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Uptake and light-induced cytotoxicity of hyaluronic acid-grafted liposomes containing porphyrin in tumor cells
Journal of Drug Delivery Science and Technology 47 (2018) 137–143
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Uptake and light-induced cytotoxicity of hyaluronic acid-grafted liposomes containing porphyrin in tumor cells
T
Qian Fenga,b, Jing Wangb,d, Hu Songb, Lian-gang Zhuob,c,d, Guanquan Wangb,d, Wei Liaob,d, Yue Fenga,d, Hongyuan Weib,c,d,∗∗, Yue Chena,d,∗∗∗, Yuchuan Yangb,c,d, Xia Yangb,c,d,∗ a
Department of Nuclear Medicine, The Affiliated Hospital Southwest of Medical University, 646000, Luzhou, PR China Institute of Nuclear Physics and Chemistry (INPC), China Academy of Engineering Physics (CAEP), 621999, Mianyang, PR China c Collaborative Innovation Center of Radiation Medicine of Jiangsu, Higher Education Institutions, 215123, Suzhou, PR China d Key Laboratory of Nuclear Medicine and Molecular Imaging of Sichuan Province, 621999, Mianyang, PR China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Porphyrin Liposome “Click” chemistry Hyaluronic acid CD44
Porphyrins are used in clinic as the photosensitizers for cancer treatment. However, most porphyrins tend to form aggregates in aqueous solution due to their hydrophobicity, which results in significantly reduced photocytotoxicities. Therefore, their applications in clinic are limited. To overcome these disadvantages, drugcarriers are designed for the delivery of porphyrins. In this study, we designed the surface modified liposomes for porphyrin delivery. Four types of porphyrins were loaded in liposome separately. We systemically investigated the porphyrin-containing liposomes in terms of their physicochemical properties as well as light-responsive properties. The hyaluronic acid derivative with aldehyde group was used to modify the liposome-porphyrin via the “click” reaction in order to enhance the uptake in CD44 overexpressed cancer cells. The results showed that 10,15,20-tetrakis(4-hydroxyphenyl) porphyrin (p-OH) had highest loading efficiency. The liposomes with p-OH showed highest toxicity to the cells with or without light exposure. The uptake of hyaluronic acid (HA) coated Lip-p-OH was proved to be specific to MDA-MB-231 cells by pretreating the cells with natural HA polymer as the block agent. This study provided an efficient strategy to enhance the cytotoxicity of porphyrin in aqueous solution, and to increase the affinity of porphyrin to the cancer cells.
1. Introduction Porphyrins are photosensitive compounds which can generate reactive oxygen species (ROS) upon light activation to destroy cancer cells. They are widely used in diagnostic imaging and therapy, especially photo-dynamic therapy (PDT) [10]; [26]. PDT is a binary cancer therapy which requires the activation of sensitizer in tissue with light locally. Compared to other cancer treatments (e.g. surgery, radiotherapy, and chemotherapy), the side-effects can be minimized by employing PDT therapeutic methologies [3]. The strategy of using targeted nanomaterials for PDT becomes more and more popular as it can also significantly improve the PDT treatment [5]. Porphyrins can accumulate in cancer cells more than in surrounding healthy tissues [17]. Some porphyrins are partly soluble in aqueous solutions. For example, the FDA approved drug, porfimer sodium, can be dissolved in alkaline condition. However, most porphyrin derivatives used in clinic
or pre-clinic are hydrophobic. The aggregation of porphyrins in aqueous solution could reduce the therapeutic efficacy significantly and limit their applications [4]. To overcome these problems, various nanocarriers, such as polymers, liposomes, micelles, and solid nanoparticles, were developed for porphyrins delivery [6] [16]; [22]; [11]. The photodynamic activity of porphyrins could be enhanced by incorporating them into nanoparticles [12] [23]; [8]. The most intensively investigated nano-carriers for porphyrin delivery are the bilayer structural liposomes composing of phospholipids [13]; [19]. Those liposomes exhibit an enhanced cellular uptake due to their cellular mimic composition and structure. Same as other nanocarriers, liposomes can also accumulate in solid tumors via the retention effect to reach higher delivery efficiency. Additionally, the higher selectivity to tumors is also required during porphyrin delivery in order to lower the side effects of porphyrins. However, liposomes are nonspecific towards tumors and most of them are less stable in vivo. Thus,
∗
Corresponding author. Institute of Nuclear Physics and Chemistry (INPC), China Academy of Engineering Physics (CAEP), 621999, Mianyang, PR China. Corresponding author. Department of Nuclear Medicine, The Affiliated Hospital Southwest of Medical University, 646000, Luzhou, PR China. ∗∗∗ Corresponding author. Department of Nuclear Medicine, The Affiliated Hospital Southwest of Medical University, 646000, Luzhou, PR China. E-mail addresses: [email protected] (H. Wei), [email protected] (Y. Chen), [email protected] (X. Yang). ∗∗
https://doi.org/10.1016/j.jddst.2018.06.024 Received 19 April 2018; Received in revised form 27 June 2018; Accepted 29 June 2018 Available online 05 July 2018 1773-2247/ © 2018 Elsevier B.V. All rights reserved.
