As shown in Supplementary Table 1 (available online), when the ratio of drug and protein increased from 0.001 to 0.100, the diameter of formed CCM-CSA-NPs had almost no significant change firstly and then gradually increased, while there was no significant change for that of Dox-CSA-NPs. This result had primarily demonstrated that unlike soluble Dox, the highly hydrophobic CCM might play an important role in the formation of CSA-NPs via this self-assembly method.
Owing to the overlap of the absorption spectrum for CCM and Dox UV-vis analysis, fluorescence analysis with good specificity had been applied to determine the level of Dox. For the fixed extraction wavelength 480 nm, there was a significant difference for the emission spectra of CCM and Dox (Supplementary Fig. 1, available online), demonstrating the feasibility to quantify the level of Dox by fluorescence spectrophotometry. Besides, recovery analysis had been performed to evaluate the accuracy of the determined level of CCM or Dox by either UV-vis or fluorescence spectrophotometry and results had been listed in Supplementary Table 2 (available online). As shown, considering the spectrum overlap for CCM, the determined level turned out to be slight higher than the added concentration. However, the recovery ranged from (104.3±2.7)% to (104.3±2.7)%, demonstrating acceptable deviation. While for Dox, the satisfactory recovery varied from (99.8±1.9)% to (101.2±1.3)% had also demonstrated the accuracy of fluorescence spectrophotometry. According to the results outlined in Supplementary Table 3 (available online), the DLE increased with the increase of drug/protein ratio. However, there was a relative sharp decrease of DEE when the ratio reached 0.1, partly resulting from the restricted encapsulation space of CSA. Therefore, we chose 0.05 as the optimized drug/protein ratio for experiments. Meanwhile, previous reports suggested molecular interactions of drug/ chemosensitizer are closely related to the weight ratio in nanocarriers with a desirable value of 1 : 1 in the case of CCM/Dox. Therefore, the weight ratio of CCM and Dox was set as 1 : 1 in this study.
As shown in Fig. 1A, a colloidal dispersion of prepared drug-loaded CSA-NPs was visualized after re-dissolving the lyophilized materials indicating its desirable stability. Meanwhile, the hydrodynamic diameters (Fig. 1B and Table 1) of obtained protein NPs were (195.2±7.5) nm, (208.6±10.9) nm, (210.5±9.8) nm, and (215.6±8.2) nm for blank CSA NPs, CCM-CSA NPs, Dox-CSA NPs and CCM/Dox-CSA NPs, respectively. Narrow particle size distribution was observed suggesting the good homogeneity, which was further corroborated by a low PDI value (<0.2) for all samples. All the samples had displayed a negative charge (Fig. 1C and Table 1) in which the average Zeta potential was (−30.8±1.3) mV, (−27.5±2.3) mV, (−24.6±2.2) mV and (−26.2±1.6) mV, respectively.
Formulation Hydrodynamic size (nm) PDI Zeta potential (mV) Blank CSA NPs 195.2±7.5 0.09±0.01 −30.8±1.3 CCM-CSA NPs 208.6±10.9 0.07±0.03 −27.5±2.3 Dox-CSA NPs 210.5±9.8 0.12±0.02 −24.6±2.2 CCM/Dox-CSA NPs 215.7±8.2 0.10±0.04 −26.2±1.6 Data represented as mean±SD (n=3).
Table 1. Hydrodynamic diameter, PDI and Zeta potential of prepared CSA-NPs
The morphology of obtained nanoparticles was evaluated using SEM and TEM. As shown in Fig. 1D and E, all NPs exhibited uniform spherical shape and homogeneous distribution with the size of ~200 nm in accordance with the results from DLS. The stability of prepared CSA-NPs was also investigated (Fig. 2A) in different media including deionized water, 0.9% NaCl, 5% glucose and phosphate buffered saline. These results indicated that the particle size almost remained unchanged during the 2 weeks which represented a good stability of prepared CSA-NPs. The calculated DLE and DEE of NPs were listed in Table 2, demonstrating satisfactory DLE and DEE.
