International Journal of Pharmaceutics 466 (2014) 307–313
Contents lists available at ScienceDirect
International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
Pharmaceutical nanotechnology
Lipid coated upconverting nanoparticles as NIR remote controlled transducer for simultaneous photodynamic therapy and cell imaging Hanjie Wang a , Chunhong Dong a , Peiqi Zhao b , Sheng Wang a , Zhongyun Liu a , Jin Chang a, * a Institute of Nanobiotechnology, School of Materials Science and Engineering, Tianjin University and Tianjin Key Laboratory of Composites and Functional Materials, Tianjin 300072, PR China b Department of Lymphoma, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center of Cancer, Sino-US Center for Lymphoma and Leukemia, Key Laboratory of Cancer Prevention and Therapy, Tianjin 300060, PR China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 28 January 2014 Received in revised form 27 February 2014 Accepted 17 March 2014 Available online 20 March 2014
The application of photodynamic therapy in deep tissue is constrained by some pending problems, such as the limited penetration depth of excitation light and lacking of targeting ability. In this paper, a new kind of lipid coated upconverting nanoparticles consisiting of upconerting nanocrystal core and targeted lipid polymer shell was first reported for NIR triggered photodynamic therapy and cell imaging simultaneously. The lipid coated upconverting nanoparticles offers advantages to overcome the problem mentioned above. The UCN core works as a transducer to convert deeply penetrating near-infrared light to visible lights for activating photosensitizer and cell fluorescence imaging simultaneously. The amphiphilic lipid polymer RGD peptide conjugated poly (maleic anhydride-alt-1-octadecene) grafted dioleoyl L-a-phosphatidylethanolamine (RGD-PMAO-DOPE) acts as a shield. It can protect the system from catching by RES and target the whole system to the lesions. The experiment results show that the lipid coated upconverting nanoparticle is individual nanosphere with an average size of 20 nm. The drug loading can reach 9%. After NIR exposed, the MC540 was activated to produce singlet oxygen (ROS) successfully by the upconverting fluorescence emitted from UCN. Importantly, compared with nanoparticle without RGD decoration, the lipid coated upconverting nanoparticle can co-deliver the MC540 and UCNs into the same cell with higher efficiency. Besides, the MC540 loaded UCN/RGD-PMAO-DOPE nanoparticles showed significant inhibitory effect on tumor cells after NIR shining. Our data suggests that MC540 loaded UCN/RGD-PMAO-DOPE nanoparticle may be a useful nanoplatform for future PDT treatment in deep-cancer therapy. ã 2014 Elsevier B.V. All rights reserved.
Keywords: Up-converting Nanocrystal Photodynamic therapy PMAO DOPE NIR
1. Introduction Recently, there is an increasing interest in developing lanthanide-doped upconverting nanoparticles (UCNs), which can absorb NIR long-wavelength excitations and convert to short-wavelength emissions (Shen et al., 2013; Wang et al., 2011a,b; 2013a,b,c). Compared to traditional fluorescent probes such as organic dyes and quantum dots (QDs), upconverting fluorescence imaging based on NIR excitation of UCNPs shows some attractive features including the minimum photodamage to living organisms, low autofluorescence, high signal to noise ratio and detection sensitivity, and high penetration depth in biological samples, all
* Corresponding author at: Tianjin University and Tianjin Key Laboratory of Composites and Functional Materials, Institute of Nanobiotechnology, School of Materials Science and Engineering, Tianjin 300072, China. E-mail address: [email protected] (J. Chang). http://dx.doi.org/10.1016/j.ijpharm.2014.03.029 0378-5173/ ã 2014 Elsevier B.V. All rights reserved.
of which make the UCNs particularly useful for sensing, bioimaging light triggered photodynamic therapy (PDT) and so on (Idris et al., 2012; Cheng et al., 2013). However, in order to keep UCNs with good properties, such as uniform particle size, highly crystalline controlled morphology, and so on, organic solvents must be used during the synthesis procedure. Besides, there are no appropriate functional groups (such as carboxyl groups or amino groups) existing on the surface of the UCNs (Wang et al., 2011a,b). So the hydrophobic UCNs can’t be used in the biomedical applications before surface hydrophilic modification. To overcome this problem, further surface modifications are necessary. In the past several decades, different strategies to improve the surface hydrophilic property of UCNs have been developed, such as inorganic shell, organic capping ligands and so on (Wang et al., 2013a,b,c; Yang et al., 2010; Ma et al., 2013; Zhou et al., 2011). Among these strategies, amphiphilic polymer coating technique has become an increasingly exciting field in academic and industrial research and shown significant potential advantages.
308
H. Wang et al. / International Journal of Pharmaceutics 466 (2014) 307–313
For example, the hydrophilic section of the amphiphilic polymer works as a shield to keep the UCNs stable and prevent protein adsorption during the transportation. The hydrophobic portion of the polymer intercalates with the hydrophobic ligands on the NP surface, which avoids the leaking problem; additionally, there are more functional groups on the amphiphilic polymer for connecting with biomolecules, in comparison with the other small molecules (Tan et al., 2012). All of these features can effectively improve the hydrophilicity of UCNs. Poly (maleic anhydride-alt-1-octadecene) (PMAO) is a of the most popular framework polymers for coating nanoparticles (Jiang et al., 2012). It has good biocomatiblity, strong interaction with the oleate chains presenting on the surface of the nanoparticles and funcational groups, anhydride rings for crossing other molecules. Nevertheless, PMAO is not hydrophilic that sometimes nanoparticles will aggregate during surface modification which limits its applications in nanoparticle modification (Janczewski et al., 2011). It is still necessary to graft it with others molecules to improve the amphiphilic property. Herein, in this work, RGD peptide functionalized PMAO grafted amphiphilic lipid, L-a-phosphatidylethanolamine, dioleoyl (RGDPMAO-DOPE) was first reported. The amphiphilic RGD-PMAO-DOPE coated UCN as nanoparticle, was designed and prepared successfully by using the hydrophobic interaction between the alkyl long chain of the RGD-PMAO-DOPE and the oleate ligand on the surface of the UCNPs for photodynamic therapy test. This new kind of amphiphilic lipid polymer has several special properties. It can form a lipid shell, which could interact with hydrophobic UCNs, provide electrostatic repulsion against particle aggregation and prevent the drug leaking during the transportation; the RGD anchored on the surface has targeting capacity for improving the cell uptake efficiency. To evaluate the photodynamic therapy (PDT) effect in vitro, a kind of hydrophobic photosensitizer MC540 was incorporated into nanoparticle. The properties, such as structure, morphology, size distribution, fluorescence spectrum, drug loading ability and singlet oxygen production were evaluated. Further to this, cell uptake efficiency and PDT effect on cell level were evaluated.
NH4F, Chloroform, 9, 10-anthracenediyl-bis (methylene) dimalonic acid (ABDA), dextran merocyanine 540 (MC540), poly (maleic anhydride-alt-1-octadecene) (PMAO) and L-a-phosphatidylethanolamine, dioleoyl (DOPE), were purchased from Sigma–Aldrich. 2.2. Preparation of NaYF4: Yb, Er (78: 20, 2) upconverting nanocrystals Upconverting nanocrystal, (NaYF4: Yb, Er) were synthesized following a previously reported protocol (Liu et al., 2013). YCl36H2O (780 mL, 1 M), YbCl36H2O (20 mL, 1 M), ErCl36H2O and (20 mL, 0.1 M) in deionized water were added to a 100 mL flask containing 15 mL oleic acid and 20 mL 1-octadecene. The solution was stirred at room temperature for 1 h. Then the mixture was slowly heated to 120 C to get rid of water under argon atmosphere and maintained at 160 C for about 1 h until a homogeneous transparent yellow solution was obtained. The system was then cooled down to room temperature. Then methanol solution of NaF (10 mL, 2.5 mM) was added and the solution was maintained at 100 C for 30 min to remove the methanol. Then the solution was heated to 300 C and kept for 1 h and then it was cooled down to room temperature. The upconverting nanoparticles were precipitated by the addition of 20 mL ethanol and purified by centrifugation. After washed 4 times, the final product was dispersed in 20 mL cyclohexane. 2.3. Synthesis of amphiphilic polymeric lipid, PMAO grafted DOPE (PMAO-DOPE) and RGD-PMAO-DOPE The synthesized process is shown in Fig. 1. Firstly, 500 mg of PMAO (Mw = 30,000–50,000 g/mol), 30 mg DOPE and 50 mL THF were added into a three-neck flask. Then the temperature of the whole system was increased to 30 C and stirred vigorously for 24 h. After that THF was evaporated and the final solution was dialyzed by water under alkaline condition for 4 days (MWCO 8000–14,000). Finally, the solution was lyophilized to give PMAODOPE as white powder. The synthesis process of RGD-PMAO-DOPE was the same as the synthesis process of PMAO-DOPE except replacing the 30 mg DOPE with 25 mg DOPE and 5 mg RGD.
