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A redox prodrug micelle co-delivering camptothecin and curcumin for synergetic B16 melanoma cells inhibition
Accepted Manuscript A redox prodrug micelle co-delivering camptothecin and curcumin for synergetic B16 melanoma cells inhibition Yunfeng Hu, Shi Wu, Yong He, Liehua Deng PII: DOI: Reference:
S1385-8947(19)30082-8 https://doi.org/10.1016/j.cej.2019.01.074 CEJ 20795
To appear in:
Chemical Engineering Journal
Received Date: Revised Date: Accepted Date:
10 November 2018 29 December 2018 12 January 2019
Please cite this article as: Y. Hu, S. Wu, Y. He, L. Deng, A redox prodrug micelle co-delivering camptothecin and curcumin for synergetic B16 melanoma cells inhibition, Chemical Engineering Journal (2019), doi: https://doi.org/ 10.1016/j.cej.2019.01.074
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A redox prodrug micelle co-delivering camptothecin and curcumin for synergetic B16 melanoma cells inhibition
Yunfeng Hu *, Shi Wu, Yong He, Liehua Deng
1
Department of Dermatology, The First Affiliated Hospital of Jinan University, Jinan University,
Guangzhou 510630, Guangdong, China
Email: [email protected]
Abstract A redox prodrug consisting of camptothecin-conjugating PEG through the diselenide bond was synthesized, and the resultant amphiphilic prodrug was then used to load the curcumin to form the co-delivery system for the synergetic B16 melanoma cells inhibition. The co-delivery system showed a good response to glutataione, and the prodrug could be degraded and the two loaded drugs were then released. Both camptothecin and curcumin could induce the obvious cytotoxicity to B16 melanoma cells. Particularly, the co-delivery strategy showed a synergetic inhibition effect to B16 melanoma cells through an in vitro assay. In vivo assay also showed that the co-delivery system could inhibit B16 tumor growth and showed much better inhibition effect than that of only camptothecin or curcumin used. Moreover, as an injectable drug delivery system, the biocompatibility including the blood compatibility of the co-delivery system was confirmed, suggesting that the redox co-delivery system have a potential application in B16 tumor therapy.
Key words: redox, diselenide, B16 melanoma, synergetic
1. Introduction Melanoma is a malignant skin cancer with the very high fatality rate due to its active metastasis [1]. The current treatment methods against the melanoma include the surgical resection of the tumor, radiation or chemotherapy. Moreover, the monoclonal anti-bodies or immunomodulators is also investigated in clinical [2]. Among them, chemotherapy is still the main strategy to tumors treatment in clinical. However, due to the drug resistance of tumor cells and the nonspecific adsorption in circulation etc., chemotherapy usually induced the serious side effects as well as the low drug bioavailability [3]. The co-delivery of multiple drugs becomes the primary strategy in cancer therapy in recent years, which could promote the synergistic effect, reduce the side effect and also deter the drug resistance [4-6]. Some traditional drug delivery systems, such as liposomes, micelles, polymers and hybrid nanoparticles etc have been explored as the co-delivery carriers and showed excellent results [7-13]. Among them, the redox drug delivery systems which are response to glutathione (GSH) have been selected for tumor therapy, because the concentrations of GSH between tumor extracellular and intracellular environments are different, which could reduce the carriers released the loaded drugs intracellularly [14]. It has been reported that the tumor tissues displays at least four-fold GSH concentrations compared to the normal tissues, and the intracellular GSH concentration is then more higher and reaches up to 2–10 mmol/L [15]. To enhance the drug release intracellularly and then result in an effective tumor therapy, the polymer carriers containing diselenide (Se-Se) bonds have been developed. Similar to the disulfide bonds, Se-Se bonds also could be broken under the redox conditions, such as in the presence of GSH [16]. More important, the Se-Se bonds could be broken more easily compared to the disulfide bonds, because of the lower bond energy (172 kJ/mol) of Se-Se bonds [17]. Moreover, the polymer carriers containing Se-Se bonds have been reported to be metabolized after the intracellular dissociation, which showed good biocompatibility [18]. Herein, a diselenide-containing amphiphilic prodrug was synthesized, of which camptothecin (CPT) molecules were conjugated to a polyethylene glycol through the selenocystamine. CPT is a widely used chemotherapy drug and also usually used for the prodrug construction [19,20]. The prodrug design not only was conducive to the cellular internalization and drug delivery, but also loaded another hydrophobic drug to form the dual drug delivery system. Then the amphiphilic
prodrug formed the micelles in aqueous solution and the other drug of curcumin (CUR) was loaded to form the dual-drug co-delivery system. Both CPT and CUR have been reported to inhibit effectively to B16 melanoma cells and used in clinical. The dual-drug nanocomplex was characterized and used for synergetic therapy exploration to B16 melanoma cell. In addition, as an injectable drug delivery system, the biocompatibility of the nanocomplex including its blood compatibility was also assayed.
2. Experimental Section 2.1 Materials Polyethylene glycol, molecular weight of 2000 (PEG-2k), was purchased from Aladdin Industrial Corporation (Shanghai, China) and dried in vacuum before use. Succinic acid anhydride, camptothecin (CPT), curcumin (CUR), glutathione (GSH), N,N'-Dicyclohexylcarbodiimide (DCC), N-Hydroxysuccinimide (NHS) and 2,2'-(1,2-Diselanediyl)diethanamine were purchased from Aladdin and used directly. The mouse B16 melanoma cell line was obtained from Military Hospital in Guangzhou. Fetal bovine serum (FBS) and dulbecco’s modified eagle’s medium (DMEM) were purchased from Invitrogen Corporation (Washington, USA). Cell Counting Kit-8 was purchased from Beyotime Institute of Biotechnology (Shanghai, China). All buffers were prepared in MilliQ ultrapure water and filtered (0.45 µm) prior to use. All other chemicals were purchased and used directly.
2.2 Synthesis of diselenide-containing prodrug 2.2.1 Synthesis of carboxylate CPT Firstly, CPT was carboxylated to conjugate with PEG-2k. In brief, CPT (170 mg, 0.5 mmol), N-(4-pyridyl)dimethylamine (60 mg, 0.5 mmol) and succinic acid anhydride (190 mg, 2 mmol) were dissolved 50 mL dried DMSO and then heated to 70 °C for 24 h under the nitrogen atmosphere. After the reaction, the product was then dialyzed in the distilled water for 3 d (MWCO = 500, USA), and the precipitation was collected and dried to obtain the carboxylate CPT with a yield of 62%. Its chemical structure was measured through 1H NMR.
2.2.2 Synthesis of diselenide-modified PEG
PEG-2k (2g, 1 mmol) was dissolved in 25 mL dried dichloromethane, and then p-toluene sulfonyl chloride (0.9 g, 4 mmol) and trimethylamine (25 mL) were added at 4 °C. After 24 h reaction, the product was extracted by 0.5 mol/L aqueous HCl and the organic phase was washed by sodium bicarbonate. After that, the excess diethyl ether was added and the precipitation was collected. After dried in vacuum overnight, the p-toluene sulfonyl-PEG was obtained. Then,
the
equal
molar
weight
of
p-toluene
sulfonyl-PEG
and
2,2'-(1,2-Diselanediyl)diethanamine were co-dissolved in DMSO and reacted for 24 h under the nitrogen atmosphere. After the reaction, the product was dialyzed in the distilled water for 3 d (MWCO = 2000, USA) and the dialytic fluid was frozen to dry to obtain the diselenide-modified PEG, with a yield of 58%.
2.2.3 Synthesis of the PEG-SeSe-CPT prodrug The above obtained carboxylate CPT (100 mg, 0.23 mmol) was mixed with DCC (60 mg, 0.27 mmol) and NHS (33 mg, 0.27 mmol) in dried DMSO. After 12 h reaction in the dark, diselenide-modified PEG (190 mg, 0.08 mmol) was added and then the reaction lasted for more 24 h. After that, the product was dialyzed in the distilled water for 3 d (MWCO = 3000, USA) and the dialytic fluid was frozen to dry, and then the prodrug of PEG-SeSe-CPT was obtained with a yield of 76%.