Formulation DLE (%) DEE (%) CCM Dox CCM Dox CCM-CSA NPs 6.9±0.4 − 67.2±3.9 − Dox-CSA NPs − 7.5±0.7 − 66.3±2.6 CCM/Dox-CSA NPs 3.4±0.5 3.8±0.6 62.2±5.7 69.4±3.5
Table 2. Drug loading efficiency and drug entrapment efficiency of prepared CSA-NPs (n=3)
The solubility of free CCM and CCM encapsulated in CSA-NPs was determined by UV-vis spectrophotometry and results were displayed in Fig. 2B. The inset was the photographs of prepared CCM solution, indicating that most of the free CCM remained insoluble while enhanced solubility was observed once formulated with CSA-NPs of high solubility. Besides, the calculated solubility of CCM-CSA-NPs was (540.67±35.72) μg/mL, a 140-fold increase compared to that of free CCM [(3.89±0.32) μg/mL], which was promising for practical uses.
During the break-down process of intramolecular disulfide bonds, the hydrophobic region of CSA was exposed in which different drugs could be encapsulated. To examine the self-assembling process of CSA, the number of free sulfhydryl groups was firstly determined by Ellman's method. As shown in Fig. 2C, when incubated with reducing GSH, the number of free sulfhydryl groups in the CSA molecule was dramatically increased from 5.1 to 24.7 and it dropped to ~6.4 after the formation of nanoparticles comparable with that of native CSA. Moreover, to examine GSH-responsive attributes of CSA nanocomposite, the hydrodynamic diameter of nanoparticles was measured in the absence or presence of 20 mmol/L GSH at 37 °C. When the GSH was added into the solution, the particle diameter decreased while the PDI increased with the incubation time according to measured DLS (Fig. 2D). The diameter decreased from 214 to 106 nm within 24 hours, suggesting the dissociation of nanoparticles into smaller fragments. This observation may verify the redox-triggered responsive behaviors of prepared nanoparticles.
Fig. 3A showed the UV-vis spectra of CCM or Dox formulated with CSA NPs. The absorption peaks around 430 and 480 nm corresponding to free CCM and Dox (Supplementary Fig. 2, available online) were observed, respectively. Moreover, for the CCM and Dox co-bound NPs, the UV spectra were also similar with that of CCM/Dox mixed solution at the same weight ratio, in which the characteristic peak of Dox at ~530 nm was also observed. Therefore, it could demonstrate the successful loading of both CCM and Dox into CSA NPs.
The UV-vis spectra of native CSA, GSH-reduced CSA and prepared CSA NPs were also recorded (Fig. 3B). The peak around 256 nm was from Dox validating the existence of Dox in the prepared NPs. Compared with native CSA, there was a slight blue shift in the reduced state, which suggested that during the self-assembly of CSA, the buried tryptophan may be more exposed to the solvent environment. Furthermore, the absorption peak of tryptophan shifted from 275 to 283 nm after NPs formation which confirmed the increased hydrophobicity.
The conformational changes of CSA NPs were also investigated by fluorescence spectroscopy. The intrinsic fluorescence signal of albumin was closely related with its compositional tryptophan (Trp) residues. As shown in Fig. 3C, the λmax of native CSA was ~340 nm and a slight red shift from 340 to 344 nm was observed for reduced form in the presence of GSH. While after the formation of nanoparticles, the λmax shifted toward lower wavelength (337 nm), and the fluorescence intensity was significantly decreased in the absence of CCM. Besides, the fluorescence quenching of CSA in the presence of CCM confirmed the binding of CCM to CSA . Notably, the superimposable fluorescence spectra of CCM-CSA-NPs with that of CSA-NPs may suggest the role of CCM in the self-assembling of proteins (Supplementary Fig. 3, available online).
As shown in Fig. 4, CCM alone showed little effect on A549 cells whereas combined use of CCM and Dox exhibited remarkably improved cytotoxicity. The IC50 value of single drug or two drugs at different concentrations after 24 hours was calculated based on MTT assay and results were listed in Table 3. The IC50 values for cells incubated with Dox alone or in combination with CCM at the dosage of 1, 5 and 10 μg/mL (24 hours) were 0.64, 0.46, 0.31 and 0.19 μg/mL, respectively. Similarly, the values for cells incubated with CCM alone or in combination with Dox at 0.1, 0.5 and 1.0 μg/mL (24 hours) were 34.9, 18.1 and 7.79 μg/mL, respectively. To quantitatively examine the synergistic effect of CCM and Dox, combination index (CI) values were calculated by CompuSyn software. Considering the weight ratio of CCM and Dox was 1 : 1 in the NPs, the calculated CI values for the assay was used with the same ratio. Notably, the obtained CI values from both assays were 0.69 and 0.71 respectively, which revealed that the combination of CCM and Dox in the CSA nanocomposite possessed strong synergistic effects on A549 cells.