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the first study about liposome-porphyrin coated with HA via the “click” reaction. The cytotoxicities of porphyrin-containing liposomes against various cell types were compared with or without light. Meanwhile, the effect of HA coating on the cytotoxicity to breast cancer cells was also investigated. 2. Materials and methods 2.1. Materials 1,2-dipalmitoylsn-glycero-2-phosphocholine (DPPC), 1,2-dioleoylsn-glycero-3[phospho-rac-(1-glycrol)] (DOPG) were purchased from Avanti, TRITC DHPE (rhodamine-lipid), fluorescein-5-thiosemicarbazide (FTSC), N-hydroxybenzotriazole (HOBt) and 1-ethyl-3-[3-dimethylaminopropyl] carbodiamide (EDC) were purchased from SigmaAldrich. Hydrazide-cholesterol (Chol-hy, MW 430.65) and hyaluronic acid-aldehyde-FTSC (HA-al-FTSC) were synthesized according to previous procedures [18]; [24], 5,10,15,20-tetrakis(4-aminophenyl) porphyrins (p-NH2), 5, 10,15,20-tetrakis(4-hydroxyphenyl) porphyrins (pOH), and 5, 10,15,20-tetraphenyl porphyrins (p) were purchased from TCI, 5,10,15,20-tetra(4-pyridyl) porphyrins (p-py) was from J&K. 2.2. Preparation of liposomes with different porphyrin derivatives The molar ratio of DPPC:DOPG:chol-hy was 13:1:6. In detail, 7.338 mg of DPPC, 0.612 mg of DOPG and 2.04 mg of chol-hy were dissolved in 1 ml of chloroform. 1 μg of TRITC DHPE was added to the above chloroform mixture in order to label the liposomes with TRITC dye. Total lipid amount was 10 mg. 1 mg of different porphyrin dissolved in 100 μL acetone was added to the lipid mixture separately. The solvent was evaporated using rotary evaporator during 2.5 h to obtain a thin lipid film. It was then hydrated at 60 °C with 1 mL of 10 mM preheated PBS buffer. After hydration, the suspension was extruded via a liposome extruder at 60 °C. Final liposome concentration was adjusted to 5 mg/ml, named as Lip-p-OH, Lip-p-P, Lip-p-NH2, Lip-p-Py, according to the type of loaded porphyrin. The liposome without porphyrin was prepared as control group and named as Lip. The unloaded porphyrin derivatives were removed by Sephedex G50. For HA-coated liposome-porphyrin, HA-al-FTSC (1 mg/ml) dissolved in PBS (10 mM, pH 7.4) was mixed with Lip-p-OH or Lip-p-NH2 (0.2 mg/ml) in 1:10 volume. This corresponded to the mass ratio of HA to liposome as 1:2, and the molar ratio of aldehyde group on HA to hydrazide group on liposomes as 1:8. The suspensions were mixed for 4 h on shaker. The unbounded HA was removed by centrifugation. The bounded HA on liposomes was determined by UV/Vis spectrometers (Biotek). The Final liposome concentration was adjusted to 1 mg/ml, named as HA-Lip-pOH and HA-Lip-p-NH2, respectively.
Fig. 1. The structures of porphyrin derivatives with various substitutes, and the strategy of enhanced intracellular delivery of porphyrin by liposomes.
the introduction of functional polymers or receptor-targeting moiety to the porphyrin delivery systems becomes more and more attractive for specific localization to a tumor tissue [7]; [20]. Previous reports confirm that coating liposomes with biocompatible polymers such as poly (ethylene glycol) (PEG) can prolong their circulation time for a higher accumulation in targeted tissue [9]. Conjugation of folic acid to the porphyrin-loaded solid-lipid nanoparticles could significantly enhance the photocytotoxicity on KB cells via the folate receptor (FR)-targeted active delivery system [21]. In this study, four types of porphyrin derivatives were selected to prepare light-sensitive liposomes (Fig. 1). We systemically investigated the effects of porphyrin-structures on the formation of liposomes. We considered that it could be hard for the cells to uptake pure porphyrin derivatives in aqueous solution due to the formation of porphyrin aggregates. Although surfactants are used in most case to increase the solubility of porphyrin, most of them are not biocompatible. The aim of this study is to exclude the use of toxic surfactants. So liposomes were used to carry porphyrins to increase the effective concentration. Packing porphyrins into the liposome bilayer could not only increase their dispersity, but also enhance their cellular uptake. In order to achieve active targeting delivery of the liposome-porphyrin, the hyaluronic acid (HA) was selected as the cancer cell specific targeting ligand for liposome surface modification. HA is a linear polysaccharide which is widely used for the preparation of cancer targeted PDT nanomaterials [25]; [14]. Previously, we reported a top-down in situ formed liposome system to generate HA-coated liposomes [24]. Now, we revise the strategy to prepare the clickable liposomes for porphyrin delivery, and further coat them with HA polymer. As mentioned above, coating the liposomes with HA could enhance their cellular uptake by CD44 over-expressed tumor cells, e.g. breast cancer cells [15]; [1]. HA natural polymer was also used as the block agent during the specific uptake test towards cancer cells. To the best of our knowledge, this is
2.3. Characterizations of liposome-porphyrin The porphyrin loading efficiency was calculated according to the corresponding porphyrin standard curves measured on UV/Vis spectrometers (Biotek). The particle size and surface potential of liposomes were analyzed by dynamic light scattering (DLS, particle size/zeta potential meter, Zetasizer Nano ZS, Malvern). The stabilities of the obtained liposomes were characterized by DLS after preparation and after 15 min light exposure. Morphologies of liposomes were observed on transmittance electron microscope (TEM, Zessie) using a negative staining method. For differential scanning calorimetry, the concentrated liposomes (centrifugation, 15000 rpm) were scanned on a differential scanning calorimeter (DSC, TA instruments) from 20 to 90 °C at a heating rate of 2 °C/min. 2.4. Cellular uptake MDA-MB-231 cells were cultured in a complete DMEM medium 138
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(containing 10% FBS and 0.1% penicillin-streptomycin) to near 80% confluence. Cells were maintained at 37 °C in 5% CO2 at 90% humidity and used at passage 4. Cells were seeded on cover glass in 6-well plates at 100000 cells/ml density and cultured in 2 mL/well of complete DMEM medium. After incubation for 24 h, the cells were cultured with 2 mL (0.2 mg/ml) of each of the following samples: 1) Lip-p-OH, 2)Lipp-NH2, 3) Lip. After 0.5 h or 2 h culturing, the cells were fixed using 4% PFA and stained with DAPI. Then, the cover glasses were sealed on microscope slides for confocal microscopy (ATRMP+, Nikon). The cellular uptake of HA coated liposomes (HA-Lip-p-OH, HA-Lip-p-NH2, HA-Lip) were performed following above process. For specificity binding test, cells were pretreated with 1 mg/ml HA natural polymer solution for 30 min to block the CD44 receptors before adding HA-Lipp-OH. Fig. 2. Glass transition temperature (Tg) of lip-p-P, Lip-p-NH2, Lip-p-py, Lip-pOH.