2.3 Characterization of PEG-SeSe-CPT prodrug 2.3.1 Critical micelle concentration (CMC) The critical micelle concentration (CMC) of the prodrug of PEG-SeSe-CPT in aqueous solution was measured by the fluorescence spectroscopy using the pyrene as a probe as reported [21]. Firstly, the pyrene was dissolved in dried acetone with a concentration of 15 mg/L, and then the solution was added to tubes for acetone evaporation. After that, the aqueous PEG-SeSe-CPT solutions with a series of concentrations was added to the tubes and then shaken at 40 °C overnight. Then, the solutions were analyzed by a fluorescent spectrometer F-7000 (Hitachi, Japan). The emission spectrum was determined as from 350 to 650 nm, and the excitation wavelength was fixed at 334 nm. The excitation and emission slit widths were set as 5 nm and 2.5 nm respectively, and the scanning speed was 500 nm/min.
2.3.2 GSH-response For GSH-response assays, GPC measurement was carried out for aqueous PEG-SeSe-CPT mixed with or without GSH. Briefly, 10 mg PEG-SeSe-CPT was dissolved in 10 mL distilled water or 10 mL aqueous GSH solution with a concentration of 0.01 mmol/L. At the predetermined time, 1 mL of the solution was collected for GPC trance. GPC measurements were equipped with a Model 270 RI detector, using the polystyrene as a standard. The eluent was tetrahydrofuran, and the measurement was carried out at 35 °C with 1 mL/min flow rate. The change of micelle sizes of aqueous PEG-SeSe-CPT with or without GSH was characterized using the Malvern Zetasizer Nano-ZS, and the monochromatic coherent He-Ne laser (633 nm) was the light source. The scattered light was recorded at a fixed angle of 90° and three measurements were carried out for each sample at room temperature. All values derived from CONTIN program. The morphology of aqueous PEG-SeSe-CPT was observed by a transmission electron microscopy (TEM) (JEM-2010HT, JEOL, Japan) operated at 80 kV. The 1 mg/mL samples were stained with 0.2 wt% phosphotungstic acid for 30 s [22]. After that, the samples were dropped onto the carbon-coated copper grids and dried in air.
2.4 CUR encapsulated into PEG-SeSe-CPT The typical procedure for the CUR loading on PEG-SeSe-CPT micelles to form the PEG-SeSe-CPT/CUR complex in aqueous solution was carried out using a dialysis method [23]. PEG-SeSe-CPT (100 mg) and CUR (15 mg) were co-dissolved in 5.0 mL DMF, and then 3 mL deionized water was added dropwise. After stirring for 12 h at room temperature, the resultant mixture was transferred into the dialysis bag (MWCO = 500) against deionized water for another 48 h. After that, the unloaded CUR was removed by filtering the dialysate through a syringe filter (0.45 µm pore size), and the PEG-SeSe-CPT/CUR complex was then obtained. The CUR loading amount in PEG-SeSe-CPT/CUR was determined by HPLC (Agilent 1200, USA). The lyophilized complex was distilled in methanol and then analyzed by HPLC equipped with a reverse phase C-18 column (Zorbax Eclipse XDB-C18). The mixed acetonitrile and deionized water (45/55, v/v) was used as the mobile phase with the flow rate of 1 mL/min. The detection wavelength and column temperature were set as 430 nm and 30°C, respectively. The standard CUR
in mixed acetonitrile and deionized water (45/55, v/v) with the concentrations from 1 to 100 μg/mL were also recorded as the standard curve (R2 > 0.99).
2.5 In vitro drug release For in vitro CPT and CUR release assays, 5 mL PEG-SeSe-CPT/CUR solution (1 mg/mL) were trapped into a dialysis tube (MWCO = 2000) and then immersed into a 20 mL phosphate buffered saline (PBS, pH = 7.4) which contained GSH with different concentrations. All samples were incubated with the water bath at 37 °C. At predetermined time points, 5.0 mL of the released medium solution was taken out and then replaced by 5.0 mL fresh medium solution to maintain the same total volume of medium solution. The cumulative released amounts of CPT and CUR were calculated through the HPLC analysis.
2.6 Cytotoxicity B16 cells were incubated using the complete DMEM in a humidified atmosphere of 5% CO2 at 37°C and then seeded onto a 96-well plate with the density of 1×104 cells/well. After 24 h incubation, the fresh DMEM containing the desired amount of PEG-SeSe-CPT/CUR was added, and each sample was set for five multiple holes. The cells incubated with PBS, PEG-SeSe-CPT or CUR was set as the control groups. After another 24 h, the medium was replaced by 10% CCK-8 DMEM solution and then incubated for 2 h. Thereafter, the cell viability was determined by the holes absorbance recorded by an MRX-Microplate Reader at 450 nm. Each sample was set for five holes (n = 5). The synergistic effect of CUR and CPT was analyzed through the isobologram analysis, and the IC50 of CUR and PEG-SeSe-CPT were obtained from the above cytotoxicity assays [24].
2.7 In vivo assays For in vivo anti-tumor test of the PEG-SeSe-CPT/CUR co-delivery system, Balb/c mice were injected with B16 cells (3×106 cells per mouse) under the armpit. After two weeks modeled, the B16 tumor-bearing mice were divided into 4 groups randomly (5 mice per group). Subsequently, the mice were injected with 200 μL aqueous PEG-SeSe-CPT/CUR (10 mg). PBS (200 μL), PEG-SeSe-CPT (200 μL, 9.8 mg) and CUR (200 μL, 0.2 mg) were set as the control groups. Particularly, all formulations were given every day. After 3 weeks treatments, the mice were sacrificed, and the
tumor volume was calculated (V = (L × W2)/2, where L and W are the longest and shortest diameters of tumors respectively). For in vivo toxicity study, the above mice treated with PEG-SeSe-CPT/CUR were sacrificed, and their main organs such as heart, liver, spleen, lung and kidney were separated and washed twice with PBS. The organs were fixed using 4% formaldehyde, and then the histological examination was performed.
2.8 Blood compatibility 2.8.1 Hemolysis The fresh whole blood obtained from mice was collected using the sodium citrate as an anti-coagulant (blood/anticoagulant = 9:1). The whole blood was then centrifuged at 500 rmp for 8 min, and the plasma and buffy coat layer were then removed. The resultant red blood cells (RBCs) were washed with PBS for three times. RBCs were then re-suspended in PBS at 16% hematocrit (v/v), and then 5 mL aqueous PEG-SeSe-CPT/CUR with different concentrations was mixed and incubated for 12 h at 37 °C. The positive (100% hemolysis induced by 0.1% Na2CO3 solution) and negative (0% hemolysis treated with pure PBS) controls were also set up. Each sample was recorded for five times (n = 5). After that, the RBC suspensions were centrifuged at 500 rmp for 10 min, and the supernatants were measured using a microplate reader at 540 nm. The percentage hemolysis was calculated as the following formula [25]: Hemolysis (%) = [(OD of the test sample – OD of negative control) × 100]/OD of positive control.
2.8.2 Activated partial thromboplastin time (APTT) and prothrombin time (PT) The APTT and PT index were recorded from an SF-8000 automatic coagulation analyzer (Beijing Succeeder Company, Beijing, China) as reported [26]. Platelet-poor plasma (PPP) was obtained by centrifuging the citrated whole blood at 800 rmp for 10 min, and then 180 μL of that was mixed with PEG-SeSe-CPT/CUR at different concentrations. The analysis was carried out at 37°C and each sample was repeated for five times (n = 5). The sample mixing with only PBS was set as control.
