Framboidal Nanoparticles Containing A Curcumin-Phenylboronic Acid

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Framboidal Nanoparticles Containing A Curcumin-Phenylboronic Acid Complex with Antiangiogenic and Anticancer Activities Andre J van der Vlies, Manami Morisaki, Hoi I Neng, Emma Hansen, and Urara Hasegawa Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.9b00006 • Publication Date (Web): 24 Jan 2019 Downloaded from http://pubs.acs.org on January 26, 2019

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Bioconjugate Chemistry

Framboidal Nanoparticles Containing A CurcuminPhenylboronic Acid Complex with Antiangiogenic and Anticancer Activities André J. van der Vlies§,†, Manami Morisaki§,‡, Hoi I Neng‡, Emma M. Hansen‖ and Urara Hasegawa*,‖ †Department of Chemistry, Kansas State University, 213 CBC Building, 1212 Mid-Campus Dr North, Manhattan, KS 66506, USA. ‡

Department of Applied Chemistry, Graduate School of Engineering, Osaka University, 2-1

Yamadaoka, Suita, Osaka 565-0871, Japan. ‖

Tim Taylor Department of Chemical Engineering, Kansas State University, 1005 Durland Hall,

1701A Platt St, Manhattan, KS 66506, USA. §

These authors contributed equally.

KEYWORDS: curcumin, polymeric nanoparticles, phenylboronic acid, antiangiogenic activity, anticancer activity, chicken chorioallantoic membrane assay

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Bioconjugate Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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ABSTRACT

Curcumin (Cur) has a wide range of bioactivities that show potential for the treatment of cancer as well as chronic diseases associated with inflammation and aging. However, therapeutic efficacy of Cur has been hampered by its rapid degradation under physiological conditions and low aqueous solubility. To address these problems, we prepared Cur-loaded polymeric nanoparticles (CNPs), in which Cur was complexed with phenylboronic acid-containing framboidal nanoparticles (NPs), by simple mixing of Cur and NPs in an aqueous solution. CNPs improved chemical stability of Cur and released it in a sustained manner under physiological conditions. Furthermore, CNPs significantly enhanced the antiangiogenic and anticancer activities of Cur in chicken chorioallantoic membrane models.

INTRODUCTION Curcumin (Cur) is found in the spice turmeric and has a wide range of bioactivities including antiangiogenic,1 antioxidative,2 anti-inflammatory,3 anticancer,4 immunomodulatory5 and neuroprotective activities.6 Numerous reports have shown that Cur is a potential therapeutic agent for treating lung, breast, skin and gastrointestinal cancers.7 In addition, Cur is also reported to alleviate chronic diseases like cystic fibrosis,8 inflammatory bowel disease,9 arthritis,10 Parkinson’s11 and Alzheimer’s diseases.12 Despite the interesting pharmacological effects, therapeutic use of Cur has not been fully explored mainly due to its chemical instability and low aqueous solubility.13 Especially, Cur is known to degrade rapidly under physiological conditions, which significantly reduces its bioavailability.14 One of the suggested mechanisms for this degradation reaction is


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auto-oxidation of the keto-enol structure in the presence of oxygen resulting in the formation of a bicyclopentadione and other products.15 Several reports have appeared concerning the chemical modification of Cur to improve its therapeutic efficacy. For example, Cur was conjugated to hydrophilic amino acid-based building blocks via an hydrolysis-sensitive ester bond to increase its solubility.16 17 Furthermore, its metal complexes in which the keto-enol structure of Cur was complexed with metal cations showed much higher stability than Cur alone.18 In another approach, nanocarrier systems, such as solid lipid nanoparticles, liposomes, polymeric micelles, cyclodextrin and dendrimers, have been used to encapsulate Cur within a hydrophobic milieu.19-24 These nanocarriers improved both stability and solubility of Cur and prolonged its circulation time by preventing renal clearance. Recently, Cur-loaded polymeric nanoparticles have been developed by combining the chemical modification and nanocarrier approaches.25 A polymer containing phenylboronic acid (PBA) groups was complexed with Cur and dispersed in water to prepare nanoparticles by self-assembly. These nanoparticles significantly improved the stability of Cur under physiological conditions and released Cur upon the oxidative degradation of the boron-carbon bond. It is well-known that PBA and its derivatives interact with Cur to form a bright red 1:1 complex.26 According to the crystal structure of the Cur-PBA complex prepared in dichloromethane/methanol, the keto-enol structure of Cur complexes with PBA by forming a six-membered ring with a tetrahedral symmetry at the boron center (Scheme 1).27 Since the keto-enol structure of Cur is involved in its oxidative degradation, the high stability observed for the Cur-loaded polymeric nanoparticles is possibly due to the complexation of the keto-enol structure with PBA, which slowed down the degradation of Cur.


