3.1. Characterizations of mPEG2000-PBAE
In the FT-IR spectrum (Fig. 1a), the peak at 2889 cm− 1 is the stretching vibration of -CH2- and the peak at 1244 cm− 1 is the bending vibration of -CH2-. The peak located at 1736 cm− 1 is the carbonyl absorption peak. The disappearance of the characteristic peak of the stretching vibration of the double bond (-C = C-) at 1718 cm− 1 indicates that the double bond was broken and the mPEG2000-PBAE copolymer was successfully prepared. The structure of mPEG2000-PBAE was determined by 1HNMR spectroscopy (Fig. 1b) in CDCl3. The chemical shift signals 2.86 ppm, 2.66 ppm, 1.94 ppm, and 1.63 ppm represent the chemical shifts of the hydrogen protons on the piperidine ring in mPEG2000-PBAE, respectively. 4.05 ppm, 3.63 ppm and 2.86 ppm denote the chemical shifts of hydrogen protons in -O-CH2CH2-O-C = O-CH2-, respectively.
In Fig. 1c, when the concentration of mPEG2000-PBAE was in the range of 1×10− 4 ~ 5×10− 2 mg/mL, the I338/I336 ratio remained basically constant. When the concentration was greater than 5×10− 2 mg/mL the I338/I336 ratio gradually decreased with increasing concentration. In Fig. 1d, the intersection of the two lines is the CMC of mPEG2000-PBAE, and the CMC of mPEG2000-PBAE was calculated to be 4.83×10− 2 mg/ml. The low CMC of the copolymer suggests that mPEG2000-PBAE has the ability to self-assemble in water and still form micellar solutions at small concentrations, facilitating the loading and delivery of lipid-soluble drugs.
3.2. Preparation of TPL/PTX-PMs
Previous studies have shown that TPL and PTX synergize against tumors, but the ratio of TPL and PTX in combination is uncertain [13, 27]. Therefore, we used MDA-MB-231 cells as a model to examine the ratio of the two drugs in combination. First, the inhibitory effects of TPL and PTX on the proliferation of MDA-MB-231 cells were examined by CCK8 assay, respectively. The results showed that both TPL and PTX had significant growth inhibitory effects on MDA-MB-231 cells, and the degree of inhibition showed a positive correlation with the single drug concentration (Fig. 2a,2b). Moreover, it is clear from Fig. 2c that the minimum effect concentration of TPL is approximately twice that of PTX. TPL at 1.25 µg/mL could inhibit cell growth by 10–20%, while PTX required 2.5-5 µg/mL to achieve the same inhibitory effect. It further indicates that TPL has a stronger inhibitory effect on MDA-MB-231 cells compared to PTX. Based on the above results, the ratio of 1:2, 1:4, and 1:6 combination dosing of TPL and PTX were examined. In Fig. 2d, it is shown that each group of the combination drug showed higher inhibition of MDA-MB-231 breast cancer cells than TPL alone, and the degree of inhibition was also correlated with the concentration of the combination drug group. It can be indicated that the combination of the two drugs could better inhibit the proliferation of tumor cells.
Based on the above experimental results of CCK-8, the IC50 of single drug was fitted using GraphPad Prism 8.0, and the IC50 of different ratios of combined drugs was fitted using Calcusyn statistical. Based on the median-effect equation, regression equations and correlation coefficients (r) were obtained for the single-drug group and the group administered in different ratios in combination (Table S1). It can be seen that the inhibitory effect of PTX alone on MDA-MB-231 cells is stronger than that of TPL alone. The IC50 for both TPL and PTX combined administration was significantly lower than that of the group using PTX alone, and the IC50 values decreased significantly with an increasing proportion of PTX. This indicates that PTX enhances the proliferation inhibitory effect of TPL on MDA-MB-231 cells. Calcusyn statistical software was applied to derive the association index (CI) when different ratios of TPL and PTX were combined to act on MDA-MB-231 cells (Table S2). The CI curves for the combination of TPL and PTX at different ratios are shown in Fig. 2e. In the figure, CI > 1 indicates antagonism, CI = 1 indicates addition effect, and CI < 1 indicates synergistic effect. The fa was between 0.5 and 0.8 to indicate the strong synergistic effect of the drug combination. Therefore, when the combination drug ratio of TPL and PTX is 1:4, the CI curve is mostly in the high effect area (CI < 1), so the combination drug at this ratio can exert a better anti-tumor effect. In overview, we chose to load TPL and PTX into PMs in a 1:4 ratio for subsequent studies.