2. Materials and methods 2.1. Materials
2.4. Preparation of MC540 loaded UCN/RGD-PMAO-DOPE nanoparticles by reverse phase evaporation method
YCl36H2O (99.99%), YbCl36H2O (99.99%), ErCl36H2O (99.99%), oleic acid (90%, technical grade), octadecene (90%, technical grade),
Targeted nanoparticles were prepared by reverse-phase evaporation (seen in Fig. 2) (Wang et al., 2010a,b). RGD-PMAO-DOPE,
Fig. 1. The synthesis process of PMAO grafted DOPE and RGD functionalized PMAO-g-DOPE.
H. Wang et al. / International Journal of Pharmaceutics 466 (2014) 307–313
309
Fig. 2. The scheme about the preparation of MC540 loaded UCN/RGD-PMAO-DOPE nanoparticles by reverse-phase evaporation method.
hydrophobic upconverting nanoparticles (weight ratio: 1:5, gross mass 30 mg) and different volume of MC540 (1 mg/mL) were dissolved in 2 mL chloroform at room temperature to obtain the organic phase. Then, aqueous phase (4 mL deionized water) was mixed with organic phase under sonication for 120 s at 100 W output. The organic solvents were evaporated on a rotary evaporator to form a gel-like MPL suspension. After centrifugation 3 times for removing the free MC540, the final samples were redispersed into 4 mL deionized water. UCN/PMAO-DOPE nanoparticles was prepared followed the same procedure except replacing the RGD-PMAO-DOPE with PMAO-DOPE. 2.5. Morphology and particle size test The shape and morphology of the NPs investigated were observed via transmission electron microscopy (TEM). TEM observation of the NPs was carried out at an operating voltage of 200 kV with a 2010F in bright-field mode. Dilute suspensions of samples in water were dropped onto a carbon-coated copper grid and then air dried. The effective particle size was determined by zetasizer nano series (Malvern instrument) at room temperature. About 0.2 mL of each sample suspension was diluted with 2.5 mL of water immediately after preparation. 2.6. Chemical structure test FTIR spectra were recorded with KBr pellets on a IRPrestige-21 spectrometer. A spatula full of KBr was added into an agate mortar and grinded to fine powder until crystallites can no longer be. Then a small amount of powder samples (of about 0.1–2% of the KBr amount, or just enough to cover the tip of spatula) was taken to mix with the KBr powder. Subsequently the mixture was grinded for 3– 5 min. After that the collar together with the pellet was put onto the sample holder. 2.7. Drug loading efficiency test The unencapsulated MC540 was collected by centrifugation and the amount was calculated based on the UV absorbance at 540 nm. The fluorescence spectrum was used to detect the emission peak strength change of different samples. The loading efficiency (LE) of the process was calculated from the formats below: LE ¼
M M1 100% MþN
M is the total amount of MC540 added, M1 is the amount of unencapsulated MC540, and N is the total weight of the UCN and polymers added.
2.8. Singlet oxygen production test Generation of singlet oxygen is usually detected by singlet oxygen sensors such as 9, 10-anthracenediyl-bis (methylene) dimalonic acid (ABDA). Here the ABDA method was chosen to monitor the amount of singlet oxygen (Tao et al., 2013; Zhao et al., 2010). In this method, ABDA can react with singlet oxygen to yield an endoperoxide and causes a decrease of ABDA absorption centered at 380 nm. Generally, different samples were mixed with 1 mM ABDA and then irradiated by a 980 nm laser for different periods of time. The generation of singlet oxygen by different samples would result in the bleaching of ABDA absorption at 380 nm. The reduction of optical density at 380 nm thus reflects the production of 1O2. 2.9. Reactive oxygen species (ROS) production and cell uptake in vitro MCF-7 cells were cultured in a confocal dish for 24 h different samples with the same concentration were added to the dish and the cells were incubated at 37 C. Afterward, the cells were washed three times with phosphate buffered solution (PBS). Cell imaging of MCF-7 cells was performed by laser confocal scanning microscope with an external 980 nm NIR laser. ROS generated in cells treated with different samples and then exposed to NIR 980 nm laser detected using the Image-iT LIVE Reactive Oxygen Species. Confocal images show that green fluorescence indicated for ROS; the positions of the cells are indicated by red upconverting fluorescence emitted from UCNs taken up by the cells and blue fluorescence indicative of nuclear counterstaining with DAPI. 2.10. Cancer cell inhibition experiments based on photodynamic therapy After establishing its efficacy as a 1O2 producer, we tested the nanoparticles for their PDT efficiency in inducing cell death. MCF-7 cells were seeded onto 96-well plates and incubated for 24 h. After cell attachment, different samples were added into the wells. After the culture of 24 h, the cells in each well were exposed to 980 nm NIR light. Cell viability was measured by MTT assay. 3. Results and discussion 3.1. The structures and self-assembly properties characterizations of the amphiphilic lipid polymers, PMAO-DOPE and RGD-PMAO-DOPE In order to improve the amphiphilic property of PMAO, DOPE is a common lipid molecule for preparation of liposome, which is similar to 1,2-didodecanoyl-sn-glycero-3-phosphoethanolamine (DLPE)
310
H. Wang et al. / International Journal of Pharmaceutics 466 (2014) 307–313
(Wang et al., 2013a,b,c; 2010a,b). It was grafted on the side chain of PMAO by reaction between the anhydride rings in the PMAO chains and the amino groups in the DOPE. Then targeting RGD peptide was grafted on PMAO-DOPE (seen in Fig. 1). The chemical structure and assembly property of PMAO-DOPE was characterized by FTIR and dynamic light scattering (DLS) (seen in Fig. S1 and Table S1). The FTIR spectrum of RGD-PMAO-DOPE shows some new peaks at 1712.79 cm1 and 1558.48 cm1, representing CQC binding on DOPE and NH binding on RGD, which suggested the RGD-PMAODOPE was prepared successfully. As we known, the polymer with good amphiphilic property is much easier to self-assemble into nanoparticles in water (Weissleder, 2001). In order to test the self-assembly properties, particle size was measured by DLS after different polymers dissolved into water. The DLS data show that comparison with the particle size in pure PMAO group (7000 nm, PDI: 1), the particle size of PMAO-DOPE group becomes very small (269 nm, PDI: 0.393). These results indicated that the pure PMAO is very difficult to form the nanoparticle due to the poor amphiphilic property; with the help of DOPE, the hydrophilic property of PMAO significantly increased and self-assembled into nanoparticles easily. 3.2. Self-assembly of MC540 loaded UCN/RGD-PMAO-DOPE nanoparticles The nanoparticles consisting of UCNs and RGD-PMAO-DOPE were prepared by reverse phase evaporation method (Fig. 2). Briefly amphiphilic polymers, PMAO-DOPE and RGD-PMAO-DOPE were dissolved into 2 mL of water to form the water phase. Hydrophobic UCNs and MC540 were dissolved in 0.5 mL of dichloromethane to form the organic phase. After that oil phase was mixed with water phase to form the emulsion under
sonication. Then, the dichloromethane in the emulsion was evaporated under high vacuum. After removing all the solvent, the nanoparticles were prepared. Near-infrared (NIR) light has the deepest tissue penetration compared to visible and UV light (Nowak-Sliwinska et al., 2006). The nanoparticles consisting of UCN core and targeted lipid polymer shell is designed to transduce NIR into visible light for exciting the MC540. The whole process is shown in Fig. 3. Firstly, the nanoparticles get into the cells with the help of RGD receptormediated; After NIR shining at the lesions, the nanoparticles transfer the NIR light (980 nm) into visible light (540 nm and 650 nm) by upcoverting method. The fluorescence at 540 nm emission, well matching the absorption of MC540, was used to excite MC540 to produce reactive singlet oxygen for PDT and the fluorescence at 650 nm was used as for tracking the nanoparticles in cells, all of which can simultaneously realize imaging and PDT treatment. Finally the cancer cell was killed by these ROS species. The morphology and particle size of these nanoparticles was examined by TEM and DLS (Fig. 3 and Table S2). As seen in Fig. 3B, the UCNs have a good monodispersity with an average diameter of about 20 nm. Amphiphilic PMAO-DOPE and RGD-PMAO-DOPE were used to transfer UCNs from organic solvent into water, respectively. As seen in Fig. 3C and D, these UCNs decorated with polymers still dispersed as individuals with the small particle size, which indicated that the UCNs was transferred into water successfully without changing the size and morphology of the UCNPs. Table S2 shows that the effective hydrodynamic diameter of the UCNs, MC540 loaded nanoparticles without RGD decoration and MC540 loaded nanoparticles with RGD decoration. After coating with polymeric lipids, the size increased from 52.6 nm to 147 nm, indicating the surface modification is successful. The whole systems further increased to 225 nm after loading MC540.