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Scheme 1. Complexation of Curcumin (Cur) and phenylboronic acid (PBA) in dichloromethane containing methanol.27 We recently reported phenylboronic acid (PBA)-containing nanoparticles (NPs) with a unique framboidal morphology, prepared by aqueous dispersion polymerization of N-acryloyl-3aminophenylboronic acid using methoxy poly(ethylene glycol) acrylamide as the polymerizable dispersant and N,N’-methylenebis(acrylamide) as the crosslinker.28 Due to the chemical properties of PBA groups as well as the high surface area-to-volume ratio of the framboidal structure, these NPs are expected to be a promising platform for drug delivery, biosensing and biomedical diagnosis applications. We recently showed that these NPs can be used as a vehicle for a catecholcontaining carbon monoxide (CO)-releasing drug. This drug was loaded to the NPs via the PBAdiol complexation upon mixing in aqueous buffer solutions.29 The CO-releasing drug-loaded NPs reduced toxic side effects and exerted anti-inflammatory activity in murine macrophages. To further explore the potential of NPs in drug delivery applications, we present herein Curloaded NPs (CNPs) prepared by complexation of Cur with PBA groups in NPs (Figure 1). The chemical stability and the release profiles of Cur were evaluated in phosphate buffered saline (PBS) as well as in human colon cancer (HT29) and human umbilical vein endothelial cells (HUVECs). Antiangiogenic effect of CNPs was assessed in the in vitro tube formation and gap closure migration assays as well as the in ovo chicken chorioallantoic membrane (CAM) assay of angiogenesis. Furthermore, anticancer activity was evaluated in the in vitro cancer cell proliferation assay and an in ovo CAM cancer model.


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Figure 1. Schematic illustration of Curcumin (Cur)-loaded nanoparticles (CNPs). Cur was loaded to phenylboronic acid (PBA)-containing framboidal nanoparticles (NPs) via the PBA-Cur complexation.

RESULTS AND DISCUSSION Preparation of Cur-loaded NPs (CNPs) via Cur-PBA complexation. Complexation of Cur with boric acid or PBA is well known and has been used for detecting boron-containing compounds.26 Boric acid interacts with the keto-enol structure of Cur to form a bright red 2:1 complex,30 also known as rosocyanine complex,31 which has been used for photometric quantification of boron.32 Similarly, PBAs, where one of the OH groups of boric acid has been replaced with a phenyl (Ph) substituent, have also been shown to interact with Cur in dichloromethane/methanol27 and DMSO/methanol.25 While the complexation of Cur with PBA has been explored in different organic solvents, to the best of our knowledge, there is no report on the Cur-PBA complexation in aqueous media. Here, we explored whether Cur can be complexed with the PBA groups of NPs in phosphate buffered saline (PBS) at pH 7.4. NPs were prepared by aqueous dispersion polymerization as reported previously,28 and mixed with Cur in PBS to prepare Cur-loaded NPs (CNPs). Upon mixing of Cur with NPs, a color change


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from yellow to red-orange was observed as shown in Figure 2a. To confirm Cur-PBA complexation, UV-Vis spectra of the mixtures of Cur and NPs in PBS at different molar ratios ([Cur] : [PBA]) were measured (Figure 2b). The UV-Vis spectrum of Cur shows absorbance at around 425 nm, which has been assigned to the -* transition.33 In the presence of NPs, the absorbance maximum shifted from 425 to 480 nm. Since similar spectral changes have been reported for Cur-PBA complexes prepared in organic solvents,25, 27 this red shift indicates the complexation of the keto-enol structure of Cur with the PBA groups of NPs. Furthermore, the absorbance at 480 nm increased with the increase of the PBA concentration and reached a plateau when 10 equivalent of the PBA groups was added relative to Cur. This shows that 10 fold excess of PBA groups is required for the complete complexation of Cur. To further confirm that the red shift is due to the formation of Cur-PBA complex, we also measured UV-Vis spectra of Cur after mixing with 3-acetamidophenylboronic acid (PBAAc, Figure S1a), a model compound, in PBS. As similar to the mixture of Cur and NPs, we observed a color change from yellow to orange after mixing Cur with PBAAc (Figure S1b). Interestingly, much higher concentration of PBAAc was required to prepare Cur-PBAAc complex compared to NPs. According to UV-Vis spectra, complexation was not completed even after adding 400 equivalent of PBAAc relative to Cur (Figure S1c). This result shows that complexation between Cur and PBA does not proceed efficiently in aqueous media. On the other hand, the previous reports showed that Cur complexes with 1 equivalent of PBA in dichloromethane.27 Therefore, it is likely that the hydrophobic microenvironment within NPs favors Cur-PBA complexation.