2.9 Statistical analysis
Comparison among different groups was analyzed by the one-tailed Student’s t-test using statistical software SPSS 11.5. All data are presented as means ± S.D. Differences with P < 0.05 (*) and P < 0.01 (**) were considered statistically significant difference.
3. Results and discussion 3.1 Synthesis of prodrug of PEG-SeSe-CPT For PEG-SeSe-CPT synthesis, the PEG containing the diselenide bonds was first synthesized as shown in Scheme 1, and its chemical structure was characterized by 1H NMR shown as Figure S1 (Supporting Information). CPT was carboxylated as shown in Scheme 2, and its chemical structure was also characterized by 1H NMR (Figure S2, Supporting Information). Then, the PEG containing the diselenide bonds was conjugated with carboxylate CPT to form the PEG-SeSe-CPT prodrug through the aminification reaction as shown in Scheme 3. The chemical structure of PEG-SeSe-CPT was characterized by 1H NMR as shown in Figure 1. For a clear structural analysis, all peaks shown had been marked and the result indicated that PEG-SeSe-CPT was synthesized successfully. Moreover, the molar ratio of CPT/PEG in PEG-SeSe-CPT was also calculated to be 1.76 by calculating the integral ratio of the proton resonance signals, I2/I1 (belonged to CPT and PEG segments respectively), implying that each PEG molecule was conjugated with 1.76 CPT molecules in average. The CPT content in PEG-SeSe-CPT was further determined by UV-Vis analysis and calculated as 18.3%, which was accordance with the result from 1H NMR. To explore whether PEG-SeSe-CPT could load the hydrophobic drugs, the critical micelle concentration (CMC) of aqueous PEG-SeSe-CPT was determined using the pyrene as the fluorescent probe [27]. The value of I1/I3 intensity ratio was shown in Figure 2, and it displayed that with the increase of PEG-SeSe-CPT concentration, the I1/I3 value showed a substantial decrease at the initial stage and then changed little later. At the initial stage of low PEG-SeSe-CPT concentrations, the micelles were formed from the aqueous amphiphilic PEG-SeSe-CPT, and pyrene molecule entered into the hydrophobic core of the micelles, which induced the substantial decrease of I1/I3. After all pyrene was encapsulated by micelles, the value of I1/I3 changed little. The cross-point of the I1/I3 values at different stages was considered as the CMC of PEG-SeSe-CPT. Then, its CMC was determined to be 0.068 mg/mL, much lower than some reported results and suggesting a good
loading ability to hydrophobic drugs [28].
3.2 GSH-response of PEG-SeSe-CPT The prodrug of PEG-SeSe-CPT containing the diselenide bonds may be easily degraded under the reductive conditions, such as aqueous GSH solution. Then, the GSH-sensitivity of the prodrug was studied by adding it in aqueous 10 mmol/L GSH solution, mimicking the tumor intracellular environment [29]. The GPC trance results of PEG-SeSe-CPT changes in the presence of GSH were given in Fig. 3, which displayed the changes in molecular weight. It was found that PEG-SeSe-CPT showed its elution peak at 28.2 min. After incubated with 10 mmol/L GSH for 4 h, its peak shifted to 29.5 min. The extension of the elution time indicated that the molecular weight of polymers decreased. This result confirmed that the 10 mmol/L GSH could make PEG-SeSe-CPT degraded, which was useful in drug delivery to tumors [30]. The changes in morphology and size of PEG-SeSe-CPT micelles were also studied to further confirm the responsibility of PEG-SeSe-CPT to GSH. As shown in Fig. 4a observed by TEM, in aqueous solution, PEG-SeSe-CPT could self-assembled into compact spherical micelles and their diameters ranged from 160 to 200 nm, and the DLS result in Fig. 4c gave the statistical diameter of 255.3 nm. After incubated with 10 mmol/L GSH, the PEG-SeSe-CPT micellar structure disassembled and then converted into irregular aggregates with a great diversity of morphology (shown in Fig. 4b), and the DLS result from Fig. 4d showed a larger diameter and wider polydisperse. These results further confirmed that PEG-SeSe-CPT micelles could response to GSH and had the potential application in drug delivery to tumors [31].
3.3 In vitro CPT and CUR release CUR was encapsulated into the PEG-SeSe-CPT micelles to form the co-delivery system of PEG-SeSe-CPT/CUR, and the CUR content in PEG-SeSe-CPT/CUR was determined to be 2.3% (w/w). Such a CUR loading amount was not high compared with some reported micelles. Song et al synthesized an amphiphilic poly(ethylene glycol)-b-poly(ϵ-caprolactone) copolymer with a high CUR loading content [32]. The hydrophobic drug loading amount in micelles was dependent on the nature of hydrophobic segments and the ratio of hydrophilic segments to hydrophobic segments.
After the optimization, the CUR content in this micelle could reach higher than 12%. In this work, PEG-SeSe-CPT micelle without chemical structural optimization was used as the CUR carrier and the
CUR
content
in
PEG-SeSe-CPT/CUR
was
2.3%,
which
was
enough
for
the
synergistic inhibition of B16 melanoma cells in this work. To explore the release kinetics of CPT and CUR in vitro as well as the GSH-sensitive release behaviors, the aqueous PEG-SeSe-CPT/CUR complex was immersed in the release media with or without GSH. The release profiles were given in Fig. 5. It can be seen that CPT showed a slow release rate and a lower cumulative release amount (less than 10% over 12 h) in the absence of GSH. It may be resulted from that the released CPT was mainly attributed to the unavoidable drug leakage [33]. After incubated with GSH, CPT could be release much faster, and the release rate increased with the increase of GSH concentration, and about 75% CPT was released from the complex during the first 12 h when incubated with 10 mmol/L GSH. This result indicated that the PEG-SeSe-CPT/CUR complex was responsible to GSH. Moreover, the controlled CPT release could be realized by modulating the concentrations of GSH. For CUR, it also displayed the slow release in the absence of GSH while the faster release in the presence of GSH, also implying a GSH-controlled release behavior. In particular, in the absence of GSH, about 43% CUR has been released from the complex during the first 12 h, which was much higher than that of CPT. This result may be attributed from that CUR was released due to the diffusion mechanism [34], which was different with CPT which was chemically conjugated to PEG.
3.4 Cytotoxicity For B16 tumor treatment, CPT and CUR were combined used to expect a synergistic therapy, and then the cytotoxicity assays were carried out [35]. For the co-delivery system of PEG-SeSe-CPT/CUR, the loading contents of CPT and CUR in that were 18.3% and 2.3% respectively. Fig. 6 gave the cytotoxicity results of B16 cells when incubated with the different CPT concentrations. It can be seen that B16 cells were sensitive to CPT as well as CPT prodrug, and the cell viability was concentration-dependent. In particular, when CPT was used under the same concentration, the co-delivery system of PEG-SeSe-CPT/CUR displayed obviously better inhibition effect to B16 cells than that of only CPT used. This result indicated that CUR combined with CPT to
inhibit B16 cells. Moreover, the results of B16 cells viability treated with different CUR concentrations were also studied. As shown in Fig. 7, B16 cells were also sensitive to CUR, and its cell viability was also concentration-dependent. Under the same CUR concentration, the co-delivery system of PEG-SeSe-CPT/CUR also displayed much better inhibition effect than free CUR. The IC50 of PEG-SeSe-CPT and CUR to B16 cells after 24 h treatment were obtained through the results from Fig 6 and 7, and were determined as 4.83 mg/mL and 4.79 mg/mL respectively. Using the isobologram analysis, the IC50 of PEG-SeSe-CPT was used as the intercepts of the X axe and the IC50 of CUR was used as the Y axe. Then, an additive effect line was obtained which was shown in Fig. 8. Under this line, it represents that two formulations showed the synergistic effect [36]. In this work, the IC50 of the co-delivery system of PEG-SeSe-CPT/CUR was under this line, indicating that CPT and CUR showed the synergistic effect to B16 cells. To make the synergistic result more rigorous, the non-toxicity hydrophobic PCL (polycaprolactone) segment was used and then the PEG-SeSe-PCL/CUR was prepared for the cytotoxicity assays. The drug of CUR was loaded in PEG-SeSe-CPT micelles in this work, and then the micellization may be affecting the cytotoxicity of the free CUR itself. Then, the CUR-loaded micelles should also be prepared for the synergistic cytotoxicity assay to replace of free CUR. The cytotoxicity results were shown as Fig. S3 and S4 (Supporting Information). It was found that the same conclusion as the above used free CUR was obtained and the synergistic effect was reflected. This result further confirmed that CPT and CUR showed the synergistic effect to B16 cells.