Fig. 3. (A) Scheme of lipid coated unconverting nanoparticles getting into cells for NIR triggered photodynamic therapy and cell imaging simultaneously and (B)–(E) TEM images of pure UCN, UCN/PMAO-DOPE nanoparticles, UCN/RGD-PMAO-DOPE nanoparticles and MC540 loaded UCN/RGD-PMAO-DOPE.
H. Wang et al. / International Journal of Pharmaceutics 466 (2014) 307–313
3.3. MC540 loading efficiency test and singlet oxygen generation (ROS) test Merocyanine 540 (MC540) is a kind of photosensitizer used for PDT in clinic, which can be excited by visible light at 540 nm (Lin et al., 2012). However, MC540 is only suitable for treatment of skin disease or other superficial tissue lesions, because the excitation light source at 540 nm with short wavelength and high energy that not only has a poor tissue penetration depth, but also has a serious damage to the normal tissue, all of which made it unacceptable for clinical use in deep tissue directly. MC540 is hydrophobic substance containing sodium ion, which show very strong contrast under TEM. When MC540 was loaded into the hydrophobic interlayer between UCN core and RGD-PMAO-DOPE shell, it improved the contrast of the polymer shell. As seen in Fig. 3E inserted image, a circle outside the UCNs indicated that the RGDPMAO-DOPE shell absorb onto the surface of the UCNs. In order to confirm whether the energy can be transferred from UCNs to MC540, the fluorescence emission spectra and UV spectra were measured (Fig. 4). As seen in Fig. 4A and C, the emission peak of the UCNs at 540 nm corresponding to green fluorescence overlaps with the absorption peak of MC540, suggesting that the green light emitted from the UCNs upon excitation can be absorbed by MC540. As seen in Fig. 3C, the absorption peak strength of MC540 at 540 nm increased with the increase of MC540 added during the assembly process. As the same time, the green fluorescence at 540 nm of UCNs quenched significantly with the increase of MC540 added, which indicated that the resonance energy transferred from the UCNs to the loaded MC540. Drug loading ability of vectors is an important factor which determines the utilization rate. So it was calculated based on the UV spectra. Along with the increase of MC540 added, the loading efficiency increased and reached 9.3% (w/w) at 80 mL. In order to keep a balance between drug loading and drug utilization, we choose to add 80 mL of MC540 in the next experiment.
311
An important requirement for application of photosensitizers in photodynamic therapy is high singlet oxygen generation efficiency. To assess the capability of singlet oxygen generation of MC540 loaded UCNs/lipid micelle nanoparticles, ABDA was employed as a probe molecule to monitor the singlet oxygen generation. In this method, ABDA can react with the newly generated singlet oxygen to yield an endoperoxide, which causes a decrease of ABDA absorption centered at 380 nm. Same amount of ABDA was added into different samples to monitor the absorbance change within 160 min under the irradiation at 980 nm (Fig. 3D). As seen in Fig. 4D, in pure MC540 group, the absorbance of ABDA at 380 nm nearly no change with the irradiation time increase and the similar phenomenon is found in pure UCN/PMAO-DOPE nanoparticle group, all of which indicated that pure UCNs or MC540 are not able to generate 1O2 under the 980 nm light excitation. Meanwhile at the MC540 loaded UCN/PMAO-DOPE nanoparticles group, the changes of ABDA absorption is obvious, which means that the combination of MC540 and UCN/PMAO-DOPE can generate the singlet oxygen effectively under irradiation at 980 nm. 3.4. Cytotoxicity study The biocompatibility of these materials was studied by MTT assay and fluorescence images (Fig. 5). The PMAO-DOPE group and RGD-PMAO-DOPE group did not cause significant cytotoxicity against the cell line under the concentration of 200 mg/mL, suggesting PMAO-DOPE and RGD-PMAO-DOPE have good biocompatibility. After assembling into UCN/PMAO-DOPE and UCN/RGDPMAO-DOPE, the cell viability still nearly kept the same, which indicated there is nearly no influence on the cell viability after UCNs assembled into the polymeric lipid shell. With increase of the concentration, the cell viability decreased in all these samples, but cell viability is still very high even at 250 mg/mL. Calcein-AM and propidium iodide were used to label to live cells and dead cells
Fig. 4. (A) Upconverting fluorescence emission spectrum, (B) MC540 loading efficiency and (C) UV absorption spectrum of different samples with various MC540 added (D) ROS production test of pure MC540, UCN/PMAO-DOPE nanoparticles and MC540 loaded UCN/PMAO-DOPE nanoparticles based on ABDA method.
312
H. Wang et al. / International Journal of Pharmaceutics 466 (2014) 307–313
Fig. 5. (A) Cell toxicity; live and dead cell imaging of (concentration of samples: 200 mg/mL) (B) PMAO-DOPE group, (C) RGD-PMAO-DOPE group, (D). UCN/PMAO-DOPE nanoparticles group and (D) UCN/RGD-PMAO-DOPE nanoparticles group.
(Fig. 5A). At 200 mg/mL of these four samples, nearly all the cells show green fluorescence emited from calcein-AM, suggesting that the cells are alive. 3.5. Cell uptake and singlet oxygen production of different samples Singlet oxygen (1O2) is a higher energy state molecular oxygen species. It is one of the most active intermediates involved in chemical and biochemical reactions. MC540 can be excited to produce reactive singlet oxygen species which caused detrimental oxidation of biomolecules in cancer cells. To monitor the intracellular uptake and singlet oxygen production, cancer cells were incubated with the different samples for 4 h (Fig. 6). In order to evaluate the ability of the UCN to generate reactive oxygen species (ROS), the dye 9, 10-anthracenediyl-bis (methylene) dimalonic acid as an acceptor of 1O2 was added into the cells. The blue fluorescence, green florescence and red florescence represent the cell nuclear, ROS and upconverting nanoparticles, respectively. Obvious red upconverting fluorescence and green fluorescence of ROS were simultaneously observed in the cancer cells.
The merged images indicate that the red upconverting fluorescence and green fluorescence of ROS co-exist in the cells, mainly appearing in the cytoplasmic regions, all of which indicated that the energy was transferred from UCN to MC540 for producing the ROS successfully. Arginine-glycine-aspartic acid (RGD) are well-known to bind preferentially to the avb3 integrin that is a promising approach for delivering anticancer drugs or contrast agents for cancer therapy and diagnosis. As some reported, expression level of the integrin receptor on MCF-7 cells is overexpressed [18]. Compared with MC540 loaded UCN/PMAO-DOPE without RGD decoration, there is much stronger green fluorescence and red fluorescence in the MC540 loaded UCN/RGDPMAO-DOPE nanoparticle group, which proved that the cell uptake efficiency improved by RGD receptor mediated endocytosis. 3.6. PDT effects test on cancer cells by MTT The main goal of nanoparticles is to prevent cancer cells from progressing by PDT. So PDT treatment effect in vitro by exposing
Fig. 6. Cell uptake and ROS generation test. The positions of the cells are indicated by blue fluorescence indicative of nuclear counterstaining with DAPI (blue color); green fluorescence represents carboxy-H2DCFDA marker for detection of ROS. Red fluorescence represents UCNs.