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Figure 2. Complexation of Cur and NPs. (a) Photographs of Cur and Cur mixed with NPs (CNP). The molar ratio of Cur and PBA groups, [Cur] : [PBA] = 1:20. (b) UV-Vis spectra of the mixtures of Cur and NPs. Cur and NPs were mixed in PBS (pH7.4 containing 1% DMSO) at different [Cur] : [PBA] ratios and kept at 20oC for 30 min. The UV-Vis spectrum of Cur (without NPs, [Cur] : [PBA] = 1 : 0) was recorded immediately after dissolving Cur in PBS. Cur concentration: 50 µM. Size and morphology of CNPs. The effect of Cur loading on the NP structure was investigated by dynamic light scattering (DLS) and transmission electron microscopy (TEM). According to DLS, the Z-average diameter of NPs was 153±20 nm and that of CNPs was 147±12 nm (Figure 3a). Both NPs and CNPs were monodisperse with the polydispersity index (PDI) below 0.1. The morphology of NPs and CNPs were observed by TEM after staining negatively with 2wt% sodium dodecatungstato(VI) phosphate solution. As reported previously, NPs showed the characteristic framboidal structure (Figure 3b, left). This morphology was maintained after Cur loading. (Figure 3b, right). These results clearly show that the Cur loading did not affect the size and morphology of NPs significantly.


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Figure 3. Structural characterization of CNPs. (a) Z-average diameter as measured by DLS. [Cur] : [PBA] = 1:20, n=4. (b) TEM images of NPs (left) and CNPs (right). Samples were negatively stained with 2wt% sodium dodecatungstato(VI) phosphate solution. Scale bars: 100 nm. Chemical stability of Cur loaded to CNPs. The chemical stability of Cur against auto-oxidation in PBS at 20oC was studied by UV-Vis spectroscopy. As shown in Figure 4a, the absorbance at λmax =425 nm decreased to 55 and 10 % within 10 min and 1 d, respectively, showing the rapid decomposition of Cur in PBS. On the other hand, the spectral change was much slower for CNPs and the absorbance at λmax=480 nm, which is attributed to Cur-PBA complex, decreased to 81% after 1 d and 60% after 5 d (Figure 4b). These results clearly show that the complexation of Cur with NPs inhibit oxidative degradation of Cur. A similar result has been reported by Luo et al. that Cur loaded to the polymeric nanoparticles via the Cur-PBA complexation showed the half-life of 11 h while the half-life of free Cur was less than 15 min.25 Importantly, the stability of CNPs was much higher compared to this polymeric nanoparticle system. We also investigated the stability of the Cur-PBAAc complex in PBS (Figure S2). While the complexation of Cur with PBAAc ([Cur] : [PBA] = 1 : 400) improved stability, the absorbance decreased to 38% after 1 d. Therefore, the high chemical stability of CNPs might be due to the hydrophobic microenvironment that stabilizes Cur-PBA complex thereby protecting Cur from oxidative degradation.


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Figure 4. Chemical stability of Cur and CNPs in PBS (pH7.4). UV-Vis spectra of (a) Cur and (b) CNP ([Cur]: [PBA] = 1:20) at 20 oC.

Cur release from CNPs. The Cur release profile from CNPs was measured in PBS. CNPs were dialyzed against PBS containing bovine serum albumin (BSA) as a carrier protein. Since the complexation of Cur and PBA is reversible, Cur is expected to be released from CNPs by removing free Cur from the solution by dialysis. As expected, we observed the sustained Cur release from CNPs in PBS and the amount of released Cur after 1, 3 and 24 h was 21, 30 and 47%, respectively (Figure 5).