Formulation IC50 value Free CCM 34.94 CCM+0.1 μg/mL Dox 18.12 CCM+0.5 μg/mL Dox 7.79 CCM+1.0 μg/mL Dox N.A. Free Dox 0.64 Dox+1 μg/mL CCM 0.46 Dox+5 μg/mL CCM 0.31 Dox+10 μg/mL CCM 0.19 CCM-CSA-NP 867.28 Dox-CSA-NP 32.49 CCM/Dox-CSA-NP 26.50
Table 3. IC50 of different combinations and prepared nanoparticles in A549 cells
The cytotoxicity of blank CSA-NPs was firstly investigated to evaluate its biocompatibility. As shown in Supplementary Fig. 4 (available online), there was almost no effect on the cell viabilities for A549 cells even at the concentration of NPs up to 500 μg/mL, verifying an ideal biocompatible CSA NPs prepared via the self-assembly method as nano-carrier.
Then the possible synergistic effects against A540 cells of the encapsulated CCM/Dox in the CSA nanoparticles were further evaluated by MTT assay. As shown in Fig. 5, CCM-CSA-NPs showed virtually little effect on the cell viability even at a relative high concentration pointing to the low toxicity of CCM. However, for the Dox-CSA-NPs and CCM/Dox-CSA-NPs, a concentration-dependent decrease in cell viability was apparently observed. Compared with Dox-CSA-NPs, CCM/Dox co-bound CSA NPs displayed higher cytotoxicity consistent with results of combination index analysis. The calculated IC50 for CCM-CSA NP, Dox-CSA NP and CCM/Dox-CSA NP were 867.28, 32.49 and 26.50 μg/mL, respectively, demonstrating the co-bound CSA were more effective than those single drug-loaded NPs. Taken together, results of both CI values of drugs and cytotoxicity evaluations of drug-loaded CSA NPs have identified a synergistic effect of CCM and Dox.
As the DLS analysis revealed the redox responsiveness of prepared nanoparticles, the in vitro release kinetic files of CCM/Dox from CSA NPs were investigated in the presence of different concentrations of GSH (Fig. 6A and B). Unlike free drugs (Supplementary Fig. 5A), there was no obvious burst release for all NPs and the drugs were released from CSA NPs in a mainly sustained manner. The relatively rapid drug release at the beginning (~2 hours) was likely attributed to the desorption of drugs at the surface of the formed nanoparticles.
Next, in the presence of 5 mmol/L GSH, the calculated releasing amounts of CCM and Dox over 48-hour incubation were (43.7±2.7)% and (41.6±3.3)%, respectively. At 10 mmol/L GSH, it reached to (60.5±5.2)% and (67.1±4.1)%, respectively. By contrast, only (39.7±4.4)% and (35.2±5.5)% were released in the absence GSH. Furthermore, for the nanoparticles incubated with PBS with 10 mmol/L GSH mimicking the reducing environmental conditions of cytosols, a significantly increased release was observed, demonstrating the prepared CCM/Dox-CSA NPs was a suitable drug delivery system for cellular uptake.
Similar with that of pH 7.4, a burst phase (~51.5%) was observed for the release profile of CCM and Dox in acidic condition (Supplementary Fig. 5B). As for drugs encapsulated CSA NP, similar GSH-dependent release behavior was identified (Fig. 6C and D). Notably, in the absence of GSH, 39.5% and 36.4% of CCM and Dox was released. By contrast, at 10 mmol/L GSH, it increased to 66.1% and 71.1%, respectively. Although there was little pH-responsiveness for the constructed delivery system, the slightly improved drug release might be attributed to the pH effect on the stability of the formed disulfide bonds.
To further explore the release mechanism, the in vitro release data were fitted by different models including first order, Higuchi and Korsmeyer-Peppas models. The obtained correlation coefficient was listed in Table 4. According to the correlation coefficient, the release of CCM and Dox from CSA-NPs was well fitted by first order model with the higher r2-values. Moreover, based on the n value obtained from Korsmeyer-Peppas model which was less than 0.45, the release mechanism was likely involved with the Fickian diffusion process.