H. Wang et al. / International Journal of Pharmaceutics 466 (2014) 307–313
313
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ijpharm.2014.03.029. References
Fig. 7. PDT treatment efficiency on MCF-7 cells. Untreated cells as control group, cells treated with UCN/PMAO-DOPE nanoparticle plus NIR laser, cells treated with MC540 loaded UCN/PMAO-DOPE nanoparticles plus NIR laser and cells treated with MC540 loaded UCN/ RGD-PMAO-DOPE nanoparticles plus NIR laser as experiment group.
cells to 980 nm laser for 30 min was measured by MTS assay (Fig. 7). After exposed to 980 nm laser in 30 min, the cell viability is 65% in MC540 loaded UCN/PMAO-DOPE nanoparticle group, which implied that UCNs has increased treatment effect after combining with MC540. More importantly, there was only 35% cell viability in MC540 loaded UCN/RGD-PMAO-DOPE nanoparticle group, which confirmed that targeting modification on the surface increased PDT efficiency greatly. 4. Conclusions In summary, UCN/RGD-PMAO-DOPE nanoparticle, as new photosensitizer carriers for PDT, was prepared successfully. It has been demonstrated that these nanoparticles have core-shell structure with small particle size and narrow size distribution. Hydrophobic MC540 was loaded into these nanoparticles through hydrophobic interactions. The cell uptake results suggested that the nanoparticles transported the MC540 into cells and the NIR energy was transferred from UCNs to MC540 for generating cytotoxic ROS successfully. In PDT treatment effect test, the MC540 loaded UCN/RGD-PMAO-DOPE nanoparticle has much better treatment effect compared with the nanoparticles without RGD decoration. In conclusion, the UCN/RGD-PMAO-DOPE nanoparticles may be suitable as a potential drug delivery system for PDT. Acknowledgments The authors gratefully acknowledge National High Technology Program of China (863 Program) (2012AA022603), Nature Science Foundation of China (51303126, 51373117, 81171372), Key Project of Tianjin Applied Basic Research Program (13JCZDJC33200) and Doctoral Base Foundation of Educational Ministry of China (20120032110027).
Cheng, L., Wang, C., Liu, Z., 2013. Upconversion nanoparticles and their composite nanostructures for biomedical imaging and cancer therapy. Nanoscale 5, 23–37. Idris, N.M., Gnanasammandhan, M.K., Zhang, J., Ho, P.C., Mahendran, R., Zhang, Y., 2012. In vivo photodynamic therapy using upconversion nanoparticles as remote-controlled nanotransducers. Nature Medicine 18, 1580–U190. Janczewski, D., Tomczak, N., Han, M.Y., Vancso, G.J., 2011. Synthesis of functionalized amphiphilic polymers for coating quantum dots. Nature Protocols 6, 1546–1553. Liu, J.N., Bu, W.B., Pan, L.M., Shi, J.L., 2013. NIR-triggered anticancer drug delivery by upconverting nanoparticles with integrated azobenzene-modified mesoporous silica. Angewandte Chemie-International Edition 52, 4375–4379. Jiang, G.C., Pichaandi, J., Johnson, N.J.J., Burke, R.D., van Veggel, F., 2012. An effective polymer cross-linking strategy to obtain stable dispersions of upconverting NaYF4 nanoparticles in buffers and biological growth media for biolabeling applications. Langmuir 28, 3239–3247. Lin, R.Y., Dayananda, K., Chen, T.J., Chen, C.Y., Liu, G.C., Lin, K.L., Wang, Y.M., 2012. Targeted RGD nanoparticles for highly sensitive in vivo integrin receptor imaging. Contrast Media Molecular Imaging 7, 7–18. Ma, P.a., Xiao, H., Li, X., Li, C., Dai, Y., Cheng, Z., Jing, X., Lin, J., 2013. Rational design of multifunctional upconversion nanocrystals/polymer nanocomposites for cisplatin (IV) delivery and biomedical imaging. Advanced Materials 25, 4898– 4905. Nowak-Sliwinska, P., Karocki, A., Elas, M., Pawlak, A., Stochel, G., Urbanska, K., 2006. Verteporfin, photofrin II, and merocyanine 540 as PDT photosensitizers against melanoma cells. Biochemical and Biophysical Research Communications 349, 549–555. Shen, J., Zhao, L., Han, G., 2013. Lanthanide-doped upconverting luminescent nanoparticle platforms for optical imaging-guided drug delivery and therapy. Advanced Drug Delivery Reviews 65, 744–755. Tan, L., Wan, A., Li, H., Lu, Q., 2012. Novel quantum dots-carboxymethyl chitosan nanocomposite nitric oxide donors capable of detecting release of nitric oxide in situ. Acta Biomaterialia 8, 3744–3753. Tao, X., Yang, Y.-J., Liu, S., Zheng, Y.-Z., Fu, J., Chen, J.-F., 2013. Poly(amidoamine) dendrimer-grafted porous hollow silica nanoparticles for enhanced intracellular photodynamic therapy. Acta Biomaterialia 9, 6431–6438. Wang, H.J., Zhang, S.N., Liao, Z.Y., Wang, C.Y., Liu, Y., Feng, S.Q., Jiang, X.G., Chang, J., 2010a. PEGlated magnetic polymeric liposome anchored with TAT for delivery of drugs across the blood-spinal cord barrier. Biomaterials 31, 6589–6596. Wang, H.J., Zhao, P.Q., Liang, X.F., Gong, X.Q., Song, T., Niu, R.F., Chang, J., 2010b. Folate-PEG coated cationic modified chitosan – cholesterol liposomes for tumor-targeted drug delivery. Biomaterials 31, 4129–4138. Wang, H.-W., Jiang, P.-L., Lin, S.-F., Lin, H.-J., Ou, K.-L., Deng, W.-P., Lee, L.-W., Huang, Y.-Y., Liang, P.-H., Liu, D.-Z., 2013a. Application of galactose-modified liposomes as a potent antigen presenting cell targeted carrier for intranasal immunization. Acta Biomaterialia 9, 5681–5688. Wang, M., Abbineni, G., Clevenger, A., Mao, C., Xu, S., 2011a. Upconversion nanoparticles: synthesis, surface modification and biological applications. Nanomedicine: Nanotechnology, Biology and Medicine 7, 710–729. Wang, C., Cheng, L., Liu, Z., 2013c. Upconversion nanoparticles for photodynamic therapy and other cancer therapeutics. Theranostics 3, 317–330. Wang, C., Tao, H., Cheng, L., Liu, Z., 2011b. Near-infrared light induced in vivo photodynamic therapy of cancer based on upconversion nanoparticles. Biomaterials 32, 6145–6154. Wang, C., Cheng, L., Liu, Y., Wang, X., Ma, X., Deng, Z., Li, Y., Liu, Z., 2013b. Imagingguided pH-sensitive photodynamic therapy using charge reversible upconversion nanoparticles under near-infrared light. Advanced Functional Materials 23, 3077–3086. Weissleder, R., 2001. A clearer vision for in vivo imaging. Nature Biotechnology 19, 316–317. Yang, J., Deng, Y., Wu, Q., Zhou, J., Bao, H., Li, Q., Zhang, F., Li, F., Tu, B., Zhao, D., 2010. Mesoporous silica encapsulating upconversion luminescence rare-earth fluoride nanorods for secondary excitation. Langmuir 26, 8850–8856. Zhao, T.T., Wu, H., Yao, S.Q., Xu, Q.H., Xu, G.Q., 2010. Nanocomposites containing gold nanorods and porphyrin-doped mesoporous silica with dual capability of twophoton imaging and photosensitization. Langmuir 26, 14937–14942. Zhou, J., Yu, M., Sun, Y., Zhang, X., Zhu, X., Wu, Z., Wu, D., Li, F., 2011. Fluorine-18labeled Gd3+/Yb3+/Er3+ co-doped NaYF4 nanophosphors for multimodality PET/MR/UCL imaging. Biomaterials 32, 1148–1156.