3.3. Characterization of PMs and TPL/PTX-PMs
3.3.1. Stability and pH sensitivity of PMs
Colloidal storage stability is one of the important factors to evaluate the merits of nano-delivery systems. We investigated the stability of PMs, and the results showed that the particle size and PDI of PMs hardly changed within two weeks (Figure S1a), indicating good stability of PMs. The changes in the particle size of PMs at different pH conditions are shown in Fig. 3a. When the pH was higher than 7.4, the change in particle size was not obvious. pH between 7.4 and 6.0 showed a significant increase in the particle size of PMs up to about 500 nm. In this pH range, the tertiary amine group in PBAE undergoes protonation, causing a gradual change in the hydrophobic segment of the amphiphilic material, which results in a swelling of the carrier structure and leads to an increase in particle size. After the pH was reduced to below 6.0, the particle size basically stopped increasing because the protonation of tertiary amine was close to saturation. The results indicate that the prepared PMs are pH sensitive.
3.3.2. Biocompatibility of PMs in different PH environments
The biocompatibility of nano-delivery carriers is one of the important factors to be considered for the future clinical application of novel drug formulations. The proliferation inhibition of MDA-MB-231 cells by self-assembled micellar carrier materials was assessed by CCK-8 assay. We tested the effect of PMs at concentrations ranging from 15.625 to 500 µg/mL on cell viability at pH 6.5 and 7.4, respectively. The results are shown in Fig. 3b. Cell viability was still close to 100% after 48 h of co-culture of MDA-MB-231 with PMs ranging from 15.625 to 500 µg/mL at pH 6.5 and pH 7.4. This indicates that the carrier materials self-assembled into micelles have good biocompatibility in both pH 6.5 and pH 7.4 environments.
3.3.3. Particle Size, Zeta Potential and Morphology of TPL/PTX-PMs
The average particle size of TPL/PTX-PMs prepared by the thin film dispersion method was 97.29 ± 1.63 nm, with a PDI of 0.237 ± 0.003 and a zeta potential of 9.57 ± 0.80 mV (Fig. 3c, S1b, S1c). The micelles were observed under scanning electron microscopy, and the micelles were spherical in shape and had a three-dimensional appearance (Figure S1d).
3.3.4. Drug loading and encapsulation efficiency of TPL/PTX-PMs
Calculated according to the formula, LC% was 6.19 ± 0.21%. EE% was 88.67 ± 3.06%. The change in the mean particle size and drug loading of TPL/PTX-PMs over 14 days are shown in Fig. 3d. The micelle particle size changed very little over the two weeks, while the drug loading decreased slightly from day 8 to day 14 by 4.79%. The decrease in drug loading may be due to a slight drug leakage of TPL/PTX-PMs in the second week, corresponding to a slight decrease in micelle size from 96.67 nm to 95.74 nm.