3.5 In vivo assays Besides the in vitro assays, the tumor inhibition assay was also performed using the nude mice bearing B16 tumors. As shown in Fig. 9a and b, it was found that PBS control showed little effect on B16 tumors growth, and the tumor grew with time. After 21 days cultivation, the tumors increased more than 40 times in volume compared with that at the initial stage. For CUR and PEG-SeSe-CPT groups, both of them showed the significant inhibition effect to B16 tumors. In volume, CUR group decreased about 19% compared with PBS control after 21 days, and PEG-SeSe-CPT displayed a much better result and the tumor volume reduced more than 50%. This result indicated that CUR and CPT
could
inhibit
B16
tumors
growth
effectively.
For
the
co-delivery
system
of
PEG-SeSe-CPT/CUR, it showed the best inhibition effect to B16 tumor and the tumor volume
reduced about 78%, much better than that of CUR or CPT used only, suggesting a synergistic therapy effect. The body weight changes and cumulative survival ratios of the treated mice were recorded as shown in Fig. 9C and 9D. It was found that the body weights of mice remained constant for most treatment mice during the experiment, only the mice treated with CUR showed a slight decrease in body weight after 12 days treatments. This result suggested that free CUR formulation could result into the obvious toxicity and side effects. The cumulative survival ratios of treated mice also indicated this. The mice treated with PEG-containing formulations (PEG-SeSe-CPT and PEG-SeSe-CPT/CUR) showed a relative high survival ratios and the mice treated with free CUR showed a relative low survival ratio. These results indicated that the drug carrier could deliver drug and also reduce the toxicity and side effects of drugs. In vivo toxicity assay was carried out through a histological analysis to confirm the safety of PEG-SeSe-CPT/CUR [37]. As shown in Figure 10, there was no visible difference between the sample (bottom row) and the control (top row) histologically. The toxicity of carriers is mainly influenced by their chemical structures, particle sizes, exposure duration and biodistribution, as well as the nature of the surface and terminal groups of the carriers [38]. In this work, the non-observed toxicity of PEG-SeSe-CPT/CUR could be attributed to PEG at a certain point with its excellent biocompatibility [39].
3.6 Blood compatibility For drug delivery systems, their blood stability is a concern. Their interactions with the blood composition are considered as the serious limitation in clinical, and their nonspecific interactions could also severely diminish the half-life and targeting of drugs [40]. The blood compatibility of PEG-SeSe-CPT/CUR was studied by the spectrophotometric record of hemoglobin release from erythrocytes.
Fig.
12A
gave
the
percentage
hemolysis
after
treated
with
different
PEG-SeSe-CPT/CUR concentrations. It was found that PEG-SeSe-CPT/CUR showed good blood compatibility. After 24 h incubation, 10 mg/mL PEG-SeSe-CPT/CUR still showed non-hemolytic, with the extent of hemolysis lower than the permissible level of 5% [41]. Another important concern on blood compatibility for drug carriers is the effect on the blood coagulation [42]. Coagulation at the right time and location is very important and necessary to preserve the normal metabolism. On the contrary, inappropriate coagulation will cause severe effect,
even risks to the living system. As reported, the blood coagulation includes three pathways: intrinsic, extrinsic and common pathway. The indicator, APTT, reflecting the intrinsic and common coagulation pathways, refers to the time from a partial thromboplastin reagent (or CaCl2) is added to a fibrin clot form. PT reflects the extrinsic and common coagulation pathways, and refers to the time from tissue thromboplastin is added to a fibrin clot from [43]. The effects of PEG-SeSe-CPT/CUR on APTT and PT indicators were shown as Fig. 12B. It could be seen that compared to the PBS control, PEG-SeSe-CPT/CUR did not significant affect the APTT and PT within the concentration of 0.01 to 10 mg/mL. These results suggested the blood safety of PEG-SeSe-CPT/CUR in this work and the promising application in cancer therapy.
4. Conclusion To realize the synergetic therapy effect to B16 tumor, a redox prodrug of containing the diselenide bond was synthesized and two antitumor drugs of CPT and CUR were co-loaded. The co-delivery system showed the rapid response to GSH. In the presence of GSH similar to the intracellular environment of tumor cells, the prodrug could be degraded and the two loaded drugs could be released. The cytotoxicity results indicated that both CPT and CUR could inhibit the viability of B16 melanoma cells effectively. Particularly, the co-delivery system showed a much more cytotoxicity to B16 cells, and the synergetic inhibition effect was confirmed by the isobologram analysis. In vivo assay further indicated that the co-delivery system could inhibit B16 tumor growth and showed much better inhibition effect than that of only CPT or CUR was used. Moreover, as an injectable drug carrier, the blood compatibility of the co-delivery system was also confirmed, suggesting that this redox co-delivery system have a potential application in B16 tumor therapy.
Acknowledgement This work was financially supported by Science and Technology Planning Project of Guangdong Province (2017ZC0003) and the Special Fund for Scientific Research of the First Clinical Medical College of Jinan University (2017308).
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Scheme 1. Synthesis routes to diselenide-modified PEG.
Scheme 2. Synthesis route to carboxylate CPT.
Scheme 3. Chemical structure of PEG-SeSe-CPT.
Figure 1. 1H NMR spectrum of PEG-SeSe-CPT (DMSO-d6, 25 °C).
Figure 2. Effect of PEG-SeSe-CPT concentration on I1/I3 ratio of pyrene in aqueous PEG-SeSe-CPT solution.
Figure 3. GPC trances of PEG-SeSe-CPT in the absence and presence of 10 mM GSH.
Figure 4. TEM images (a and b) and particle sizes (c and d) of PEG-SeSe-CPT in the absence and presence of 10 mM GSH.
Figure 5. In vitro release profiles of CPT (a) and CUR (b) from PEG-SeSe-CPT/CUR in the absence and presence of GSH (n = 3).
Figure 6. Cytotoxicity of PEG-SeSe-CPT/CUR compared with free CPT and PEG-SeSe-CPT (n = 5).
Figure 7. Cytotoxicity of PEG-SeSe-CPT/CUR compared with free CUR (n = 5).
Figure 8. Isobologram analysis of the antiproliferation effect of PEG-SeSe-CPT and CUR on B16 cells.
Figure 9．Representative image of B16 tumors at 21 days treatment (A) and the tumor growth profiles (B) treated with various formulations. (C) The change of body weight of the mice with the experimental time. (D) The cumulative survival rate for different formulations. (1: PBS; 2: CUR; 3: PEG-SeSe-CPT; 4: PEG-SeSe-CPT/CUR) (n = 5).
(B)
(A) 1
(C)
2
3
4
(D)
Figure 10. Representative HE staining of organ histology by PEG-SeSe-CPT/CUR (top row) and PBS (bottom row).
Figure 11. The hemolysis and clotting analysis of PEG-SeSe-CPR/CUR (n = 5).
Graphical abstract
aqueous
CUR loading
(PEG-SeSe-CPT prodrug) (PEG-SeSe-CPT micelles)
(PEG-SeSe-CPT/CUR)
GSH PEG SeSe CPT
CUR
Highlight
1. A redox prodrug co-loading camptothecin and curcumin was prepared. 2. The co-delivery system showed a synergetic inhibition effect to B16 melanoma cells. 3. The co-delivery system showed a good biocompatibility.