Contents lists available at ScienceDirect
International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
Pharmaceutical nanotechnology
Lipid coated upconverting nanoparticles as NIR remote controlled transducer for simultaneous photodynamic therapy and cell imaging Hanjie Wang a , Chunhong Dong a , Peiqi Zhao b , Sheng Wang a , Zhongyun Liu a , Jin Chang a, * a Institute of Nanobiotechnology, School of Materials Science and Engineering, Tianjin University and Tianjin Key Laboratory of Composites and Functional Materials, Tianjin 300072, PR China b Department of Lymphoma, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center of Cancer, Sino-US Center for Lymphoma and Leukemia, Key Laboratory of Cancer Prevention and Therapy, Tianjin 300060, PR China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 28 January 2014 Received in revised form 27 February 2014 Accepted 17 March 2014 Available online 20 March 2014
The application of photodynamic therapy in deep tissue is constrained by some pending problems, such as the limited penetration depth of excitation light and lacking of targeting ability. In this paper, a new kind of lipid coated upconverting nanoparticles consisiting of upconerting nanocrystal core and targeted lipid polymer shell was first reported for NIR triggered photodynamic therapy and cell imaging simultaneously. The lipid coated upconverting nanoparticles offers advantages to overcome the problem mentioned above. The UCN core works as a transducer to convert deeply penetrating near-infrared light to visible lights for activating photosensitizer and cell fluorescence imaging simultaneously. The amphiphilic lipid polymer RGD peptide conjugated poly (maleic anhydride-alt-1-octadecene) grafted dioleoyl L-a-phosphatidylethanolamine (RGD-PMAO-DOPE) acts as a shield. It can protect the system from catching by RES and target the whole system to the lesions. The experiment results show that the lipid coated upconverting nanoparticle is individual nanosphere with an average size of 20 nm. The drug loading can reach 9%. After NIR exposed, the MC540 was activated to produce singlet oxygen (ROS) successfully by the upconverting fluorescence emitted from UCN. Importantly, compared with nanoparticle without RGD decoration, the lipid coated upconverting nanoparticle can co-deliver the MC540 and UCNs into the same cell with higher efficiency. Besides, the MC540 loaded UCN/RGD-PMAO-DOPE nanoparticles showed significant inhibitory effect on tumor cells after NIR shining. Our data suggests that MC540 loaded UCN/RGD-PMAO-DOPE nanoparticle may be a useful nanoplatform for future PDT treatment in deep-cancer therapy. ã 2014 Elsevier B.V. All rights reserved.
Keywords: Up-converting Nanocrystal Photodynamic therapy PMAO DOPE NIR
1. Introduction Recently, there is an increasing interest in developing lanthanide-doped upconverting nanoparticles (UCNs), which can absorb NIR long-wavelength excitations and convert to short-wavelength emissions (Shen et al., 2013; Wang et al., 2011a,b; 2013a,b,c). Compared to traditional fluorescent probes such as organic dyes and quantum dots (QDs), upconverting fluorescence imaging based on NIR excitation of UCNPs shows some attractive features including the minimum photodamage to living organisms, low autofluorescence, high signal to noise ratio and detection sensitivity, and high penetration depth in biological samples, all
* Corresponding author at: Tianjin University and Tianjin Key Laboratory of Composites and Functional Materials, Institute of Nanobiotechnology, School of Materials Science and Engineering, Tianjin 300072, China. E-mail address: [email protected] (J. Chang). http://dx.doi.org/10.1016/j.ijpharm.2014.03.029 0378-5173/ ã 2014 Elsevier B.V. All rights reserved.
of which make the UCNs particularly useful for sensing, bioimaging light triggered photodynamic therapy (PDT) and so on (Idris et al., 2012; Cheng et al., 2013). However, in order to keep UCNs with good properties, such as uniform particle size, highly crystalline controlled morphology, and so on, organic solvents must be used during the synthesis procedure. Besides, there are no appropriate functional groups (such as carboxyl groups or amino groups) existing on the surface of the UCNs (Wang et al., 2011a,b). So the hydrophobic UCNs can’t be used in the biomedical applications before surface hydrophilic modification. To overcome this problem, further surface modifications are necessary. In the past several decades, different strategies to improve the surface hydrophilic property of UCNs have been developed, such as inorganic shell, organic capping ligands and so on (Wang et al., 2013a,b,c; Yang et al., 2010; Ma et al., 2013; Zhou et al., 2011). Among these strategies, amphiphilic polymer coating technique has become an increasingly exciting field in academic and industrial research and shown significant potential advantages.
308
H. Wang et al. / International Journal of Pharmaceutics 466 (2014) 307–313
For example, the hydrophilic section of the amphiphilic polymer works as a shield to keep the UCNs stable and prevent protein adsorption during the transportation. The hydrophobic portion of the polymer intercalates with the hydrophobic ligands on the NP surface, which avoids the leaking problem; additionally, there are more functional groups on the amphiphilic polymer for connecting with biomolecules, in comparison with the other small molecules (Tan et al., 2012). All of these features can effectively improve the hydrophilicity of UCNs. Poly (maleic anhydride-alt-1-octadecene) (PMAO) is a of the most popular framework polymers for coating nanoparticles (Jiang et al., 2012). It has good biocomatiblity, strong interaction with the oleate chains presenting on the surface of the nanoparticles and funcational groups, anhydride rings for crossing other molecules. Nevertheless, PMAO is not hydrophilic that sometimes nanoparticles will aggregate during surface modification which limits its applications in nanoparticle modification (Janczewski et al., 2011). It is still necessary to graft it with others molecules to improve the amphiphilic property. Herein, in this work, RGD peptide functionalized PMAO grafted amphiphilic lipid, L-a-phosphatidylethanolamine, dioleoyl (RGDPMAO-DOPE) was first reported. The amphiphilic RGD-PMAO-DOPE coated UCN as nanoparticle, was designed and prepared successfully by using the hydrophobic interaction between the alkyl long chain of the RGD-PMAO-DOPE and the oleate ligand on the surface of the UCNPs for photodynamic therapy test. This new kind of amphiphilic lipid polymer has several special properties. It can form a lipid shell, which could interact with hydrophobic UCNs, provide electrostatic repulsion against particle aggregation and prevent the drug leaking during the transportation; the RGD anchored on the surface has targeting capacity for improving the cell uptake efficiency. To evaluate the photodynamic therapy (PDT) effect in vitro, a kind of hydrophobic photosensitizer MC540 was incorporated into nanoparticle. The properties, such as structure, morphology, size distribution, fluorescence spectrum, drug loading ability and singlet oxygen production were evaluated. Further to this, cell uptake efficiency and PDT effect on cell level were evaluated.
NH4F, Chloroform, 9, 10-anthracenediyl-bis (methylene) dimalonic acid (ABDA), dextran merocyanine 540 (MC540), poly (maleic anhydride-alt-1-octadecene) (PMAO) and L-a-phosphatidylethanolamine, dioleoyl (DOPE), were purchased from Sigma–Aldrich. 2.2. Preparation of NaYF4: Yb, Er (78: 20, 2) upconverting nanocrystals Upconverting nanocrystal, (NaYF4: Yb, Er) were synthesized following a previously reported protocol (Liu et al., 2013). YCl36H2O (780 mL, 1 M), YbCl36H2O (20 mL, 1 M), ErCl36H2O and (20 mL, 0.1 M) in deionized water were added to a 100 mL flask containing 15 mL oleic acid and 20 mL 1-octadecene. The solution was stirred at room temperature for 1 h. Then the mixture was slowly heated to 120 C to get rid of water under argon atmosphere and maintained at 160 C for about 1 h until a homogeneous transparent yellow solution was obtained. The system was then cooled down to room temperature. Then methanol solution of NaF (10 mL, 2.5 mM) was added and the solution was maintained at 100 C for 30 min to remove the methanol. Then the solution was heated to 300 C and kept for 1 h and then it was cooled down to room temperature. The upconverting nanoparticles were precipitated by the addition of 20 mL ethanol and purified by centrifugation. After washed 4 times, the final product was dispersed in 20 mL cyclohexane. 2.3. Synthesis of amphiphilic polymeric lipid, PMAO grafted DOPE (PMAO-DOPE) and RGD-PMAO-DOPE The synthesized process is shown in Fig. 1. Firstly, 500 mg of PMAO (Mw = 30,000–50,000 g/mol), 30 mg DOPE and 50 mL THF were added into a three-neck flask. Then the temperature of the whole system was increased to 30 C and stirred vigorously for 24 h. After that THF was evaporated and the final solution was dialyzed by water under alkaline condition for 4 days (MWCO 8000–14,000). Finally, the solution was lyophilized to give PMAODOPE as white powder. The synthesis process of RGD-PMAO-DOPE was the same as the synthesis process of PMAO-DOPE except replacing the 30 mg DOPE with 25 mg DOPE and 5 mg RGD.