2.5. Light-induced cytotoxicity of porphyrin containing liposomes 3. Results and discussion MCF-10A and A375 cells were cultured in a complete DMEM medium (containing 10% FBS and 0.1% penicillin-streptomycin) to near 80% confluence. Cells were maintained at 37 °C in 5% CO2 at 90% humidity and used at passage 5. Cells were seeded in 96-well plates at 10000 cells/ml density and cultured in 100 μL/well of complete DMEM medium. After incubation for 24 h, the cells were cultured with 100 μL of each of the following samples for 24h: 1) Lip-p-OH, 2) Lip-p-NH2, 3) Lip, 4) p-OH, 5) p-NH2. The solutions/suspensions were diluted with complete DMEM medium, so that the final concentrations of liposomes were 1 mg/ml, 0.5 mg/ml, 0.25 mg/ml, 0.125 mg/ml and 0.0625 mg/ ml. Since the liposome concentration was fixed, the concentrations of porphyrins inside liposomes were various according to the loading efficiency. The porphyrin (p-OH) concentrations of Lip-p-OH were 35.6 μg/ml, 17.8 μg/ml, 8.9 μg/ml, 4.45 μg/ml, and 2.225 μg/ml, respectively. The porphyrin (p-NH2) concentrations of lip-p-NH2 were 12.7 μg/ml, 6.35 μg/ml, 3.175 μg/ml, 1.587 μg/ml, and 0.794 μg/ml, respectively. The concentrations of each porphyrin without liposome carriers were the same as the corresponding porphyrin inside liposomes. The cells grown in 100 μL of complete DMEM medium were used as positive control. For the light-induced cytotoxicity groups, the cells were exposed to visible light for 15 min (wavelength: 400–600 nm, intensity: 20 mw/cm2) after addition of the corresponding samples, then continued the incubation for 24 h under dark condition. Cell viability was determined using MTS assay. 20 μL of MTS/PMS solution (CellTiter 96® Aqueous Assay) was added into each well of 96well plate without removing the medium. The plate was incubated for 4 h at 37 °C, 5% CO2. The absorbance was measured at 450 nm and subtracted from the blank taken at 595 nm. The relative cell viabilities were calculated by comparing with the control wells. All experiments were performed in triplicates.
3.1. Preparation of porphyrin derivatives containing liposomes The final liposome concentration after purification was 1.14 mg/ml. The loading efficiencies of four types of porphyrin derivatives were 35.6 μg/mg (mass of liposomes) for Lip-p-OH, 12.7 μg/mg for Lip-pNH2, 3.45 μg/mg for Lip-p-P, 2.82 μg/mg for Lip-p-py. The loading efficiency varies according to the types of porphyrins. More p-OH and pNH2 could be loaded in liposomes comparing with the amounts of loaded p-P and p-py due to the better solubility of p-OH and p-NH2 in the solvent mixture. During the liposome hydration process, samples with p-P and p-py tended to precipitate leading to the broken of liposomes. The broken liposomes and precipitates were removed after passing through the size-extruder. Hence, only little amount of porphyrins remained in the samples. Meanwhile, samples with p-OH and pNH2 could form nano-sized liposomes without precipitation, so that the porphyrins were well dispersed and protected. Fig. 2 showed the glass transition temperature (Tg) of liposomes with four types of porphyrins measured by DSC. It is reported that liposomes composed of DPPC/ DOPG/cholesterol exhibit the Tg in the range of 55–60 °C depending on the ratio of lipid to cholesterol. Introduction of porphyrins to the lipidbilayers could significantly increase the Tg of the liposomes. Lip-p-py exhibited highest Tg at 87 °C. Lip-p-NH2 showed Tg at 79 °C. The Tg of Lip-p-OH was 77 °C. However, no Tg was observed with sample Lip-p-P (or Tg was higher than 90 °C).
3.2. Characterizations and in vitro stability of liposome-porphyrin The particle sizes of all liposomes were comparable regardless of porphyrin type, which is due to that all samples were extruded via 200 nm-filters during the preparation process (Fig.S1). Considering the higher loading efficiency of porphyrins in liposomes, we finally chose Lip-p-OH and Lip-p-NH2 for further study. We first investigated the stability of Lip-p-OH against light exposure. As shown in Fig. 3, the mean size of the Lip-p-OH increased from 153 ± 14 nm to 413 ± 99 nm. The mean size of Lip without porphyrins (control group) increased from 159 ± 10 nm to 471 ± 12 nm. Those results indicated that the sizes of the original liposomes were sensitive to light as well. But the size distribution of the lip-p-OH became bounder. The number of Lip-p-OH with smaller particle sizes also increased after light exposure, while the size distribution of Lip still followed the Gaussian distribution. The original liposomes composed of the lipid DOPG with double bonds in the structure. They could form aggregates due to the light additional reaction of double bonds. Once the porphyrins were involved, the light-sensitivity of the liposomes increased. As a result, the liposomes could aggregate into large clusters or be destroyed into smaller liposome fragments. It is reported that porphyrin could transfer
2.6. Light-induced cytotoxicity of HA-Lip-p-OH in MDA-MB-231 cells MDA-MB-231 cells were seeded in 96-well plates at 10000 cells/ml density and cultured in 100 μL/well of complete DMEM medium. After incubation for 24 h, the cells were cultured with 100 μL of the following samples, respectively: 1) HA-Lip-p-OH, 2) Lip-p-OH, 3) Lip, 4) p-OH, 5) HA. The solutions/suspensions were diluted with complete DMEM medium, so that the final concentrations of liposomes were 0.2 mg/ml, 0.1 mg/ml, 0.05 mg/ml, 0.025 mg/ml and 0.0125 mg/ml, corresponding to the p-OH concentration as 7.12 μg/ml, 3.56 μg/ml, 1.78 μg/ml, 0.89 μg/ml and 0.45 μg/ml. Concentrations of HA polymer for the test were calculated to be the same as the HA modified on the surface of HA-Lip-p-OH. The cell viability was determined using MTS assay as described above.