S1385-8947(19)30082-8 https://doi.org/10.1016/j.cej.2019.01.074 CEJ 20795
To appear in:
Chemical Engineering Journal
Received Date: Revised Date: Accepted Date:
10 November 2018 29 December 2018 12 January 2019
Please cite this article as: Y. Hu, S. Wu, Y. He, L. Deng, A redox prodrug micelle co-delivering camptothecin and curcumin for synergetic B16 melanoma cells inhibition, Chemical Engineering Journal (2019), doi: https://doi.org/ 10.1016/j.cej.2019.01.074
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A redox prodrug micelle co-delivering camptothecin and curcumin for synergetic B16 melanoma cells inhibition
Yunfeng Hu *, Shi Wu, Yong He, Liehua Deng
1
Department of Dermatology, The First Affiliated Hospital of Jinan University, Jinan University,
Guangzhou 510630, Guangdong, China
Email: [email protected]
Abstract A redox prodrug consisting of camptothecin-conjugating PEG through the diselenide bond was synthesized, and the resultant amphiphilic prodrug was then used to load the curcumin to form the co-delivery system for the synergetic B16 melanoma cells inhibition. The co-delivery system showed a good response to glutataione, and the prodrug could be degraded and the two loaded drugs were then released. Both camptothecin and curcumin could induce the obvious cytotoxicity to B16 melanoma cells. Particularly, the co-delivery strategy showed a synergetic inhibition effect to B16 melanoma cells through an in vitro assay. In vivo assay also showed that the co-delivery system could inhibit B16 tumor growth and showed much better inhibition effect than that of only camptothecin or curcumin used. Moreover, as an injectable drug delivery system, the biocompatibility including the blood compatibility of the co-delivery system was confirmed, suggesting that the redox co-delivery system have a potential application in B16 tumor therapy.
Key words: redox, diselenide, B16 melanoma, synergetic
1. Introduction Melanoma is a malignant skin cancer with the very high fatality rate due to its active metastasis [1]. The current treatment methods against the melanoma include the surgical resection of the tumor, radiation or chemotherapy. Moreover, the monoclonal anti-bodies or immunomodulators is also investigated in clinical [2]. Among them, chemotherapy is still the main strategy to tumors treatment in clinical. However, due to the drug resistance of tumor cells and the nonspecific adsorption in circulation etc., chemotherapy usually induced the serious side effects as well as the low drug bioavailability [3]. The co-delivery of multiple drugs becomes the primary strategy in cancer therapy in recent years, which could promote the synergistic effect, reduce the side effect and also deter the drug resistance [4-6]. Some traditional drug delivery systems, such as liposomes, micelles, polymers and hybrid nanoparticles etc have been explored as the co-delivery carriers and showed excellent results [7-13]. Among them, the redox drug delivery systems which are response to glutathione (GSH) have been selected for tumor therapy, because the concentrations of GSH between tumor extracellular and intracellular environments are different, which could reduce the carriers released the loaded drugs intracellularly [14]. It has been reported that the tumor tissues displays at least four-fold GSH concentrations compared to the normal tissues, and the intracellular GSH concentration is then more higher and reaches up to 2–10 mmol/L [15]. To enhance the drug release intracellularly and then result in an effective tumor therapy, the polymer carriers containing diselenide (Se-Se) bonds have been developed. Similar to the disulfide bonds, Se-Se bonds also could be broken under the redox conditions, such as in the presence of GSH [16]. More important, the Se-Se bonds could be broken more easily compared to the disulfide bonds, because of the lower bond energy (172 kJ/mol) of Se-Se bonds [17]. Moreover, the polymer carriers containing Se-Se bonds have been reported to be metabolized after the intracellular dissociation, which showed good biocompatibility [18]. Herein, a diselenide-containing amphiphilic prodrug was synthesized, of which camptothecin (CPT) molecules were conjugated to a polyethylene glycol through the selenocystamine. CPT is a widely used chemotherapy drug and also usually used for the prodrug construction [19,20]. The prodrug design not only was conducive to the cellular internalization and drug delivery, but also loaded another hydrophobic drug to form the dual drug delivery system. Then the amphiphilic
prodrug formed the micelles in aqueous solution and the other drug of curcumin (CUR) was loaded to form the dual-drug co-delivery system. Both CPT and CUR have been reported to inhibit effectively to B16 melanoma cells and used in clinical. The dual-drug nanocomplex was characterized and used for synergetic therapy exploration to B16 melanoma cell. In addition, as an injectable drug delivery system, the biocompatibility of the nanocomplex including its blood compatibility was also assayed.
2. Experimental Section 2.1 Materials Polyethylene glycol, molecular weight of 2000 (PEG-2k), was purchased from Aladdin Industrial Corporation (Shanghai, China) and dried in vacuum before use. Succinic acid anhydride, camptothecin (CPT), curcumin (CUR), glutathione (GSH), N,N'-Dicyclohexylcarbodiimide (DCC), N-Hydroxysuccinimide (NHS) and 2,2'-(1,2-Diselanediyl)diethanamine were purchased from Aladdin and used directly. The mouse B16 melanoma cell line was obtained from Military Hospital in Guangzhou. Fetal bovine serum (FBS) and dulbecco’s modified eagle’s medium (DMEM) were purchased from Invitrogen Corporation (Washington, USA). Cell Counting Kit-8 was purchased from Beyotime Institute of Biotechnology (Shanghai, China). All buffers were prepared in MilliQ ultrapure water and filtered (0.45 µm) prior to use. All other chemicals were purchased and used directly.
2.2 Synthesis of diselenide-containing prodrug 2.2.1 Synthesis of carboxylate CPT Firstly, CPT was carboxylated to conjugate with PEG-2k. In brief, CPT (170 mg, 0.5 mmol), N-(4-pyridyl)dimethylamine (60 mg, 0.5 mmol) and succinic acid anhydride (190 mg, 2 mmol) were dissolved 50 mL dried DMSO and then heated to 70 °C for 24 h under the nitrogen atmosphere. After the reaction, the product was then dialyzed in the distilled water for 3 d (MWCO = 500, USA), and the precipitation was collected and dried to obtain the carboxylate CPT with a yield of 62%. Its chemical structure was measured through 1H NMR.
2.2.2 Synthesis of diselenide-modified PEG
PEG-2k (2g, 1 mmol) was dissolved in 25 mL dried dichloromethane, and then p-toluene sulfonyl chloride (0.9 g, 4 mmol) and trimethylamine (25 mL) were added at 4 °C. After 24 h reaction, the product was extracted by 0.5 mol/L aqueous HCl and the organic phase was washed by sodium bicarbonate. After that, the excess diethyl ether was added and the precipitation was collected. After dried in vacuum overnight, the p-toluene sulfonyl-PEG was obtained. Then,
the
equal
molar
weight
of
p-toluene
sulfonyl-PEG
and
2,2'-(1,2-Diselanediyl)diethanamine were co-dissolved in DMSO and reacted for 24 h under the nitrogen atmosphere. After the reaction, the product was dialyzed in the distilled water for 3 d (MWCO = 2000, USA) and the dialytic fluid was frozen to dry to obtain the diselenide-modified PEG, with a yield of 58%.
2.2.3 Synthesis of the PEG-SeSe-CPT prodrug The above obtained carboxylate CPT (100 mg, 0.23 mmol) was mixed with DCC (60 mg, 0.27 mmol) and NHS (33 mg, 0.27 mmol) in dried DMSO. After 12 h reaction in the dark, diselenide-modified PEG (190 mg, 0.08 mmol) was added and then the reaction lasted for more 24 h. After that, the product was dialyzed in the distilled water for 3 d (MWCO = 3000, USA) and the dialytic fluid was frozen to dry, and then the prodrug of PEG-SeSe-CPT was obtained with a yield of 76%.