3.4. pH-responsive Release of the Drug
The rapid growth of the tumor results in a lack of nutrients and oxygen in some tissues, producing acid metabolites. The accumulation of these acid metabolites causes the pH of the tumor microenvironment to be slightly lower than that of normal tissues [28–29]. pH-responsive nano-drug delivery systems loaded with antitumor drugs can be released at an acidic pH to achieve targeted drug release in the tumor microenvironment. The results of in vitro release showed that the drug release of TPL/PTX-PMs was pH-dependent. Under acidic (pH 5.5) conditions, TPL/PTX-PMs undergo ionically disassembling, so that the drug is released abruptly, with 65.57% of the drug released from TPL/PTX-PMs in the first 6 hours, and the percentage of drug released reaches a maximum in the following 6 to 12 hours. Abrupt release of TPL/PTX-PMs was also observed under mildly acidic conditions (pH 6.5), but the maximum percentage of drug release was only 64%, which was lower than that under acidic conditions. Under neutral conditions (pH 7.4), because the structure of TPL/PTX-PMs is basically stable, the drug can only be released through leakage, so the drug release curve showed a natural slow-release trend. In addition, the cumulative release profile of the drug amount (Fig. 3F) also showed a similar trend to the cumulative percentage release profile. Because the content of PTX in TPL/PTX-PMs was higher than that in TPL, the release amount of PTX was also higher than that in TPL, which was consistent with the drug delivery ratio when the micelles were initially prepared. From the above results, it can be seen that TPL/PTX-PMs has a fast pH response, and TPL and PTX burst out from TPL/PTX-PMs under acidic or mildly acidic conditions, while under normal physiological conditions, the release rate of TPL and PTX was not high, and the cumulative release amount was also low. It shows that it has certain biological safety and will not kill normal cells.
3.5. Cellular uptake
Cellular uptake experiments use the green fluorescence of C6 to localize the distribution of micelles within the cell, and the nucleus is localized by DAPI (blue) staining, thus simulating the distribution of the drug being loaded after being endocytosed into the cell. The fluorescence distribution of Free-C6 and C6-PMs in MDA-MB-231 cells was observed by a confocal laser scanning microscope.
Figure 4a shows that the green fluorescence intensity of C6-PMs was obviously higher than that of Free-C6 in MDA-MB-231 cells. The fluorescence intensity of C6-PMs reached the maximum after 6 hours of co- incubation, and this time coincided with the drug release time of TPL/PTX-PMs, i.e., the drug release of TPL/PTX-PMs increased steeply from 6 hours under acidic conditions. This result indicated that C6-PMs increased the uptake of MDA-MB-231 cells and increased drug accumulation in tumor tissue sites.
As shown by the quantitative fluorescence analysis (Fig. 4b, Table S3), the Free-C6 fluorescence intensity at 0.5 h incubation was very low and almost not observed. At 6 h incubation time, although the Free-C6 fluorescence intensity reached 49.7 ± 0.56, it was only half of that of C6-PMs incubated for 0.5 h. The fluorescence intensity of C6-PMs was always about 4 times higher than that of Free-C6 under the same incubation time. In summary, the co-delivery of TPL and PTX with PMs significantly enhanced the intracellular concentration of the drugs and thus will have strong antitumor activity.
3.6. In vitro cytotoxicity
The cytotoxicity of PTX and TPL synergistically on MDA-MB-231 cells was determined by CCK-8 assay. As shown in Fig. 4c, TPL/PTX-PMs had the greatest cytotoxicity and the strongest inhibitory proliferation effect on MDA-MB-231 cells. At the same dose, the cytotoxicity of TPL/PTX-PMs was about 15 times greater than that of the free TPL, 8 times greater than that of the free PTX, and 2 times greater than that of the free TPL/PTX. Therefore, only a low dose of TPL/PTX-PMs is needed to achieve the proliferation inhibition effect on tumor cells that can only be achieved with a high dose of free drug. It is also for this reason that TPL/PTX -PMs can significantly reduce the dose of the drug, and thus can effectively mitigate the damage to normal cells caused by the drug itself in clinical applications.
In order to investigate the optimal dose of TPL/PTX-PMs to inhibit the proliferation of tumor cells, we tested the inhibitory rate of TPL/PTX-PMs on the proliferation of MDA-MB-231 cells in a concentration range of 1. 25–40 µg/mL. As shown in Fig. 4d, the proliferation inhibition rate of TPL/PTX-PMs and Free-TPL/PTX on MDA-MB-231 cells increased with the increase of drug concentration, and the anti-tumor effect was dose-dependent. However, we can clearly find that only 5 µg/mL of TPL/PTX-PMs can reduce the viability of tumor cells to less than 20%, indicating that the anticancer activity of TPL/PTX-PMs, although dose-dependent, has a strong anticancer effect at low doses.