhibited VEGF-induced migration and invasion of HUVECs, but the ability of [email protected](POP) was the highest. As shown in Figures 4C and S3, tube formation assay was performed to further evaluate the antiangiogenic ability of [email protected](POP). As expected, VEGF promoted the formation of elongated tube-like structures, but Ru(POP), [email protected](POP), and [email protected](POP) inhibited the tube formation. Moreover, [email protected](POP) had the strongest inhibition. Overall, the relative reduced numbers of the migrated cells and invaded cells, as well as the relative decreased length of capillary tube, suggested the significant antiangiogenic ability of [email protected](POP).
Figure 4 (A) Ru(POP), [email protected](POP), and [email protected](POP) (1 μM) inhibited VEGF-induced HUVEC (2×105 cells/mL) migration. (B) [email protected](POP) (1 μM) inhibited VEGF-induced HUVEC (5×104 cells/mL) invasion. (C) Antiangiogenesis assay [email protected](POP) (1 μM) on HUVECs (5×104 cells/mL). (D) Representative images of angiogenesis inhibition of Ru(POP), [email protected](POP), and [email protected](POP) (30 μM) in CAM assay with VEGF.
[email protected](POP) inhibited VEGF-induced CAM angiogenesis
The chick CAM assay is a powerful in vivo angiogenesis model and permits a realistic evaluation of the angiogenic response.22 The abovementioned results showed that [email protected](POP) inhibited VEGF-induced tube formation in vitro. Therefore, CAM assay was used to further assess the antiangiogenesis of [email protected](POP) in vivo. As shown in Figures 4D and S4, the formation of branched blood vessels was promoted by VEGF, but [email protected](POP) showed a dose-dependent reduction in the branched vessels. Compared to Ru(POP) and [email protected](POP), [email protected](POP) demonstrated the highest activity. These results suggested that [email protected](POP) inhibited VEGF-induced CAM angiogenesis.
Apoptosis induced by [email protected](POP)
The [email protected](POP) nanosystem showed strong suppressive effects against VEGF-induced angiogenesis. So, the effects of synthesized drugs on the cell cycle and apoptosis were analyzed by flow cytometry. After 72 h of incubation with 7.5 μM, 15 μM and 30 μM of [email protected](POP), the representative DNA histograms (Figure 5) showed a significant increase from 21.9% and 46.7% to 58.8% in the sub-G1 peaks, respectively (Figure S5). When comparing the activity of Ru(POP), [email protected](POP), and [email protected](POP) at the same concentration of 30 μM, [email protected](POP) was the highest. These results indicated that suppressive effects induced by [email protected](POP) were mainly through apoptosis.
Figure 5 Effects of Ru(POP), [email protected](POP), and [email protected](POP) on HUVEC cycle distribution for 72 h.
We successfully synthesized an antiangiogenic [email protected](POP) nanosystem with the decoration of mPEG and RGD peptide, which improved both the pH response and the cellular uptake of the [email protected](POP) nanosystem. The targeted [email protected](POP) could swell in an acidic environment and efficiently release Ru(POP) drug in the lysosome of HUVECs. Moreover, the [email protected](POP) had strong suppressive effects against VEGF-induced angiogenesis through apoptosis. The [email protected](POP) significantly inhibited VEGF-induced HUVEC migration and tube formation both in vitro and in vivo. These results have provided new and meaningful insights into the antiangiogenic effects of [email protected](POP) nanosystem.
This work was supported by the Natural Science Foundation of China (21401076, 21271002), National High Technology Research and Development Program of China (863 Program, SS2014AA020538), Science Foundation for Distinguished Young Scholars (S2013050014667) of Guangdong Province, Foundation for High-level Talents in Higher Education of Guangdong, YangFan Innovative & Entrepreneurial Research Team Project (201312H05), Guangdong Special Support Program, and Guangdong Frontier and Key Technological Innovation Special Funds.
The authors report no conflicts of interest in this work.
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Figure S1 The particle size of [email protected](POP) in 40 days.
Figure S2 (A) Ru(POP), [email protected](POP), and [email protected](POP) (1 μM) inhibited VEGF-induced HUVEC (5×104 cells/mL) invasion. (B) Antiangiogenesis assay of Ru(POP), [email protected](POP), and [email protected](POP) (1 μM) on HUVECs (5×104 cells/mL).
Figure S3 The relative reduction in the migrated cell numbers, invaded cell numbers, and capillary tube length suggested remarkable anti-metastasis effect of Ru(POP), [email protected](POP), and [email protected](POP).
Figure S4 (A) Representative images of angiogenesis inhibition of Ru(POP), [email protected](POP), and [email protected](POP) (30 μM) in CAM assay without VEGF. (B) The relative quantitation of vascular density based on the CAM images.
Figure S5 Analysis of sub-G1 value obtained from flow cytometry.
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