2.3 Characterization of PEG-SeSe-CPT prodrug 2.3.1 Critical micelle concentration (CMC) The critical micelle concentration (CMC) of the prodrug of PEG-SeSe-CPT in aqueous solution was measured by the fluorescence spectroscopy using the pyrene as a probe as reported [21]. Firstly, the pyrene was dissolved in dried acetone with a concentration of 15 mg/L, and then the solution was added to tubes for acetone evaporation. After that, the aqueous PEG-SeSe-CPT solutions with a series of concentrations was added to the tubes and then shaken at 40 °C overnight. Then, the solutions were analyzed by a fluorescent spectrometer F-7000 (Hitachi, Japan). The emission spectrum was determined as from 350 to 650 nm, and the excitation wavelength was fixed at 334 nm. The excitation and emission slit widths were set as 5 nm and 2.5 nm respectively, and the scanning speed was 500 nm/min.
2.3.2 GSH-response For GSH-response assays, GPC measurement was carried out for aqueous PEG-SeSe-CPT mixed with or without GSH. Briefly, 10 mg PEG-SeSe-CPT was dissolved in 10 mL distilled water or 10 mL aqueous GSH solution with a concentration of 0.01 mmol/L. At the predetermined time, 1 mL of the solution was collected for GPC trance. GPC measurements were equipped with a Model 270 RI detector, using the polystyrene as a standard. The eluent was tetrahydrofuran, and the measurement was carried out at 35 °C with 1 mL/min flow rate. The change of micelle sizes of aqueous PEG-SeSe-CPT with or without GSH was characterized using the Malvern Zetasizer Nano-ZS, and the monochromatic coherent He-Ne laser (633 nm) was the light source. The scattered light was recorded at a fixed angle of 90° and three measurements were carried out for each sample at room temperature. All values derived from CONTIN program. The morphology of aqueous PEG-SeSe-CPT was observed by a transmission electron microscopy (TEM) (JEM-2010HT, JEOL, Japan) operated at 80 kV. The 1 mg/mL samples were stained with 0.2 wt% phosphotungstic acid for 30 s [22]. After that, the samples were dropped onto the carbon-coated copper grids and dried in air.
2.4 CUR encapsulated into PEG-SeSe-CPT The typical procedure for the CUR loading on PEG-SeSe-CPT micelles to form the PEG-SeSe-CPT/CUR complex in aqueous solution was carried out using a dialysis method [23]. PEG-SeSe-CPT (100 mg) and CUR (15 mg) were co-dissolved in 5.0 mL DMF, and then 3 mL deionized water was added dropwise. After stirring for 12 h at room temperature, the resultant mixture was transferred into the dialysis bag (MWCO = 500) against deionized water for another 48 h. After that, the unloaded CUR was removed by filtering the dialysate through a syringe filter (0.45 µm pore size), and the PEG-SeSe-CPT/CUR complex was then obtained. The CUR loading amount in PEG-SeSe-CPT/CUR was determined by HPLC (Agilent 1200, USA). The lyophilized complex was distilled in methanol and then analyzed by HPLC equipped with a reverse phase C-18 column (Zorbax Eclipse XDB-C18). The mixed acetonitrile and deionized water (45/55, v/v) was used as the mobile phase with the flow rate of 1 mL/min. The detection wavelength and column temperature were set as 430 nm and 30°C, respectively. The standard CUR
in mixed acetonitrile and deionized water (45/55, v/v) with the concentrations from 1 to 100 μg/mL were also recorded as the standard curve (R2 > 0.99).
2.5 In vitro drug release For in vitro CPT and CUR release assays, 5 mL PEG-SeSe-CPT/CUR solution (1 mg/mL) were trapped into a dialysis tube (MWCO = 2000) and then immersed into a 20 mL phosphate buffered saline (PBS, pH = 7.4) which contained GSH with different concentrations. All samples were incubated with the water bath at 37 °C. At predetermined time points, 5.0 mL of the released medium solution was taken out and then replaced by 5.0 mL fresh medium solution to maintain the same total volume of medium solution. The cumulative released amounts of CPT and CUR were calculated through the HPLC analysis.
2.6 Cytotoxicity B16 cells were incubated using the complete DMEM in a humidified atmosphere of 5% CO2 at 37°C and then seeded onto a 96-well plate with the density of 1×104 cells/well. After 24 h incubation, the fresh DMEM containing the desired amount of PEG-SeSe-CPT/CUR was added, and each sample was set for five multiple holes. The cells incubated with PBS, PEG-SeSe-CPT or CUR was set as the control groups. After another 24 h, the medium was replaced by 10% CCK-8 DMEM solution and then incubated for 2 h. Thereafter, the cell viability was determined by the holes absorbance recorded by an MRX-Microplate Reader at 450 nm. Each sample was set for five holes (n = 5). The synergistic effect of CUR and CPT was analyzed through the isobologram analysis, and the IC50 of CUR and PEG-SeSe-CPT were obtained from the above cytotoxicity assays [24].
2.7 In vivo assays For in vivo anti-tumor test of the PEG-SeSe-CPT/CUR co-delivery system, Balb/c mice were injected with B16 cells (3×106 cells per mouse) under the armpit. After two weeks modeled, the B16 tumor-bearing mice were divided into 4 groups randomly (5 mice per group). Subsequently, the mice were injected with 200 μL aqueous PEG-SeSe-CPT/CUR (10 mg). PBS (200 μL), PEG-SeSe-CPT (200 μL, 9.8 mg) and CUR (200 μL, 0.2 mg) were set as the control groups. Particularly, all formulations were given every day. After 3 weeks treatments, the mice were sacrificed, and the
tumor volume was calculated (V = (L × W2)/2, where L and W are the longest and shortest diameters of tumors respectively). For in vivo toxicity study, the above mice treated with PEG-SeSe-CPT/CUR were sacrificed, and their main organs such as heart, liver, spleen, lung and kidney were separated and washed twice with PBS. The organs were fixed using 4% formaldehyde, and then the histological examination was performed.
2.8 Blood compatibility 2.8.1 Hemolysis The fresh whole blood obtained from mice was collected using the sodium citrate as an anti-coagulant (blood/anticoagulant = 9:1). The whole blood was then centrifuged at 500 rmp for 8 min, and the plasma and buffy coat layer were then removed. The resultant red blood cells (RBCs) were washed with PBS for three times. RBCs were then re-suspended in PBS at 16% hematocrit (v/v), and then 5 mL aqueous PEG-SeSe-CPT/CUR with different concentrations was mixed and incubated for 12 h at 37 °C. The positive (100% hemolysis induced by 0.1% Na2CO3 solution) and negative (0% hemolysis treated with pure PBS) controls were also set up. Each sample was recorded for five times (n = 5). After that, the RBC suspensions were centrifuged at 500 rmp for 10 min, and the supernatants were measured using a microplate reader at 540 nm. The percentage hemolysis was calculated as the following formula [25]: Hemolysis (%) = [(OD of the test sample – OD of negative control) × 100]/OD of positive control.
2.8.2 Activated partial thromboplastin time (APTT) and prothrombin time (PT) The APTT and PT index were recorded from an SF-8000 automatic coagulation analyzer (Beijing Succeeder Company, Beijing, China) as reported [26]. Platelet-poor plasma (PPP) was obtained by centrifuging the citrated whole blood at 800 rmp for 10 min, and then 180 μL of that was mixed with PEG-SeSe-CPT/CUR at different concentrations. The analysis was carried out at 37°C and each sample was repeated for five times (n = 5). The sample mixing with only PBS was set as control.
2.9 Statistical analysis
Comparison among different groups was analyzed by the one-tailed Student’s t-test using statistical software SPSS 11.5. All data are presented as means ± S.D. Differences with P < 0.05 (*) and P < 0.01 (**) were considered statistically significant difference.