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3.3. Cellular uptake It is known that most porphyrin derivatives are hydrophobic and could not be well dispersed in aqueous solution. This property significantly reduces the efficiency of cellular uptake of porphyrins and limits their applications. To determine the applicability of the liposomes as the intracellular drug delivery carriers for porphyrins towards cancer cells, we investigated the cellular uptake of Lip-p-OH and Lip-pNH2. The liposomes without porphyrins were used as the control group. The MBA-MB-231 cells were incubated with the corresponding sample for 0.5 h and 2 h. Due to the presence of TRITC DHPE in lipid membranes, the liposomes can be easily traced by confocal fluorescence microscopy at the emission wavelength of TRITC dye (red channel). As shown in Fig S3, the cells did not show any fluorescence at 0.5 h. After 2 h incubation, we observed cellular uptake of each sample by MDAMB-231 cells. Since MDA-MB-231 cell belong to the breast cancer cell lines that can overexpress CD44 receptor, we further modified the surface of Lipp-OH and Lip-p-NH2 with the CD44 targeting polymer (HA) to improve the specific cellular uptake ability of liposomes. The FTSC labeled HA (HA-FTSC) could show green fluorescence under fluorescence microscopy. We confirmed the success of HA coating on liposomes via UV/Vis spectrometer as shown in Fig.S5, where the wavelength peak at 490 nm corresponded to HA-FTSC absorbance. After cellular uptake, the green fluorescence signals should be coincident with the red fluorescence signals in terms of both shape and location under the confocal fluorescence microscopy. As expected, The uptake of HA-Lip-p-OH or HALip-p-NH2 by MDA-MB-231 cells was observed from Fig. 5. Most of them existed in cytoplasm around the nucleus of the cells. As we mentioned previously, Most of the porphyrin free HA-liposomes were removed during the centrifugation processes. Hence, we only found the red signals for sample of HA-Lip as the HA signal was too weak to be observed on microscope. In order to prove that the HA-coated liposomes interact with the MDA-MB-231 cells specifically, we performed a blocking test by pre-treating the cells with natural HA polymer for 30 min before addition of HA-Lip-p-OH. The natural HA should bind to the surface of MDA-MB-231 cells and block CD44 receptors. The uptake of HA-Lip-p-OH was indeed diminished by the blocking procedure (Fig. 5). It proved that the uptake of the HA-coated liposome-porphyrin was mainly due to the specific interaction between HA polymer on
Fig. 3. Size distributions of lip and lip-p-OH before (A, C) and after 15 min of light exposure (B, D).
to serum when it is only physically incorporated [2]. However, since the membranes of our liposomes were relatively rigid, the potential for porphyrin leaking out was low. We further investigated the light-induced changes in the morphologies of liposomes with or without porphyrins by TEM image. According to Fig. 4, the Lip showed multi-layer structure. No broken Lip was observed after light exposure. The increased particle size measured on DLS could be due to the aggregation of Lip. On the contrary, the morphology changed dramatically for Lip-p-OH after light exposure (Fig. 4D). The membranes of liposomes had collapsed together. It is known that the singlet oxygen could oxide the phospholipids leading to the destruction of liposomes. Similar change in morphology was also observed for Lip-p-NH2 (Fig.S2) after light exposure.
Fig. 5. Confocal microscope imaging of MDA-MB-231 cellular uptake of HA-lipp-OH, HA-lip-p-NH2 and HA-lip. For HA blocking, the cells were pretreated with HA polymer (1 mg/ml) for 30 min.
Fig. 4. TEM images of Lip and Lip-p-OH before (A, C) and after 15 min of light exposure (B, D). 140
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Similar to the cytotoxicity test using Lip-p-OH, all samples did not show toxicity to MDA-MB-231 cells under dark conditions (Fig. 6A). For the cells that were exposed to visible light for 15 min (Fig. 6B), the HALip-p-OH exibited higher toxicity than other groups (Lip-p-OH, p-OH, Lip and HA). The uptake of HA-Lip-p-OH should be higher than the uptake of Lip-p-OH by MDA-MB-231 cells due to the specific binding of HA to the cell surfaces. The HA-Lip-p-OH could release p-OH within the cells resulting in the increasing toxicity(see Fig. 7).
liposomes and the CD44 receptors on MDA-MB-231 cells. The HAcoated liposomes could be used as the targeting carriers for the delivery of porphyrins.
3.4. Light induced cytotoxicity study The cytotoxicities of liposome-porphyrins were performed against three cell lines: MCF-10A (normal human breast cell), A375 (human melanoma cell) and MDA-MB-231 (human breast cancer cell). We selected the liposome concentrations of Lip-p-OH and Lip-p-NH2 in the range from 0.0625 to 1 mg/ml. As shown in Fig.S4A and Fig.S4C, only Lip-p-OH showed toxicity towards A375 under dark condition, and the cell viability of A375 dropped to 7 ± 0.3% when the concentration of Lip-p-OH was 1 mg/ml, (the corresponding concentration of p-OH in liposomes was35.6 μg/ml). Interestingly, the p-OH without liposomes did not show any toxicity to the A375 cells at the same concentration (35.6 μg/ml). Meanwhile, no toxicity was observed for all samples towards MCF-10A cells under dark condition, which indicated that A375 cells are more sensitive to the toxicity of porphyrin. On the other hand, Fig.S4B and Fig.S4D showed that the cell viabilities were significantly reduced after incubation with Lip-p-OH and Lip-p-NH2 for 24 h after 15 min light exposure. The A375 cell viabilities were lower than 15% when the concentrations were higher than 0.125 mg/ml for both Lip-p-OH and Lip-p-NH2. Surprisingly, the p-OH did not show light-induced toxicity to A375 when the concentrations were higher than17.8 μg/mlor lower than 8.9 μg/ml. The lower solubility and large aggregation of porphyrins in aqueous solutions should be responsible for the lower toxicity. Based on the results of the porphyrin-loading efficiency and the cytotoxicity, we selected Lip-p-OH as the ideal sample to coat with HA polymer for specific light-induced cytotoxicity experiment against the CD44 overexpressed breast cancer cell line, MDA-MD-231.
4. Conclusion In conclusion, we have investigated the loading efficiencies and light-stabilities of the porphyrin-loaded liposomes. The liposomes containing porphyrin-OH (Lip-p-OH) showed the highest loading efficiency as well as the highest cytotoxicity against tumor cells. We also demonstrated the method to coat the porphyrin-loaded liposomes with HA polymer via “click” hydrazone reaction in order to target CD44 receptor overexpressed cells. The uptake of the HA-Lip-p-OH by MDAMB-231 cells was specific and could be block by natural HA polymer. This system provided an efficient strategy to introduce the cancer targeting property to porphyrins. The light-induced cytotoxicity of porphyrin was significantly enhanced due to the increased solubility of porphyrin and the higher cellular uptake by using liposomes as the ideal delivery carriers. The presented HA-Lip-p-OH showed high potential to be used in PDT.
Conflicts of interest The authors declared that they do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted.
Fig. 6. Cell viabilities of A375 and MCF-10A cells after incubation for 24 h with various concentrations of Lip-p-OH, Lip-p-NH2, p-OH, p-NH2 and Lip. For lightinduced cytotoxicity test, the cells were exposed to visible light for 15 min after addition of each sample, and then incubation for 24 h under dark condition. 141
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Fig. 7. Cell viabilities of MDA-MB-231 cells after incubation for 24 h with various concentrations of HA-Lip-p-OH, Lip-p-OH, p-OH, Lip and HA. For light-induced cytotoxicity test, the cells were exposed to visible light for 15 min after addition of each sample, and then incubation for 24 h under dark condition.