Condition First order r2 Higuchi r2 Korsmeyer-Peppas r2 n CCM PBS pH 7.4 0.9863 0.8885 0.9757 0.3820 5 mmol/L GSH in PBS pH 7.4 0.9911 0.88786 0.9170 0.3610 10 mmol/L GSH in PBS pH 7.4 0.9752 0.9038 0.9242 0.4176 NaAc-HAc pH 5.0 0.9909 0.9061 0.9366 0.3231 5 mmol/L GSH in NaAc-HAc pH 5.0 0.9844 0.8994 0.9465 0.3186 10 mmol/L GSH in NaAc-HAc pH 5.0 0.9829 0.9152 0.9492 0.3719 Dox PBS pH 7.4 0.9882 0.8651 0.9064 0.4011 5 mmol/L GSH in PBS pH 7.4 0.9779 0.8774 0.9148 0.3701 10 mmol/L GSH in PBS pH 7.4 0.9906 0.9152 0.9211 0.4127 NaAc-HAc pH 5.0 0.9902 0.8789 0.9427 0.3905 5 mmol/L GSH in NaAc-HAc pH 5.0 0.9853 0.8910 0.9472 0.3424 10 mmol/L GSH in NaAc-HAc pH 5.0 0.9932 0.8951 0.9036 0.3318
Table 4. The fitting parameters of release kinetic profiles of CCM and Dox
The cell uptake and synergistic cytotoxicity of prepared nanoparticles were firstly investigated with a fluorescence microscope. Initially, after 6 hours incubation, red signals resulted from the intrinsic fluorescence of Dox were observed (Supplementary Fig. 6), suggesting the cellular uptake of nanoparticles. After 24 hours incubation, considerable amounts of Dox-CSA-NPs and CCM/Dox-CSA-NPs were observed to enter A549 cells judged from the Dox fluorescence intensity. Accordingly, TUNEL assay was employed to visualize the apoptosis induced by different nanoparticles. The cells treated with CCM-CSA-NPs showed almost none or week green signals while those treated with Dox-CSA-NPs and CCM/Dox-CSA NPs displayed improved green fluorescence. After 24 hours incubation, the drug-loaded CSA-NPs were absorbed by cancer cells to induce the apoptosis as demonstrated for both Dox-CSA-NPs and CCM/Dox-CSA-NPs. These results were also consistent with the cytotoxicity evaluation results in which combinational use of CCM/Dox was more effective than the single drug. Furthermore, to quantitate the synergistic cytotoxicity, high content screening assay was performed. As shown in Fig. 7, the percentage of positive cells which met the threefold pre-set in the TUNEL method in the group incubated by CCM/Dox-CSA-NPs were significantly higher than that treated by single drug loaded CSA-NPs. The mean fluorescence intensity also showed the similar results, and more broadly, apoptosis induced by CCM/Dox co-bound CSA-NPs was much higher than that of CCM or Dox alone.
GSH-responsive curcumin/doxorubicin encapsulated Bactrian camel serum albumin nanocomposites with synergistic effect against lung cancer cells
- Received Date: 2019-03-05
- Accepted Date: 2019-04-28
Abstract: The aim of this study was to prepare camel serum albumin (CSA) nanoparticles using a self-assembly strategy to co-immobilize curcumin (CCM) and doxorubicin (Dox) which was in favor of combined chemotherapy and biomedical applications of bactrian (Camelus bactrianus) CSA. The constructed CSA nanoparticles with the size around 200 nm displayed a high degree of polydispersity and further encapsulation of CCM and Dox caused no apparent morphological changes to the nanocomposite (CCM/Dox-CSA NPs). The synergistic cytotoxic effect of CCM and Dox on cancer cell A549 was observed with the calculated combination index less than 1.0. Moreover, the release kinetic profile of encapsulated drugs showed a concentration dependence of glutathione (GSH) originating from the GSH used in nanoparticle formation to break the intramolecular disulfide bonds. In vitro cytotoxicity evaluations also revealed that CCM/Dox-CSA NPs showed higher cytotoxicity than that of single drug loaded CSA-NPs, which was also validated by high content screening assay. Taken together, the CCM/Dox-CSA NPs with redox-responsive attributes provided an integrated protein-based combinational drug-delivery matrix to exert synergistic effects.