3. Results and discussion 3.1 Synthesis of prodrug of PEG-SeSe-CPT For PEG-SeSe-CPT synthesis, the PEG containing the diselenide bonds was first synthesized as shown in Scheme 1, and its chemical structure was characterized by 1H NMR shown as Figure S1 (Supporting Information). CPT was carboxylated as shown in Scheme 2, and its chemical structure was also characterized by 1H NMR (Figure S2, Supporting Information). Then, the PEG containing the diselenide bonds was conjugated with carboxylate CPT to form the PEG-SeSe-CPT prodrug through the aminification reaction as shown in Scheme 3. The chemical structure of PEG-SeSe-CPT was characterized by 1H NMR as shown in Figure 1. For a clear structural analysis, all peaks shown had been marked and the result indicated that PEG-SeSe-CPT was synthesized successfully. Moreover, the molar ratio of CPT/PEG in PEG-SeSe-CPT was also calculated to be 1.76 by calculating the integral ratio of the proton resonance signals, I2/I1 (belonged to CPT and PEG segments respectively), implying that each PEG molecule was conjugated with 1.76 CPT molecules in average. The CPT content in PEG-SeSe-CPT was further determined by UV-Vis analysis and calculated as 18.3%, which was accordance with the result from 1H NMR. To explore whether PEG-SeSe-CPT could load the hydrophobic drugs, the critical micelle concentration (CMC) of aqueous PEG-SeSe-CPT was determined using the pyrene as the fluorescent probe [27]. The value of I1/I3 intensity ratio was shown in Figure 2, and it displayed that with the increase of PEG-SeSe-CPT concentration, the I1/I3 value showed a substantial decrease at the initial stage and then changed little later. At the initial stage of low PEG-SeSe-CPT concentrations, the micelles were formed from the aqueous amphiphilic PEG-SeSe-CPT, and pyrene molecule entered into the hydrophobic core of the micelles, which induced the substantial decrease of I1/I3. After all pyrene was encapsulated by micelles, the value of I1/I3 changed little. The cross-point of the I1/I3 values at different stages was considered as the CMC of PEG-SeSe-CPT. Then, its CMC was determined to be 0.068 mg/mL, much lower than some reported results and suggesting a good
loading ability to hydrophobic drugs [28].
3.2 GSH-response of PEG-SeSe-CPT The prodrug of PEG-SeSe-CPT containing the diselenide bonds may be easily degraded under the reductive conditions, such as aqueous GSH solution. Then, the GSH-sensitivity of the prodrug was studied by adding it in aqueous 10 mmol/L GSH solution, mimicking the tumor intracellular environment [29]. The GPC trance results of PEG-SeSe-CPT changes in the presence of GSH were given in Fig. 3, which displayed the changes in molecular weight. It was found that PEG-SeSe-CPT showed its elution peak at 28.2 min. After incubated with 10 mmol/L GSH for 4 h, its peak shifted to 29.5 min. The extension of the elution time indicated that the molecular weight of polymers decreased. This result confirmed that the 10 mmol/L GSH could make PEG-SeSe-CPT degraded, which was useful in drug delivery to tumors [30]. The changes in morphology and size of PEG-SeSe-CPT micelles were also studied to further confirm the responsibility of PEG-SeSe-CPT to GSH. As shown in Fig. 4a observed by TEM, in aqueous solution, PEG-SeSe-CPT could self-assembled into compact spherical micelles and their diameters ranged from 160 to 200 nm, and the DLS result in Fig. 4c gave the statistical diameter of 255.3 nm. After incubated with 10 mmol/L GSH, the PEG-SeSe-CPT micellar structure disassembled and then converted into irregular aggregates with a great diversity of morphology (shown in Fig. 4b), and the DLS result from Fig. 4d showed a larger diameter and wider polydisperse. These results further confirmed that PEG-SeSe-CPT micelles could response to GSH and had the potential application in drug delivery to tumors [31].
3.3 In vitro CPT and CUR release CUR was encapsulated into the PEG-SeSe-CPT micelles to form the co-delivery system of PEG-SeSe-CPT/CUR, and the CUR content in PEG-SeSe-CPT/CUR was determined to be 2.3% (w/w). Such a CUR loading amount was not high compared with some reported micelles. Song et al synthesized an amphiphilic poly(ethylene glycol)-b-poly(ϵ-caprolactone) copolymer with a high CUR loading content [32]. The hydrophobic drug loading amount in micelles was dependent on the nature of hydrophobic segments and the ratio of hydrophilic segments to hydrophobic segments.
After the optimization, the CUR content in this micelle could reach higher than 12%. In this work, PEG-SeSe-CPT micelle without chemical structural optimization was used as the CUR carrier and the
CUR
content
in
PEG-SeSe-CPT/CUR
was
2.3%,
which
was
enough
for
the
synergistic inhibition of B16 melanoma cells in this work. To explore the release kinetics of CPT and CUR in vitro as well as the GSH-sensitive release behaviors, the aqueous PEG-SeSe-CPT/CUR complex was immersed in the release media with or without GSH. The release profiles were given in Fig. 5. It can be seen that CPT showed a slow release rate and a lower cumulative release amount (less than 10% over 12 h) in the absence of GSH. It may be resulted from that the released CPT was mainly attributed to the unavoidable drug leakage [33]. After incubated with GSH, CPT could be release much faster, and the release rate increased with the increase of GSH concentration, and about 75% CPT was released from the complex during the first 12 h when incubated with 10 mmol/L GSH. This result indicated that the PEG-SeSe-CPT/CUR complex was responsible to GSH. Moreover, the controlled CPT release could be realized by modulating the concentrations of GSH. For CUR, it also displayed the slow release in the absence of GSH while the faster release in the presence of GSH, also implying a GSH-controlled release behavior. In particular, in the absence of GSH, about 43% CUR has been released from the complex during the first 12 h, which was much higher than that of CPT. This result may be attributed from that CUR was released due to the diffusion mechanism [34], which was different with CPT which was chemically conjugated to PEG.
3.4 Cytotoxicity For B16 tumor treatment, CPT and CUR were combined used to expect a synergistic therapy, and then the cytotoxicity assays were carried out [35]. For the co-delivery system of PEG-SeSe-CPT/CUR, the loading contents of CPT and CUR in that were 18.3% and 2.3% respectively. Fig. 6 gave the cytotoxicity results of B16 cells when incubated with the different CPT concentrations. It can be seen that B16 cells were sensitive to CPT as well as CPT prodrug, and the cell viability was concentration-dependent. In particular, when CPT was used under the same concentration, the co-delivery system of PEG-SeSe-CPT/CUR displayed obviously better inhibition effect to B16 cells than that of only CPT used. This result indicated that CUR combined with CPT to
inhibit B16 cells. Moreover, the results of B16 cells viability treated with different CUR concentrations were also studied. As shown in Fig. 7, B16 cells were also sensitive to CUR, and its cell viability was also concentration-dependent. Under the same CUR concentration, the co-delivery system of PEG-SeSe-CPT/CUR also displayed much better inhibition effect than free CUR. The IC50 of PEG-SeSe-CPT and CUR to B16 cells after 24 h treatment were obtained through the results from Fig 6 and 7, and were determined as 4.83 mg/mL and 4.79 mg/mL respectively. Using the isobologram analysis, the IC50 of PEG-SeSe-CPT was used as the intercepts of the X axe and the IC50 of CUR was used as the Y axe. Then, an additive effect line was obtained which was shown in Fig. 8. Under this line, it represents that two formulations showed the synergistic effect [36]. In this work, the IC50 of the co-delivery system of PEG-SeSe-CPT/CUR was under this line, indicating that CPT and CUR showed the synergistic effect to B16 cells. To make the synergistic result more rigorous, the non-toxicity hydrophobic PCL (polycaprolactone) segment was used and then the PEG-SeSe-PCL/CUR was prepared for the cytotoxicity assays. The drug of CUR was loaded in PEG-SeSe-CPT micelles in this work, and then the micellization may be affecting the cytotoxicity of the free CUR itself. Then, the CUR-loaded micelles should also be prepared for the synergistic cytotoxicity assay to replace of free CUR. The cytotoxicity results were shown as Fig. S3 and S4 (Supporting Information). It was found that the same conclusion as the above used free CUR was obtained and the synergistic effect was reflected. This result further confirmed that CPT and CUR showed the synergistic effect to B16 cells.