Acknowledgment
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Uptake and light-induced cytotoxicity of hyaluronic acid-grafted liposomes containing porphyrin in tumor cells
T
Qian Fenga,b, Jing Wangb,d, Hu Songb, Lian-gang Zhuob,c,d, Guanquan Wangb,d, Wei Liaob,d, Yue Fenga,d, Hongyuan Weib,c,d,∗∗, Yue Chena,d,∗∗∗, Yuchuan Yangb,c,d, Xia Yangb,c,d,∗ a
Department of Nuclear Medicine, The Affiliated Hospital Southwest of Medical University, 646000, Luzhou, PR China Institute of Nuclear Physics and Chemistry (INPC), China Academy of Engineering Physics (CAEP), 621999, Mianyang, PR China c Collaborative Innovation Center of Radiation Medicine of Jiangsu, Higher Education Institutions, 215123, Suzhou, PR China d Key Laboratory of Nuclear Medicine and Molecular Imaging of Sichuan Province, 621999, Mianyang, PR China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Porphyrin Liposome “Click” chemistry Hyaluronic acid CD44
Porphyrins are used in clinic as the photosensitizers for cancer treatment. However, most porphyrins tend to form aggregates in aqueous solution due to their hydrophobicity, which results in significantly reduced photocytotoxicities. Therefore, their applications in clinic are limited. To overcome these disadvantages, drugcarriers are designed for the delivery of porphyrins. In this study, we designed the surface modified liposomes for porphyrin delivery. Four types of porphyrins were loaded in liposome separately. We systemically investigated the porphyrin-containing liposomes in terms of their physicochemical properties as well as light-responsive properties. The hyaluronic acid derivative with aldehyde group was used to modify the liposome-porphyrin via the “click” reaction in order to enhance the uptake in CD44 overexpressed cancer cells. The results showed that 10,15,20-tetrakis(4-hydroxyphenyl) porphyrin (p-OH) had highest loading efficiency. The liposomes with p-OH showed highest toxicity to the cells with or without light exposure. The uptake of hyaluronic acid (HA) coated Lip-p-OH was proved to be specific to MDA-MB-231 cells by pretreating the cells with natural HA polymer as the block agent. This study provided an efficient strategy to enhance the cytotoxicity of porphyrin in aqueous solution, and to increase the affinity of porphyrin to the cancer cells.
1. Introduction Porphyrins are photosensitive compounds which can generate reactive oxygen species (ROS) upon light activation to destroy cancer cells. They are widely used in diagnostic imaging and therapy, especially photo-dynamic therapy (PDT) [10]; [26]. PDT is a binary cancer therapy which requires the activation of sensitizer in tissue with light locally. Compared to other cancer treatments (e.g. surgery, radiotherapy, and chemotherapy), the side-effects can be minimized by employing PDT therapeutic methologies [3]. The strategy of using targeted nanomaterials for PDT becomes more and more popular as it can also significantly improve the PDT treatment [5]. Porphyrins can accumulate in cancer cells more than in surrounding healthy tissues [17]. Some porphyrins are partly soluble in aqueous solutions. For example, the FDA approved drug, porfimer sodium, can be dissolved in alkaline condition. However, most porphyrin derivatives used in clinic
or pre-clinic are hydrophobic. The aggregation of porphyrins in aqueous solution could reduce the therapeutic efficacy significantly and limit their applications [4]. To overcome these problems, various nanocarriers, such as polymers, liposomes, micelles, and solid nanoparticles, were developed for porphyrins delivery [6] [16]; [22]; [11]. The photodynamic activity of porphyrins could be enhanced by incorporating them into nanoparticles [12] [23]; [8]. The most intensively investigated nano-carriers for porphyrin delivery are the bilayer structural liposomes composing of phospholipids [13]; [19]. Those liposomes exhibit an enhanced cellular uptake due to their cellular mimic composition and structure. Same as other nanocarriers, liposomes can also accumulate in solid tumors via the retention effect to reach higher delivery efficiency. Additionally, the higher selectivity to tumors is also required during porphyrin delivery in order to lower the side effects of porphyrins. However, liposomes are nonspecific towards tumors and most of them are less stable in vivo. Thus,
∗
Corresponding author. Institute of Nuclear Physics and Chemistry (INPC), China Academy of Engineering Physics (CAEP), 621999, Mianyang, PR China. Corresponding author. Department of Nuclear Medicine, The Affiliated Hospital Southwest of Medical University, 646000, Luzhou, PR China. ∗∗∗ Corresponding author. Department of Nuclear Medicine, The Affiliated Hospital Southwest of Medical University, 646000, Luzhou, PR China. E-mail addresses: [email protected] (H. Wei), [email protected] (Y. Chen), [email protected] (X. Yang). ∗∗
https://doi.org/10.1016/j.jddst.2018.06.024 Received 19 April 2018; Received in revised form 27 June 2018; Accepted 29 June 2018 Available online 05 July 2018 1773-2247/ © 2018 Elsevier B.V. All rights reserved.