2. Materials and methods 2.1. Materials
2.4. Preparation of MC540 loaded UCN/RGD-PMAO-DOPE nanoparticles by reverse phase evaporation method
YCl36H2O (99.99%), YbCl36H2O (99.99%), ErCl36H2O (99.99%), oleic acid (90%, technical grade), octadecene (90%, technical grade),
Targeted nanoparticles were prepared by reverse-phase evaporation (seen in Fig. 2) (Wang et al., 2010a,b). RGD-PMAO-DOPE,
Fig. 1. The synthesis process of PMAO grafted DOPE and RGD functionalized PMAO-g-DOPE.
H. Wang et al. / International Journal of Pharmaceutics 466 (2014) 307–313
309
Fig. 2. The scheme about the preparation of MC540 loaded UCN/RGD-PMAO-DOPE nanoparticles by reverse-phase evaporation method.
hydrophobic upconverting nanoparticles (weight ratio: 1:5, gross mass 30 mg) and different volume of MC540 (1 mg/mL) were dissolved in 2 mL chloroform at room temperature to obtain the organic phase. Then, aqueous phase (4 mL deionized water) was mixed with organic phase under sonication for 120 s at 100 W output. The organic solvents were evaporated on a rotary evaporator to form a gel-like MPL suspension. After centrifugation 3 times for removing the free MC540, the final samples were redispersed into 4 mL deionized water. UCN/PMAO-DOPE nanoparticles was prepared followed the same procedure except replacing the RGD-PMAO-DOPE with PMAO-DOPE. 2.5. Morphology and particle size test The shape and morphology of the NPs investigated were observed via transmission electron microscopy (TEM). TEM observation of the NPs was carried out at an operating voltage of 200 kV with a 2010F in bright-field mode. Dilute suspensions of samples in water were dropped onto a carbon-coated copper grid and then air dried. The effective particle size was determined by zetasizer nano series (Malvern instrument) at room temperature. About 0.2 mL of each sample suspension was diluted with 2.5 mL of water immediately after preparation. 2.6. Chemical structure test FTIR spectra were recorded with KBr pellets on a IRPrestige-21 spectrometer. A spatula full of KBr was added into an agate mortar and grinded to fine powder until crystallites can no longer be. Then a small amount of powder samples (of about 0.1–2% of the KBr amount, or just enough to cover the tip of spatula) was taken to mix with the KBr powder. Subsequently the mixture was grinded for 3– 5 min. After that the collar together with the pellet was put onto the sample holder. 2.7. Drug loading efficiency test The unencapsulated MC540 was collected by centrifugation and the amount was calculated based on the UV absorbance at 540 nm. The fluorescence spectrum was used to detect the emission peak strength change of different samples. The loading efficiency (LE) of the process was calculated from the formats below: LE ¼
M M1 100% MþN
M is the total amount of MC540 added, M1 is the amount of unencapsulated MC540, and N is the total weight of the UCN and polymers added.
2.8. Singlet oxygen production test Generation of singlet oxygen is usually detected by singlet oxygen sensors such as 9, 10-anthracenediyl-bis (methylene) dimalonic acid (ABDA). Here the ABDA method was chosen to monitor the amount of singlet oxygen (Tao et al., 2013; Zhao et al., 2010). In this method, ABDA can react with singlet oxygen to yield an endoperoxide and causes a decrease of ABDA absorption centered at 380 nm. Generally, different samples were mixed with 1 mM ABDA and then irradiated by a 980 nm laser for different periods of time. The generation of singlet oxygen by different samples would result in the bleaching of ABDA absorption at 380 nm. The reduction of optical density at 380 nm thus reflects the production of 1O2. 2.9. Reactive oxygen species (ROS) production and cell uptake in vitro MCF-7 cells were cultured in a confocal dish for 24 h different samples with the same concentration were added to the dish and the cells were incubated at 37 C. Afterward, the cells were washed three times with phosphate buffered solution (PBS). Cell imaging of MCF-7 cells was performed by laser confocal scanning microscope with an external 980 nm NIR laser. ROS generated in cells treated with different samples and then exposed to NIR 980 nm laser detected using the Image-iT LIVE Reactive Oxygen Species. Confocal images show that green fluorescence indicated for ROS; the positions of the cells are indicated by red upconverting fluorescence emitted from UCNs taken up by the cells and blue fluorescence indicative of nuclear counterstaining with DAPI. 2.10. Cancer cell inhibition experiments based on photodynamic therapy After establishing its efficacy as a 1O2 producer, we tested the nanoparticles for their PDT efficiency in inducing cell death. MCF-7 cells were seeded onto 96-well plates and incubated for 24 h. After cell attachment, different samples were added into the wells. After the culture of 24 h, the cells in each well were exposed to 980 nm NIR light. Cell viability was measured by MTT assay. 3. Results and discussion 3.1. The structures and self-assembly properties characterizations of the amphiphilic lipid polymers, PMAO-DOPE and RGD-PMAO-DOPE In order to improve the amphiphilic property of PMAO, DOPE is a common lipid molecule for preparation of liposome, which is similar to 1,2-didodecanoyl-sn-glycero-3-phosphoethanolamine (DLPE)
310
H. Wang et al. / International Journal of Pharmaceutics 466 (2014) 307–313
(Wang et al., 2013a,b,c; 2010a,b). It was grafted on the side chain of PMAO by reaction between the anhydride rings in the PMAO chains and the amino groups in the DOPE. Then targeting RGD peptide was grafted on PMAO-DOPE (seen in Fig. 1). The chemical structure and assembly property of PMAO-DOPE was characterized by FTIR and dynamic light scattering (DLS) (seen in Fig. S1 and Table S1). The FTIR spectrum of RGD-PMAO-DOPE shows some new peaks at 1712.79 cm1 and 1558.48 cm1, representing CQC binding on DOPE and NH binding on RGD, which suggested the RGD-PMAODOPE was prepared successfully. As we known, the polymer with good amphiphilic property is much easier to self-assemble into nanoparticles in water (Weissleder, 2001). In order to test the self-assembly properties, particle size was measured by DLS after different polymers dissolved into water. The DLS data show that comparison with the particle size in pure PMAO group (7000 nm, PDI: 1), the particle size of PMAO-DOPE group becomes very small (269 nm, PDI: 0.393). These results indicated that the pure PMAO is very difficult to form the nanoparticle due to the poor amphiphilic property; with the help of DOPE, the hydrophilic property of PMAO significantly increased and self-assembled into nanoparticles easily. 3.2. Self-assembly of MC540 loaded UCN/RGD-PMAO-DOPE nanoparticles The nanoparticles consisting of UCNs and RGD-PMAO-DOPE were prepared by reverse phase evaporation method (Fig. 2). Briefly amphiphilic polymers, PMAO-DOPE and RGD-PMAO-DOPE were dissolved into 2 mL of water to form the water phase. Hydrophobic UCNs and MC540 were dissolved in 0.5 mL of dichloromethane to form the organic phase. After that oil phase was mixed with water phase to form the emulsion under
sonication. Then, the dichloromethane in the emulsion was evaporated under high vacuum. After removing all the solvent, the nanoparticles were prepared. Near-infrared (NIR) light has the deepest tissue penetration compared to visible and UV light (Nowak-Sliwinska et al., 2006). The nanoparticles consisting of UCN core and targeted lipid polymer shell is designed to transduce NIR into visible light for exciting the MC540. The whole process is shown in Fig. 3. Firstly, the nanoparticles get into the cells with the help of RGD receptormediated; After NIR shining at the lesions, the nanoparticles transfer the NIR light (980 nm) into visible light (540 nm and 650 nm) by upcoverting method. The fluorescence at 540 nm emission, well matching the absorption of MC540, was used to excite MC540 to produce reactive singlet oxygen for PDT and the fluorescence at 650 nm was used as for tracking the nanoparticles in cells, all of which can simultaneously realize imaging and PDT treatment. Finally the cancer cell was killed by these ROS species. The morphology and particle size of these nanoparticles was examined by TEM and DLS (Fig. 3 and Table S2). As seen in Fig. 3B, the UCNs have a good monodispersity with an average diameter of about 20 nm. Amphiphilic PMAO-DOPE and RGD-PMAO-DOPE were used to transfer UCNs from organic solvent into water, respectively. As seen in Fig. 3C and D, these UCNs decorated with polymers still dispersed as individuals with the small particle size, which indicated that the UCNs was transferred into water successfully without changing the size and morphology of the UCNPs. Table S2 shows that the effective hydrodynamic diameter of the UCNs, MC540 loaded nanoparticles without RGD decoration and MC540 loaded nanoparticles with RGD decoration. After coating with polymeric lipids, the size increased from 52.6 nm to 147 nm, indicating the surface modification is successful. The whole systems further increased to 225 nm after loading MC540.