3.5 In vivo assays Besides the in vitro assays, the tumor inhibition assay was also performed using the nude mice bearing B16 tumors. As shown in Fig. 9a and b, it was found that PBS control showed little effect on B16 tumors growth, and the tumor grew with time. After 21 days cultivation, the tumors increased more than 40 times in volume compared with that at the initial stage. For CUR and PEG-SeSe-CPT groups, both of them showed the significant inhibition effect to B16 tumors. In volume, CUR group decreased about 19% compared with PBS control after 21 days, and PEG-SeSe-CPT displayed a much better result and the tumor volume reduced more than 50%. This result indicated that CUR and CPT
could
inhibit
B16
tumors
growth
effectively.
For
the
co-delivery
system
of
PEG-SeSe-CPT/CUR, it showed the best inhibition effect to B16 tumor and the tumor volume
reduced about 78%, much better than that of CUR or CPT used only, suggesting a synergistic therapy effect. The body weight changes and cumulative survival ratios of the treated mice were recorded as shown in Fig. 9C and 9D. It was found that the body weights of mice remained constant for most treatment mice during the experiment, only the mice treated with CUR showed a slight decrease in body weight after 12 days treatments. This result suggested that free CUR formulation could result into the obvious toxicity and side effects. The cumulative survival ratios of treated mice also indicated this. The mice treated with PEG-containing formulations (PEG-SeSe-CPT and PEG-SeSe-CPT/CUR) showed a relative high survival ratios and the mice treated with free CUR showed a relative low survival ratio. These results indicated that the drug carrier could deliver drug and also reduce the toxicity and side effects of drugs. In vivo toxicity assay was carried out through a histological analysis to confirm the safety of PEG-SeSe-CPT/CUR [37]. As shown in Figure 10, there was no visible difference between the sample (bottom row) and the control (top row) histologically. The toxicity of carriers is mainly influenced by their chemical structures, particle sizes, exposure duration and biodistribution, as well as the nature of the surface and terminal groups of the carriers [38]. In this work, the non-observed toxicity of PEG-SeSe-CPT/CUR could be attributed to PEG at a certain point with its excellent biocompatibility [39].
3.6 Blood compatibility For drug delivery systems, their blood stability is a concern. Their interactions with the blood composition are considered as the serious limitation in clinical, and their nonspecific interactions could also severely diminish the half-life and targeting of drugs [40]. The blood compatibility of PEG-SeSe-CPT/CUR was studied by the spectrophotometric record of hemoglobin release from erythrocytes.
Fig.
12A
gave
the
percentage
hemolysis
after
treated
with
different
PEG-SeSe-CPT/CUR concentrations. It was found that PEG-SeSe-CPT/CUR showed good blood compatibility. After 24 h incubation, 10 mg/mL PEG-SeSe-CPT/CUR still showed non-hemolytic, with the extent of hemolysis lower than the permissible level of 5% [41]. Another important concern on blood compatibility for drug carriers is the effect on the blood coagulation [42]. Coagulation at the right time and location is very important and necessary to preserve the normal metabolism. On the contrary, inappropriate coagulation will cause severe effect,
even risks to the living system. As reported, the blood coagulation includes three pathways: intrinsic, extrinsic and common pathway. The indicator, APTT, reflecting the intrinsic and common coagulation pathways, refers to the time from a partial thromboplastin reagent (or CaCl2) is added to a fibrin clot form. PT reflects the extrinsic and common coagulation pathways, and refers to the time from tissue thromboplastin is added to a fibrin clot from [43]. The effects of PEG-SeSe-CPT/CUR on APTT and PT indicators were shown as Fig. 12B. It could be seen that compared to the PBS control, PEG-SeSe-CPT/CUR did not significant affect the APTT and PT within the concentration of 0.01 to 10 mg/mL. These results suggested the blood safety of PEG-SeSe-CPT/CUR in this work and the promising application in cancer therapy.
4. Conclusion To realize the synergetic therapy effect to B16 tumor, a redox prodrug of containing the diselenide bond was synthesized and two antitumor drugs of CPT and CUR were co-loaded. The co-delivery system showed the rapid response to GSH. In the presence of GSH similar to the intracellular environment of tumor cells, the prodrug could be degraded and the two loaded drugs could be released. The cytotoxicity results indicated that both CPT and CUR could inhibit the viability of B16 melanoma cells effectively. Particularly, the co-delivery system showed a much more cytotoxicity to B16 cells, and the synergetic inhibition effect was confirmed by the isobologram analysis. In vivo assay further indicated that the co-delivery system could inhibit B16 tumor growth and showed much better inhibition effect than that of only CPT or CUR was used. Moreover, as an injectable drug carrier, the blood compatibility of the co-delivery system was also confirmed, suggesting that this redox co-delivery system have a potential application in B16 tumor therapy.
Acknowledgement This work was financially supported by Science and Technology Planning Project of Guangdong Province (2017ZC0003) and the Special Fund for Scientific Research of the First Clinical Medical College of Jinan University (2017308).
References
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Scheme 1. Synthesis routes to diselenide-modified PEG.
Scheme 2. Synthesis route to carboxylate CPT.
Scheme 3. Chemical structure of PEG-SeSe-CPT.
Figure 1. 1H NMR spectrum of PEG-SeSe-CPT (DMSO-d6, 25 °C).
Figure 2. Effect of PEG-SeSe-CPT concentration on I1/I3 ratio of pyrene in aqueous PEG-SeSe-CPT solution.
Figure 3. GPC trances of PEG-SeSe-CPT in the absence and presence of 10 mM GSH.
Figure 4. TEM images (a and b) and particle sizes (c and d) of PEG-SeSe-CPT in the absence and presence of 10 mM GSH.
Figure 5. In vitro release profiles of CPT (a) and CUR (b) from PEG-SeSe-CPT/CUR in the absence and presence of GSH (n = 3).
Figure 6. Cytotoxicity of PEG-SeSe-CPT/CUR compared with free CPT and PEG-SeSe-CPT (n = 5).
Figure 7. Cytotoxicity of PEG-SeSe-CPT/CUR compared with free CUR (n = 5).
Figure 8. Isobologram analysis of the antiproliferation effect of PEG-SeSe-CPT and CUR on B16 cells.
Figure 9．Representative image of B16 tumors at 21 days treatment (A) and the tumor growth profiles (B) treated with various formulations. (C) The change of body weight of the mice with the experimental time. (D) The cumulative survival rate for different formulations. (1: PBS; 2: CUR; 3: PEG-SeSe-CPT; 4: PEG-SeSe-CPT/CUR) (n = 5).
(B)
(A) 1
(C)
2
3
4
(D)
Figure 10. Representative HE staining of organ histology by PEG-SeSe-CPT/CUR (top row) and PBS (bottom row).
Figure 11. The hemolysis and clotting analysis of PEG-SeSe-CPR/CUR (n = 5).
Graphical abstract
aqueous
CUR loading
(PEG-SeSe-CPT prodrug) (PEG-SeSe-CPT micelles)
(PEG-SeSe-CPT/CUR)
GSH PEG SeSe CPT
CUR
Highlight
1. A redox prodrug co-loading camptothecin and curcumin was prepared. 2. The co-delivery system showed a synergetic inhibition effect to B16 melanoma cells. 3. The co-delivery system showed a good biocompatibility.