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the first study about liposome-porphyrin coated with HA via the “click” reaction. The cytotoxicities of porphyrin-containing liposomes against various cell types were compared with or without light. Meanwhile, the effect of HA coating on the cytotoxicity to breast cancer cells was also investigated. 2. Materials and methods 2.1. Materials 1,2-dipalmitoylsn-glycero-2-phosphocholine (DPPC), 1,2-dioleoylsn-glycero-3[phospho-rac-(1-glycrol)] (DOPG) were purchased from Avanti, TRITC DHPE (rhodamine-lipid), fluorescein-5-thiosemicarbazide (FTSC), N-hydroxybenzotriazole (HOBt) and 1-ethyl-3-[3-dimethylaminopropyl] carbodiamide (EDC) were purchased from SigmaAldrich. Hydrazide-cholesterol (Chol-hy, MW 430.65) and hyaluronic acid-aldehyde-FTSC (HA-al-FTSC) were synthesized according to previous procedures [18]; [24], 5,10,15,20-tetrakis(4-aminophenyl) porphyrins (p-NH2), 5, 10,15,20-tetrakis(4-hydroxyphenyl) porphyrins (pOH), and 5, 10,15,20-tetraphenyl porphyrins (p) were purchased from TCI, 5,10,15,20-tetra(4-pyridyl) porphyrins (p-py) was from J&K. 2.2. Preparation of liposomes with different porphyrin derivatives The molar ratio of DPPC:DOPG:chol-hy was 13:1:6. In detail, 7.338 mg of DPPC, 0.612 mg of DOPG and 2.04 mg of chol-hy were dissolved in 1 ml of chloroform. 1 μg of TRITC DHPE was added to the above chloroform mixture in order to label the liposomes with TRITC dye. Total lipid amount was 10 mg. 1 mg of different porphyrin dissolved in 100 μL acetone was added to the lipid mixture separately. The solvent was evaporated using rotary evaporator during 2.5 h to obtain a thin lipid film. It was then hydrated at 60 °C with 1 mL of 10 mM preheated PBS buffer. After hydration, the suspension was extruded via a liposome extruder at 60 °C. Final liposome concentration was adjusted to 5 mg/ml, named as Lip-p-OH, Lip-p-P, Lip-p-NH2, Lip-p-Py, according to the type of loaded porphyrin. The liposome without porphyrin was prepared as control group and named as Lip. The unloaded porphyrin derivatives were removed by Sephedex G50. For HA-coated liposome-porphyrin, HA-al-FTSC (1 mg/ml) dissolved in PBS (10 mM, pH 7.4) was mixed with Lip-p-OH or Lip-p-NH2 (0.2 mg/ml) in 1:10 volume. This corresponded to the mass ratio of HA to liposome as 1:2, and the molar ratio of aldehyde group on HA to hydrazide group on liposomes as 1:8. The suspensions were mixed for 4 h on shaker. The unbounded HA was removed by centrifugation. The bounded HA on liposomes was determined by UV/Vis spectrometers (Biotek). The Final liposome concentration was adjusted to 1 mg/ml, named as HA-Lip-pOH and HA-Lip-p-NH2, respectively.
Fig. 1. The structures of porphyrin derivatives with various substitutes, and the strategy of enhanced intracellular delivery of porphyrin by liposomes.
the introduction of functional polymers or receptor-targeting moiety to the porphyrin delivery systems becomes more and more attractive for specific localization to a tumor tissue [7]; [20]. Previous reports confirm that coating liposomes with biocompatible polymers such as poly (ethylene glycol) (PEG) can prolong their circulation time for a higher accumulation in targeted tissue [9]. Conjugation of folic acid to the porphyrin-loaded solid-lipid nanoparticles could significantly enhance the photocytotoxicity on KB cells via the folate receptor (FR)-targeted active delivery system [21]. In this study, four types of porphyrin derivatives were selected to prepare light-sensitive liposomes (Fig. 1). We systemically investigated the effects of porphyrin-structures on the formation of liposomes. We considered that it could be hard for the cells to uptake pure porphyrin derivatives in aqueous solution due to the formation of porphyrin aggregates. Although surfactants are used in most case to increase the solubility of porphyrin, most of them are not biocompatible. The aim of this study is to exclude the use of toxic surfactants. So liposomes were used to carry porphyrins to increase the effective concentration. Packing porphyrins into the liposome bilayer could not only increase their dispersity, but also enhance their cellular uptake. In order to achieve active targeting delivery of the liposome-porphyrin, the hyaluronic acid (HA) was selected as the cancer cell specific targeting ligand for liposome surface modification. HA is a linear polysaccharide which is widely used for the preparation of cancer targeted PDT nanomaterials [25]; [14]. Previously, we reported a top-down in situ formed liposome system to generate HA-coated liposomes [24]. Now, we revise the strategy to prepare the clickable liposomes for porphyrin delivery, and further coat them with HA polymer. As mentioned above, coating the liposomes with HA could enhance their cellular uptake by CD44 over-expressed tumor cells, e.g. breast cancer cells [15]; [1]. HA natural polymer was also used as the block agent during the specific uptake test towards cancer cells. To the best of our knowledge, this is
2.3. Characterizations of liposome-porphyrin The porphyrin loading efficiency was calculated according to the corresponding porphyrin standard curves measured on UV/Vis spectrometers (Biotek). The particle size and surface potential of liposomes were analyzed by dynamic light scattering (DLS, particle size/zeta potential meter, Zetasizer Nano ZS, Malvern). The stabilities of the obtained liposomes were characterized by DLS after preparation and after 15 min light exposure. Morphologies of liposomes were observed on transmittance electron microscope (TEM, Zessie) using a negative staining method. For differential scanning calorimetry, the concentrated liposomes (centrifugation, 15000 rpm) were scanned on a differential scanning calorimeter (DSC, TA instruments) from 20 to 90 °C at a heating rate of 2 °C/min. 2.4. Cellular uptake MDA-MB-231 cells were cultured in a complete DMEM medium 138
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(containing 10% FBS and 0.1% penicillin-streptomycin) to near 80% confluence. Cells were maintained at 37 °C in 5% CO2 at 90% humidity and used at passage 4. Cells were seeded on cover glass in 6-well plates at 100000 cells/ml density and cultured in 2 mL/well of complete DMEM medium. After incubation for 24 h, the cells were cultured with 2 mL (0.2 mg/ml) of each of the following samples: 1) Lip-p-OH, 2)Lipp-NH2, 3) Lip. After 0.5 h or 2 h culturing, the cells were fixed using 4% PFA and stained with DAPI. Then, the cover glasses were sealed on microscope slides for confocal microscopy (ATRMP+, Nikon). The cellular uptake of HA coated liposomes (HA-Lip-p-OH, HA-Lip-p-NH2, HA-Lip) were performed following above process. For specificity binding test, cells were pretreated with 1 mg/ml HA natural polymer solution for 30 min to block the CD44 receptors before adding HA-Lipp-OH. Fig. 2. Glass transition temperature (Tg) of lip-p-P, Lip-p-NH2, Lip-p-py, Lip-pOH.