Fig. 3. (A) Scheme of lipid coated unconverting nanoparticles getting into cells for NIR triggered photodynamic therapy and cell imaging simultaneously and (B)–(E) TEM images of pure UCN, UCN/PMAO-DOPE nanoparticles, UCN/RGD-PMAO-DOPE nanoparticles and MC540 loaded UCN/RGD-PMAO-DOPE.
H. Wang et al. / International Journal of Pharmaceutics 466 (2014) 307–313
3.3. MC540 loading efficiency test and singlet oxygen generation (ROS) test Merocyanine 540 (MC540) is a kind of photosensitizer used for PDT in clinic, which can be excited by visible light at 540 nm (Lin et al., 2012). However, MC540 is only suitable for treatment of skin disease or other superficial tissue lesions, because the excitation light source at 540 nm with short wavelength and high energy that not only has a poor tissue penetration depth, but also has a serious damage to the normal tissue, all of which made it unacceptable for clinical use in deep tissue directly. MC540 is hydrophobic substance containing sodium ion, which show very strong contrast under TEM. When MC540 was loaded into the hydrophobic interlayer between UCN core and RGD-PMAO-DOPE shell, it improved the contrast of the polymer shell. As seen in Fig. 3E inserted image, a circle outside the UCNs indicated that the RGDPMAO-DOPE shell absorb onto the surface of the UCNs. In order to confirm whether the energy can be transferred from UCNs to MC540, the fluorescence emission spectra and UV spectra were measured (Fig. 4). As seen in Fig. 4A and C, the emission peak of the UCNs at 540 nm corresponding to green fluorescence overlaps with the absorption peak of MC540, suggesting that the green light emitted from the UCNs upon excitation can be absorbed by MC540. As seen in Fig. 3C, the absorption peak strength of MC540 at 540 nm increased with the increase of MC540 added during the assembly process. As the same time, the green fluorescence at 540 nm of UCNs quenched significantly with the increase of MC540 added, which indicated that the resonance energy transferred from the UCNs to the loaded MC540. Drug loading ability of vectors is an important factor which determines the utilization rate. So it was calculated based on the UV spectra. Along with the increase of MC540 added, the loading efficiency increased and reached 9.3% (w/w) at 80 mL. In order to keep a balance between drug loading and drug utilization, we choose to add 80 mL of MC540 in the next experiment.
311
An important requirement for application of photosensitizers in photodynamic therapy is high singlet oxygen generation efficiency. To assess the capability of singlet oxygen generation of MC540 loaded UCNs/lipid micelle nanoparticles, ABDA was employed as a probe molecule to monitor the singlet oxygen generation. In this method, ABDA can react with the newly generated singlet oxygen to yield an endoperoxide, which causes a decrease of ABDA absorption centered at 380 nm. Same amount of ABDA was added into different samples to monitor the absorbance change within 160 min under the irradiation at 980 nm (Fig. 3D). As seen in Fig. 4D, in pure MC540 group, the absorbance of ABDA at 380 nm nearly no change with the irradiation time increase and the similar phenomenon is found in pure UCN/PMAO-DOPE nanoparticle group, all of which indicated that pure UCNs or MC540 are not able to generate 1O2 under the 980 nm light excitation. Meanwhile at the MC540 loaded UCN/PMAO-DOPE nanoparticles group, the changes of ABDA absorption is obvious, which means that the combination of MC540 and UCN/PMAO-DOPE can generate the singlet oxygen effectively under irradiation at 980 nm. 3.4. Cytotoxicity study The biocompatibility of these materials was studied by MTT assay and fluorescence images (Fig. 5). The PMAO-DOPE group and RGD-PMAO-DOPE group did not cause significant cytotoxicity against the cell line under the concentration of 200 mg/mL, suggesting PMAO-DOPE and RGD-PMAO-DOPE have good biocompatibility. After assembling into UCN/PMAO-DOPE and UCN/RGDPMAO-DOPE, the cell viability still nearly kept the same, which indicated there is nearly no influence on the cell viability after UCNs assembled into the polymeric lipid shell. With increase of the concentration, the cell viability decreased in all these samples, but cell viability is still very high even at 250 mg/mL. Calcein-AM and propidium iodide were used to label to live cells and dead cells
Fig. 4. (A) Upconverting fluorescence emission spectrum, (B) MC540 loading efficiency and (C) UV absorption spectrum of different samples with various MC540 added (D) ROS production test of pure MC540, UCN/PMAO-DOPE nanoparticles and MC540 loaded UCN/PMAO-DOPE nanoparticles based on ABDA method.
312
H. Wang et al. / International Journal of Pharmaceutics 466 (2014) 307–313
Fig. 5. (A) Cell toxicity; live and dead cell imaging of (concentration of samples: 200 mg/mL) (B) PMAO-DOPE group, (C) RGD-PMAO-DOPE group, (D). UCN/PMAO-DOPE nanoparticles group and (D) UCN/RGD-PMAO-DOPE nanoparticles group.
(Fig. 5A). At 200 mg/mL of these four samples, nearly all the cells show green fluorescence emited from calcein-AM, suggesting that the cells are alive. 3.5. Cell uptake and singlet oxygen production of different samples Singlet oxygen (1O2) is a higher energy state molecular oxygen species. It is one of the most active intermediates involved in chemical and biochemical reactions. MC540 can be excited to produce reactive singlet oxygen species which caused detrimental oxidation of biomolecules in cancer cells. To monitor the intracellular uptake and singlet oxygen production, cancer cells were incubated with the different samples for 4 h (Fig. 6). In order to evaluate the ability of the UCN to generate reactive oxygen species (ROS), the dye 9, 10-anthracenediyl-bis (methylene) dimalonic acid as an acceptor of 1O2 was added into the cells. The blue fluorescence, green florescence and red florescence represent the cell nuclear, ROS and upconverting nanoparticles, respectively. Obvious red upconverting fluorescence and green fluorescence of ROS were simultaneously observed in the cancer cells.
The merged images indicate that the red upconverting fluorescence and green fluorescence of ROS co-exist in the cells, mainly appearing in the cytoplasmic regions, all of which indicated that the energy was transferred from UCN to MC540 for producing the ROS successfully. Arginine-glycine-aspartic acid (RGD) are well-known to bind preferentially to the avb3 integrin that is a promising approach for delivering anticancer drugs or contrast agents for cancer therapy and diagnosis. As some reported, expression level of the integrin receptor on MCF-7 cells is overexpressed [18]. Compared with MC540 loaded UCN/PMAO-DOPE without RGD decoration, there is much stronger green fluorescence and red fluorescence in the MC540 loaded UCN/RGDPMAO-DOPE nanoparticle group, which proved that the cell uptake efficiency improved by RGD receptor mediated endocytosis. 3.6. PDT effects test on cancer cells by MTT The main goal of nanoparticles is to prevent cancer cells from progressing by PDT. So PDT treatment effect in vitro by exposing
Fig. 6. Cell uptake and ROS generation test. The positions of the cells are indicated by blue fluorescence indicative of nuclear counterstaining with DAPI (blue color); green fluorescence represents carboxy-H2DCFDA marker for detection of ROS. Red fluorescence represents UCNs.