Figure 5. Release of Cur from CNPs in PBS (pH7.4). The CNP solutions were placed in a dialysis tube (MWCO: 3 kDa) and dialyzed against PBS containing 40 mg/mL BSA as a carrier protein at


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25oC. At the indicated time points, the amount of curcumin remaining in the dialysis tube was determined by UV-Vis spectroscopy. n=3. We next studied intracellular release of Cur in human colon cancer (HT29) cells. Due to its fluorescent property,34 Cur can be tracked within the cells by confocal laser scanning fluorescence microscopy (CLSFM). Furthermore, since the fluorescence of Cur is quenched upon complexation with PBA,25 CNPs, in which Cur is complexed with PBA groups of NPs, is expected to be invisible under fluorescence microscope. Therefore, CLSFM allows for monitoring of free Cur released from CNPs in cells. HT29 cells were treated with Cur and CNPs and observed by CLSFM at different time points. After 15 min, we observed a very bright green fluorescence within the cells treated with Cur, while a much weaker fluorescence was observed in those treated with CNPs (Figure 6). For Cur, the fluorescence intensity decreased gradually and became very weak after 6 h. This fluorescence decrease is probably due to the oxidative degradation of Cur. Unlike Cur, the fluorescence intensity within the CNP-treated cells did not change significantly for 3 h and became slightly weaker after 6 h. It should be noted that the cells treated with CNPs for 6 h showed a brighter fluorescence than those treated with Cur (Figure S3). These results show that CNPs enable to deliver Cur continuously to the cells over several hours and improve its chemical stability under physiological conditions. Furthermore, similar results were obtained in human umbilical vein endothelial cells (HUVECs) as shown in Figure S4.


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Figure 6. Cellular uptake of Cur and CNPs. HT29 cells were cultured in the presence of Cur and CNP (Cur concentration: 27 μM). At the indicated time points, cells were observed by CLSFM. Upper left: Hoechst 33342 (nuclei), Upper right: Cur, Lower left: DIC, Lower right: Merge (Hoechst 33342 and Cur). Scale bars: 40 μm. Antiangiogenic activity of CNPs. It has been shown that Cur exerts antiangiogenic activity in HUVECs by modulating vascular endothelial growth factor and the R2 receptor (VEGF-VEGFR2) pathway35 and by reducing the expression of metalloproteases during endothelial cell morphogenesis.36 As angiogenesis is crucial for tumor growth and metastasis, Cur has attracted attention as an angiogenesis inhibitor, which can be beneficial in cancer therapy, especially for treating rapidly growing metastatic cancer. Here, we evaluated the inhibitory effects of CNPs on angiogenesis. We first assessed the anti-tubulogenic activity of CNPs in the Matrigel tube formation assay. As shown in Figure 7a, development of complex mesh-like network structures was inhibited after treatment with Cur and CNPs. The obtained images were further analyzed to quantify the tube length, the number of loops, the number of branching points and the total capillary tube area. Both Cur and CNPs led to a significant decrease in these four quantities (Figure S5). While there was


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no obvious difference between Cur and CNPs in terms of capillary tube length and the capillary tube area, CNPs showed a significantly lower number of loops compared to Cur. Although less pronounced, this trend was also observed for the number of branching points. The effect of Cur and CNPs on endothelial cell migration was evaluated in the in vitro gap closure migration assay. In this assay, a confluent HUVECs monolayer with an artificial cell free gap (500 μm width) was created in a 24 well plate using the ibidi Culture Insert 2 Well. After removing the culture insert, cell migration to the gap was observed and the images were analyzed to determine the percentage of the gap closure area and the rate of cell migration. As shown in Figure 7b and c, both Cur and CNPs exerted an inhibitory effect on the migration of HUVECs into the cell free gap by significantly reducing the migration speed compared to non-treated cells. To verify that the observed effects are not due to the cytotoxicity of Cur and CNPs, we measured cell viability of HUVECs after culturing for 3 d in the presence of Cur and CNPs by MTT assay (Figure S6). Since CNPs did not show obvious toxicity in the concentration range tested in this experiment (up to 30 μM), it is unlikely that the antiangiogenic effects observed for CNPs was due to its toxicity. On the other hand, cell viability was significantly reduced after treatment with high concentrations of Cur for 3 d (LC50 of 5.0 μM). Therefore, the toxicity of Cur may partially contribute to the observed antiangiogenic effects. To further evaluate the antiangiogenic activity of Cur and CNPs, we used the in ovo chicken chorioallantoic membrane (CAM) assay of angiogenesis, where the term in ovo refers to in the egg.37 The CAM is a vascular membrane that forms around day 4 to 5 of embryonic development and covers the chicken embryo. During embryo development, the CAM capillary endothelium develops into a dense network of arteries and veins in the mesodermal layer. From this blood vessel network, the capillary plexus is formed that serves as the respiratory organ until the time of