2.5. Light-induced cytotoxicity of porphyrin containing liposomes 3. Results and discussion MCF-10A and A375 cells were cultured in a complete DMEM medium (containing 10% FBS and 0.1% penicillin-streptomycin) to near 80% confluence. Cells were maintained at 37 °C in 5% CO2 at 90% humidity and used at passage 5. Cells were seeded in 96-well plates at 10000 cells/ml density and cultured in 100 μL/well of complete DMEM medium. After incubation for 24 h, the cells were cultured with 100 μL of each of the following samples for 24h: 1) Lip-p-OH, 2) Lip-p-NH2, 3) Lip, 4) p-OH, 5) p-NH2. The solutions/suspensions were diluted with complete DMEM medium, so that the final concentrations of liposomes were 1 mg/ml, 0.5 mg/ml, 0.25 mg/ml, 0.125 mg/ml and 0.0625 mg/ ml. Since the liposome concentration was fixed, the concentrations of porphyrins inside liposomes were various according to the loading efficiency. The porphyrin (p-OH) concentrations of Lip-p-OH were 35.6 μg/ml, 17.8 μg/ml, 8.9 μg/ml, 4.45 μg/ml, and 2.225 μg/ml, respectively. The porphyrin (p-NH2) concentrations of lip-p-NH2 were 12.7 μg/ml, 6.35 μg/ml, 3.175 μg/ml, 1.587 μg/ml, and 0.794 μg/ml, respectively. The concentrations of each porphyrin without liposome carriers were the same as the corresponding porphyrin inside liposomes. The cells grown in 100 μL of complete DMEM medium were used as positive control. For the light-induced cytotoxicity groups, the cells were exposed to visible light for 15 min (wavelength: 400–600 nm, intensity: 20 mw/cm2) after addition of the corresponding samples, then continued the incubation for 24 h under dark condition. Cell viability was determined using MTS assay. 20 μL of MTS/PMS solution (CellTiter 96® Aqueous Assay) was added into each well of 96well plate without removing the medium. The plate was incubated for 4 h at 37 °C, 5% CO2. The absorbance was measured at 450 nm and subtracted from the blank taken at 595 nm. The relative cell viabilities were calculated by comparing with the control wells. All experiments were performed in triplicates.
3.1. Preparation of porphyrin derivatives containing liposomes The final liposome concentration after purification was 1.14 mg/ml. The loading efficiencies of four types of porphyrin derivatives were 35.6 μg/mg (mass of liposomes) for Lip-p-OH, 12.7 μg/mg for Lip-pNH2, 3.45 μg/mg for Lip-p-P, 2.82 μg/mg for Lip-p-py. The loading efficiency varies according to the types of porphyrins. More p-OH and pNH2 could be loaded in liposomes comparing with the amounts of loaded p-P and p-py due to the better solubility of p-OH and p-NH2 in the solvent mixture. During the liposome hydration process, samples with p-P and p-py tended to precipitate leading to the broken of liposomes. The broken liposomes and precipitates were removed after passing through the size-extruder. Hence, only little amount of porphyrins remained in the samples. Meanwhile, samples with p-OH and pNH2 could form nano-sized liposomes without precipitation, so that the porphyrins were well dispersed and protected. Fig. 2 showed the glass transition temperature (Tg) of liposomes with four types of porphyrins measured by DSC. It is reported that liposomes composed of DPPC/ DOPG/cholesterol exhibit the Tg in the range of 55–60 °C depending on the ratio of lipid to cholesterol. Introduction of porphyrins to the lipidbilayers could significantly increase the Tg of the liposomes. Lip-p-py exhibited highest Tg at 87 °C. Lip-p-NH2 showed Tg at 79 °C. The Tg of Lip-p-OH was 77 °C. However, no Tg was observed with sample Lip-p-P (or Tg was higher than 90 °C).
3.2. Characterizations and in vitro stability of liposome-porphyrin The particle sizes of all liposomes were comparable regardless of porphyrin type, which is due to that all samples were extruded via 200 nm-filters during the preparation process (Fig.S1). Considering the higher loading efficiency of porphyrins in liposomes, we finally chose Lip-p-OH and Lip-p-NH2 for further study. We first investigated the stability of Lip-p-OH against light exposure. As shown in Fig. 3, the mean size of the Lip-p-OH increased from 153 ± 14 nm to 413 ± 99 nm. The mean size of Lip without porphyrins (control group) increased from 159 ± 10 nm to 471 ± 12 nm. Those results indicated that the sizes of the original liposomes were sensitive to light as well. But the size distribution of the lip-p-OH became bounder. The number of Lip-p-OH with smaller particle sizes also increased after light exposure, while the size distribution of Lip still followed the Gaussian distribution. The original liposomes composed of the lipid DOPG with double bonds in the structure. They could form aggregates due to the light additional reaction of double bonds. Once the porphyrins were involved, the light-sensitivity of the liposomes increased. As a result, the liposomes could aggregate into large clusters or be destroyed into smaller liposome fragments. It is reported that porphyrin could transfer
2.6. Light-induced cytotoxicity of HA-Lip-p-OH in MDA-MB-231 cells MDA-MB-231 cells were seeded in 96-well plates at 10000 cells/ml density and cultured in 100 μL/well of complete DMEM medium. After incubation for 24 h, the cells were cultured with 100 μL of the following samples, respectively: 1) HA-Lip-p-OH, 2) Lip-p-OH, 3) Lip, 4) p-OH, 5) HA. The solutions/suspensions were diluted with complete DMEM medium, so that the final concentrations of liposomes were 0.2 mg/ml, 0.1 mg/ml, 0.05 mg/ml, 0.025 mg/ml and 0.0125 mg/ml, corresponding to the p-OH concentration as 7.12 μg/ml, 3.56 μg/ml, 1.78 μg/ml, 0.89 μg/ml and 0.45 μg/ml. Concentrations of HA polymer for the test were calculated to be the same as the HA modified on the surface of HA-Lip-p-OH. The cell viability was determined using MTS assay as described above.