H. Wang et al. / International Journal of Pharmaceutics 466 (2014) 307–313
313
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ijpharm.2014.03.029. References
Fig. 7. PDT treatment efficiency on MCF-7 cells. Untreated cells as control group, cells treated with UCN/PMAO-DOPE nanoparticle plus NIR laser, cells treated with MC540 loaded UCN/PMAO-DOPE nanoparticles plus NIR laser and cells treated with MC540 loaded UCN/ RGD-PMAO-DOPE nanoparticles plus NIR laser as experiment group.
cells to 980 nm laser for 30 min was measured by MTS assay (Fig. 7). After exposed to 980 nm laser in 30 min, the cell viability is 65% in MC540 loaded UCN/PMAO-DOPE nanoparticle group, which implied that UCNs has increased treatment effect after combining with MC540. More importantly, there was only 35% cell viability in MC540 loaded UCN/RGD-PMAO-DOPE nanoparticle group, which confirmed that targeting modification on the surface increased PDT efficiency greatly. 4. Conclusions In summary, UCN/RGD-PMAO-DOPE nanoparticle, as new photosensitizer carriers for PDT, was prepared successfully. It has been demonstrated that these nanoparticles have core-shell structure with small particle size and narrow size distribution. Hydrophobic MC540 was loaded into these nanoparticles through hydrophobic interactions. The cell uptake results suggested that the nanoparticles transported the MC540 into cells and the NIR energy was transferred from UCNs to MC540 for generating cytotoxic ROS successfully. In PDT treatment effect test, the MC540 loaded UCN/RGD-PMAO-DOPE nanoparticle has much better treatment effect compared with the nanoparticles without RGD decoration. In conclusion, the UCN/RGD-PMAO-DOPE nanoparticles may be suitable as a potential drug delivery system for PDT. Acknowledgments The authors gratefully acknowledge National High Technology Program of China (863 Program) (2012AA022603), Nature Science Foundation of China (51303126, 51373117, 81171372), Key Project of Tianjin Applied Basic Research Program (13JCZDJC33200) and Doctoral Base Foundation of Educational Ministry of China (20120032110027).
Cheng, L., Wang, C., Liu, Z., 2013. Upconversion nanoparticles and their composite nanostructures for biomedical imaging and cancer therapy. Nanoscale 5, 23–37. Idris, N.M., Gnanasammandhan, M.K., Zhang, J., Ho, P.C., Mahendran, R., Zhang, Y., 2012. In vivo photodynamic therapy using upconversion nanoparticles as remote-controlled nanotransducers. Nature Medicine 18, 1580–U190. Janczewski, D., Tomczak, N., Han, M.Y., Vancso, G.J., 2011. Synthesis of functionalized amphiphilic polymers for coating quantum dots. Nature Protocols 6, 1546–1553. Liu, J.N., Bu, W.B., Pan, L.M., Shi, J.L., 2013. NIR-triggered anticancer drug delivery by upconverting nanoparticles with integrated azobenzene-modified mesoporous silica. Angewandte Chemie-International Edition 52, 4375–4379. Jiang, G.C., Pichaandi, J., Johnson, N.J.J., Burke, R.D., van Veggel, F., 2012. An effective polymer cross-linking strategy to obtain stable dispersions of upconverting NaYF4 nanoparticles in buffers and biological growth media for biolabeling applications. Langmuir 28, 3239–3247. Lin, R.Y., Dayananda, K., Chen, T.J., Chen, C.Y., Liu, G.C., Lin, K.L., Wang, Y.M., 2012. Targeted RGD nanoparticles for highly sensitive in vivo integrin receptor imaging. Contrast Media Molecular Imaging 7, 7–18. Ma, P.a., Xiao, H., Li, X., Li, C., Dai, Y., Cheng, Z., Jing, X., Lin, J., 2013. Rational design of multifunctional upconversion nanocrystals/polymer nanocomposites for cisplatin (IV) delivery and biomedical imaging. Advanced Materials 25, 4898– 4905. Nowak-Sliwinska, P., Karocki, A., Elas, M., Pawlak, A., Stochel, G., Urbanska, K., 2006. Verteporfin, photofrin II, and merocyanine 540 as PDT photosensitizers against melanoma cells. Biochemical and Biophysical Research Communications 349, 549–555. Shen, J., Zhao, L., Han, G., 2013. Lanthanide-doped upconverting luminescent nanoparticle platforms for optical imaging-guided drug delivery and therapy. Advanced Drug Delivery Reviews 65, 744–755. Tan, L., Wan, A., Li, H., Lu, Q., 2012. Novel quantum dots-carboxymethyl chitosan nanocomposite nitric oxide donors capable of detecting release of nitric oxide in situ. Acta Biomaterialia 8, 3744–3753. Tao, X., Yang, Y.-J., Liu, S., Zheng, Y.-Z., Fu, J., Chen, J.-F., 2013. Poly(amidoamine) dendrimer-grafted porous hollow silica nanoparticles for enhanced intracellular photodynamic therapy. Acta Biomaterialia 9, 6431–6438. Wang, H.J., Zhang, S.N., Liao, Z.Y., Wang, C.Y., Liu, Y., Feng, S.Q., Jiang, X.G., Chang, J., 2010a. PEGlated magnetic polymeric liposome anchored with TAT for delivery of drugs across the blood-spinal cord barrier. Biomaterials 31, 6589–6596. Wang, H.J., Zhao, P.Q., Liang, X.F., Gong, X.Q., Song, T., Niu, R.F., Chang, J., 2010b. Folate-PEG coated cationic modified chitosan – cholesterol liposomes for tumor-targeted drug delivery. Biomaterials 31, 4129–4138. Wang, H.-W., Jiang, P.-L., Lin, S.-F., Lin, H.-J., Ou, K.-L., Deng, W.-P., Lee, L.-W., Huang, Y.-Y., Liang, P.-H., Liu, D.-Z., 2013a. Application of galactose-modified liposomes as a potent antigen presenting cell targeted carrier for intranasal immunization. Acta Biomaterialia 9, 5681–5688. Wang, M., Abbineni, G., Clevenger, A., Mao, C., Xu, S., 2011a. Upconversion nanoparticles: synthesis, surface modification and biological applications. Nanomedicine: Nanotechnology, Biology and Medicine 7, 710–729. Wang, C., Cheng, L., Liu, Z., 2013c. Upconversion nanoparticles for photodynamic therapy and other cancer therapeutics. Theranostics 3, 317–330. Wang, C., Tao, H., Cheng, L., Liu, Z., 2011b. Near-infrared light induced in vivo photodynamic therapy of cancer based on upconversion nanoparticles. Biomaterials 32, 6145–6154. Wang, C., Cheng, L., Liu, Y., Wang, X., Ma, X., Deng, Z., Li, Y., Liu, Z., 2013b. Imagingguided pH-sensitive photodynamic therapy using charge reversible upconversion nanoparticles under near-infrared light. Advanced Functional Materials 23, 3077–3086. Weissleder, R., 2001. A clearer vision for in vivo imaging. Nature Biotechnology 19, 316–317. Yang, J., Deng, Y., Wu, Q., Zhou, J., Bao, H., Li, Q., Zhang, F., Li, F., Tu, B., Zhao, D., 2010. Mesoporous silica encapsulating upconversion luminescence rare-earth fluoride nanorods for secondary excitation. Langmuir 26, 8850–8856. Zhao, T.T., Wu, H., Yao, S.Q., Xu, Q.H., Xu, G.Q., 2010. Nanocomposites containing gold nanorods and porphyrin-doped mesoporous silica with dual capability of twophoton imaging and photosensitization. Langmuir 26, 14937–14942. Zhou, J., Yu, M., Sun, Y., Zhang, X., Zhu, X., Wu, Z., Wu, D., Li, F., 2011. Fluorine-18labeled Gd3+/Yb3+/Er3+ co-doped NaYF4 nanophosphors for multimodality PET/MR/UCL imaging. Biomaterials 32, 1148–1156.