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hatching.38, 39 Due to its extensive vascularization, CAM has been used to study the efficacy and mechanism of action of antiangiogenic molecules.40 We placed a Matrigel containing Cur or CNPs on the CAM on embryonic day 9. On day 11, the CAM was fixed and stained with rhodamine-labeled lens culinaris agglutinin (LCA), which mainly binds -linked mannose residues of polysaccharide structures present on the endothelia of arteries, veins, and capillaries with similar affinity.41 The capillary plexus around the application area was observed by CLSFM. As shown in Figure 7c, the non-treated CAMs showed a dense homogeneous mesh-like structure (capillary plexus). Treatment with Cur reduced the capillary density, resulting in the formation of small non-vascularlized areas as indicated by the white arrows. Furthermore, large non-vascularlized areas were observed for the CMAs treated with CNPs showing that the CNPs exerted a stronger antiangiogenic activity than Cur.


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Figure 7. Antiangiogenic activity of CNPs. (a) Matrigel tube formation assay. HUVECs were cultured on growth factor-reduced Matrigel and treated with water (NT), Cur and CNP (Cur concentration: 3 μM) for 6 h, stained with calcein AM and observed by fluorescence microscopy. Scale bars: 200 μm. (b and c) Gap closure migration assay. HUVECs were seeded in the ibidi Culture-Insert 2 Well to create a confluent cell monolayer with an artificial gap. The cell migration was observed under a microscope at the indicated time (Cur concentration: 3 μM). The images were analyzed using MetaMorph software. (b) The percentage of gap closure area in the absence (NT, circle) or presence of Cur (square) or CNPs (triangle) (c) the rate of cell migration in the absence (NT) or presence of Cur and CNPs. n=4. (d) CAM assay. Representative images of the blood capillaries on the CAMs treated with growth factor-reduced Matrigel containing PBS (NT), NPs, Cur and CNPs (Matrigel volume: 35 μL/egg, Cur concentration in Matrigel: 43 μM). The arrows indicate some of the holes found in the capillary plexus. Scale bars: 200 μm. Anticancer activity of CNPs. Apart from the inhibitory effect on angiogenesis, Cur is also known to exert anti-proliferative effects in cancer cells.42 For example, Cur has been reported to induce growth inhibition of human colon cancer (HT29) cells by suppressing the transcription factors NF-κB and Sp1 as well as inducing cell cycle arrest and apoptosis.43 Therefore, in this study, we evaluated the anticancer effects of CNPs in the in vitro cell proliferation assay using HT29 cells as well as in ovo CAM cancer model transplanted with HT29 cells. We first investigated the anti-proliferative effect of CNPs on cell viability of HT29 cells by MTT assay. As shown in Figure 8a, Cur inhibited proliferation of HT29 cells with LC50 of 50 μM after 2 d and. Compared to Cur, CNPs exhibited a modest effect by reducing the cell viability to 65% at Cur concertation of 270 μM. As shown in Figure 8b and c, the treatment with Cur/CNPs for


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longer duration (3 and 5 d) showed lower LC50 values of 12.5 μM for Cur and 125 μM for CNPs. No significant difference was observed between 3 and 5 d of culture. We also performed cell cycle analysis of HT29 cells treated with Cur and the CNPs. As shown in Figure 8d, both Cur and CNPs induce G1 phase arrest in HT29 cells, which is in agreement with the previous reports.44, 45 The anticancer effect of the CNPs was further assessed in the CAM cancer model.46 The Matrigels containing Cur or CNPs were mixed with HT29 cells and placed on top of the CAMs on embryonic day 10. Figure 8e shows representative images of the developed tumor-like structures treated with/without Cur or CNPs observed at embryonic day 16. These tumor-like structures were removed from the CAMs and weighed gravimetrically. Interestingly, while no obvious anticancer effect was observed for the tumor-like structures treated with Cur, CNPs significantly reduced tumor weight as shown in Figure 8f. This difference may be attributed to the high chemical stability as well as the sustained release property of CNPs.

Figure 8. Anti-cancer effect in human colon cancer HT29 cells. (a-c) Anti-proliferative effect of CNPs. HT29 cells were cultured in the presence of Cur (circle), CNP (triangle) and NP (square)


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for (a) 2 d, (b) 3 d and (c) 5 d, and cell viability was measured by MTT assay. n=3. *p

Framboidal Nanoparticles Containing A Curcumin-Phenylboronic Acid

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