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3.3. Cellular uptake It is known that most porphyrin derivatives are hydrophobic and could not be well dispersed in aqueous solution. This property significantly reduces the efficiency of cellular uptake of porphyrins and limits their applications. To determine the applicability of the liposomes as the intracellular drug delivery carriers for porphyrins towards cancer cells, we investigated the cellular uptake of Lip-p-OH and Lip-pNH2. The liposomes without porphyrins were used as the control group. The MBA-MB-231 cells were incubated with the corresponding sample for 0.5 h and 2 h. Due to the presence of TRITC DHPE in lipid membranes, the liposomes can be easily traced by confocal fluorescence microscopy at the emission wavelength of TRITC dye (red channel). As shown in Fig S3, the cells did not show any fluorescence at 0.5 h. After 2 h incubation, we observed cellular uptake of each sample by MDAMB-231 cells. Since MDA-MB-231 cell belong to the breast cancer cell lines that can overexpress CD44 receptor, we further modified the surface of Lipp-OH and Lip-p-NH2 with the CD44 targeting polymer (HA) to improve the specific cellular uptake ability of liposomes. The FTSC labeled HA (HA-FTSC) could show green fluorescence under fluorescence microscopy. We confirmed the success of HA coating on liposomes via UV/Vis spectrometer as shown in Fig.S5, where the wavelength peak at 490 nm corresponded to HA-FTSC absorbance. After cellular uptake, the green fluorescence signals should be coincident with the red fluorescence signals in terms of both shape and location under the confocal fluorescence microscopy. As expected, The uptake of HA-Lip-p-OH or HALip-p-NH2 by MDA-MB-231 cells was observed from Fig. 5. Most of them existed in cytoplasm around the nucleus of the cells. As we mentioned previously, Most of the porphyrin free HA-liposomes were removed during the centrifugation processes. Hence, we only found the red signals for sample of HA-Lip as the HA signal was too weak to be observed on microscope. In order to prove that the HA-coated liposomes interact with the MDA-MB-231 cells specifically, we performed a blocking test by pre-treating the cells with natural HA polymer for 30 min before addition of HA-Lip-p-OH. The natural HA should bind to the surface of MDA-MB-231 cells and block CD44 receptors. The uptake of HA-Lip-p-OH was indeed diminished by the blocking procedure (Fig. 5). It proved that the uptake of the HA-coated liposome-porphyrin was mainly due to the specific interaction between HA polymer on
Fig. 3. Size distributions of lip and lip-p-OH before (A, C) and after 15 min of light exposure (B, D).
to serum when it is only physically incorporated [2]. However, since the membranes of our liposomes were relatively rigid, the potential for porphyrin leaking out was low. We further investigated the light-induced changes in the morphologies of liposomes with or without porphyrins by TEM image. According to Fig. 4, the Lip showed multi-layer structure. No broken Lip was observed after light exposure. The increased particle size measured on DLS could be due to the aggregation of Lip. On the contrary, the morphology changed dramatically for Lip-p-OH after light exposure (Fig. 4D). The membranes of liposomes had collapsed together. It is known that the singlet oxygen could oxide the phospholipids leading to the destruction of liposomes. Similar change in morphology was also observed for Lip-p-NH2 (Fig.S2) after light exposure.
Fig. 5. Confocal microscope imaging of MDA-MB-231 cellular uptake of HA-lipp-OH, HA-lip-p-NH2 and HA-lip. For HA blocking, the cells were pretreated with HA polymer (1 mg/ml) for 30 min.
Fig. 4. TEM images of Lip and Lip-p-OH before (A, C) and after 15 min of light exposure (B, D). 140
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Similar to the cytotoxicity test using Lip-p-OH, all samples did not show toxicity to MDA-MB-231 cells under dark conditions (Fig. 6A). For the cells that were exposed to visible light for 15 min (Fig. 6B), the HALip-p-OH exibited higher toxicity than other groups (Lip-p-OH, p-OH, Lip and HA). The uptake of HA-Lip-p-OH should be higher than the uptake of Lip-p-OH by MDA-MB-231 cells due to the specific binding of HA to the cell surfaces. The HA-Lip-p-OH could release p-OH within the cells resulting in the increasing toxicity(see Fig. 7).
liposomes and the CD44 receptors on MDA-MB-231 cells. The HAcoated liposomes could be used as the targeting carriers for the delivery of porphyrins.
3.4. Light induced cytotoxicity study The cytotoxicities of liposome-porphyrins were performed against three cell lines: MCF-10A (normal human breast cell), A375 (human melanoma cell) and MDA-MB-231 (human breast cancer cell). We selected the liposome concentrations of Lip-p-OH and Lip-p-NH2 in the range from 0.0625 to 1 mg/ml. As shown in Fig.S4A and Fig.S4C, only Lip-p-OH showed toxicity towards A375 under dark condition, and the cell viability of A375 dropped to 7 ± 0.3% when the concentration of Lip-p-OH was 1 mg/ml, (the corresponding concentration of p-OH in liposomes was35.6 μg/ml). Interestingly, the p-OH without liposomes did not show any toxicity to the A375 cells at the same concentration (35.6 μg/ml). Meanwhile, no toxicity was observed for all samples towards MCF-10A cells under dark condition, which indicated that A375 cells are more sensitive to the toxicity of porphyrin. On the other hand, Fig.S4B and Fig.S4D showed that the cell viabilities were significantly reduced after incubation with Lip-p-OH and Lip-p-NH2 for 24 h after 15 min light exposure. The A375 cell viabilities were lower than 15% when the concentrations were higher than 0.125 mg/ml for both Lip-p-OH and Lip-p-NH2. Surprisingly, the p-OH did not show light-induced toxicity to A375 when the concentrations were higher than17.8 μg/mlor lower than 8.9 μg/ml. The lower solubility and large aggregation of porphyrins in aqueous solutions should be responsible for the lower toxicity. Based on the results of the porphyrin-loading efficiency and the cytotoxicity, we selected Lip-p-OH as the ideal sample to coat with HA polymer for specific light-induced cytotoxicity experiment against the CD44 overexpressed breast cancer cell line, MDA-MD-231.
4. Conclusion In conclusion, we have investigated the loading efficiencies and light-stabilities of the porphyrin-loaded liposomes. The liposomes containing porphyrin-OH (Lip-p-OH) showed the highest loading efficiency as well as the highest cytotoxicity against tumor cells. We also demonstrated the method to coat the porphyrin-loaded liposomes with HA polymer via “click” hydrazone reaction in order to target CD44 receptor overexpressed cells. The uptake of the HA-Lip-p-OH by MDAMB-231 cells was specific and could be block by natural HA polymer. This system provided an efficient strategy to introduce the cancer targeting property to porphyrins. The light-induced cytotoxicity of porphyrin was significantly enhanced due to the increased solubility of porphyrin and the higher cellular uptake by using liposomes as the ideal delivery carriers. The presented HA-Lip-p-OH showed high potential to be used in PDT.
Conflicts of interest The authors declared that they do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted.
Fig. 6. Cell viabilities of A375 and MCF-10A cells after incubation for 24 h with various concentrations of Lip-p-OH, Lip-p-NH2, p-OH, p-NH2 and Lip. For lightinduced cytotoxicity test, the cells were exposed to visible light for 15 min after addition of each sample, and then incubation for 24 h under dark condition. 141
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Fig. 7. Cell viabilities of MDA-MB-231 cells after incubation for 24 h with various concentrations of HA-Lip-p-OH, Lip-p-OH, p-OH, Lip and HA. For light-induced cytotoxicity test, the cells were exposed to visible light for 15 min after addition of each sample, and then incubation for 24 h under dark condition.
Acknowledgment
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