The main objective of this research was to develop enzymatically bioactive polymeric nanomicelles for solubilization and efficient transportation of anticancer chemotherapies into cancer cells. To pursue such aim, we synthesized novel waterborne polyurethane with ability to form stable nanomicelles in aqueous media. The polymeric matrix was prepared based on commonly used method for preparation of waterborne polyurethane through introduction of anionic carboxylate ion in the backbone of polymers. For enhancing the stability of polymeric aqueous solution and controlling the molecular weight of polymer, the block ratio of OH and NCO containing moieties was adjusted to less than 1, and subsequently the free terminal NCO groups were blocked with phenol blocking agent. To the best of our knowledge, this is the first report on utilization of this polymeric nanocarrier for encapsulation and delivery of PTX. We speculate that the PTX loaded nanomicelles are less viable for uptake by RES. Thus, longer circulation of the PTX loaded nanomicelles in blood and greater EPR effects in tumor microenvironment are expected. And since the cosolvent mediated toxicity has been resolved, higher doses of PTX can be administered using these nanomicelles.
Having possessed suitable negative zeta potential charge, the PTX loaded PU based nanomicelles displayed no aggregation and lower level of CMC (Table 1). It has previously been reported that negatively charged nanoparticles can significantly internalize by the primary human alveolar immortal AT1 cells . Further, hydrophilic nanomicelles were shown to stay in blood stream as long circulating drug delivery system [31, 32], during which period the entrapped drug molecules can be protected from biological impacts (e.g., glomerular excretion, enzymatic degradation) and recognition of nanomicelles by RES. Also, the hydrophilic nanoparticles in range of 50-200 nm are deemed to be less prone to such biological impacts. The PTX loaded nanomicelles in our study displayed similar size range (~50 nm) (Table 1), and resulted in high physical stability without aggregation and/or sedimentation at room temperature for 9 days (Figure 3). The drug loading efficiency of the PTX loaded PU based nanomicelles was about 80%, demonstrating their higher drug encapsulation capacity. It should be stated that the high loading efficiency is generally considered as an advantage for the amphiphilic polymeric carriers because of reserving high amount of hydrophobic drug in hydrophobic core of micelle leading to an increased solubility of drug in aqueous media . Our findings appeared to be somewhat similar to the results obtained from pluronic P105/L101 mixed polymeric micelles , however the smaller size of the PU based nanomicelles (i.e., 50 nm vs. 185 nm) may result in more efficient EPR effects. Ideally, a minimal leakage of the loaded drugs from drug nanocarriers should occur during circulation of the nanocarriers in blood stream. However, once taken up by cells, such nanocarriers should liberate the loaded drugs in the cytosol of the target cells to warrant the efficiency of chemotherapy. The release of drugs from pH-sensitive polymeric nanocarriers were shown to be triggered by the lower pH of the endosomal compartments . Furthermore, biodistribution of pH-sensitive polymeric nanomicelles were shown to possess significantly longer blood circulation pattern and higher accumulation of drugs in solid tumors . We looked at the drug release profiles of the PTX loaded nanomicelles at two different pH conditions (i.e., pH 7.4 and 5.4) and witnessed a faster PTX liberation profile at pH 5.5 (Figure 5). We assume that the ionic structure of PUD may fail at the lower pH value, perhaps as a result of transformation of carboxylate ionic groups to their acidic form and subsequent separation of polymeric carrier. In these nanomicelles, the release of PTX appeared to be dependent upon both diffusion and biodegradation processes. As shown in Figure 3, we monitored the particle sizes over the release time to confirm the possibility of degradation during the release period under an aqueous condition. The results showed no noticeable changes in the sizes of the nanomicelles measured by DLS at pH 7.4. This implies that drug release was mainly due to diffusion at pH 7.4, while diffusion and slight polymer degradation seemed to be the main mechanism of drug release at pH 5.4. Among the release kinetics models used for analysis of data (Table 2), the release of PTX from the nanomicelles was best fitted by Higuchi model (R2 = 0.99) that describes the release of drugs from matrix as a square root of time dependent process based on Fickian diffusion law .
The cytotoxicity impacts of paclitaxel, polymeric carrier and drug loaded nanomicelles were also studied in the human breast cancer, MCF-7 cells. The PTX loaded nanomicelles showed a significant influence on the prevention of cell proliferation as compared to the untreated control and positive controls (i.e., PTX and polymers treated cells). Despite biologic impacts of different polymeric carriers (e.g., polyethylenimine) in target cells , the PU polymer alone showed no cytotoxic effects in the treated cells. The PTX alone induced somewhat cytotoxic effects in the treated cells; however such cytotoxicity was not comparable to that of the PTX loaded nanomicelles. Although we did not conduct a direct examination for the cellular uptake of nanomicelles, the cytotoxic effects and the gene expression changes induced by the PU nanomicelles can presumably indicate high uptake of the PTX loaded nanomicelles by the MCF-7 cells. Based upon physicochemical characteristics of these nanomicelles, we contemplate that these nanostructures can release the loaded drugs in the endosomal compartments in a pH-dependent manner. Similarly, pH-sensitive poly(2-tetrahydropyranyl methacrylate) [poly(THPMA)] nanospheres have recently been developed and shown higher cellular uptake potential with a pH-dependent release of the loaded drug (PTX) . Micellar formulation of PTX using cholesterol-grafted poly(N-isopropylacrylamide-co-N, N-dimethylacrylamide-co-undecenoic acid) was reported to provide nanomicelles (~220 nm) with low CMC (~ 20 mg/L) and fast liberation of drug at pH 5.0 . These PTX loaded nanomicelles were shown to induce toxicity against KB cells, in which a receptor-mediated endocytosis process was responsible for nanomicelles transportation . Given the fact that enzymatic oxidation is the primary mechanism of biodegradation of the PU based nanomicelles , the PTX loaded nanomicelles used in our study may function as safer long circulating nanocarriers with ability of drug liberation into the cytosol.
To reveal the cytotoxic mechanism(s) of the PTX loaded nanomicelles, we looked at the gene expression profile of some pivotal genes related to apoptosis, in which the death signal is generated inside the cell after a chemical treatment leading to release of mitochondrial factors such as cytochrome c into the cytosol. The liberated cytochrome c interacts with APAF1 then triggers caspase 9. This complex is called apoptosome and it acts as a holoenzyme resulting in caspase 9 activation and finally leading to caspase 3 activation [40, 41]. Thus, the release of cytochrome c into the cytosol of the target cells can basically be considered as a key regulatory step than can irreversibly coerce cells to commit an intrinsic apoptosis . The STAT1 gene is represented by an anti-proliferative effect and consequent extrinsic apoptosis. Subsequent studies have shown that the STAT1 activates transcription of the CASP1 gene, a member of the protease family producing apoptosis and, in addition, activates transcription of the genes FAS and FASL, activators of the caspase system [43, 44]. In our study, we examined the expression of some of these genes and witnessed significant overexpression of the genes studied (i.e., CYCS, CASP3, CASP9 and STAT1) in the treated cells with the PTX loaded nanomicelles even after 48 h. Interestingly, the expression of CYCS was not affected by the polymer itself as compared to the untreated control cells, which may be assumed as lack of intrinsic toxicity of polymer alone. However, STAT1 gene was somewhat overexpressed in the treated cells with the polymeric nanocarrier compared to the untreated control cells. We also observed overexpression of CASP3 and CASP9 induced by the polymer itself, which was not surprising since the overexpression of CASP3 and CASP9 genes may occur via CASP1 pathway activated by STAT1 gene (Figure 7). It should also be stated that a urethane compound diethyl-4,4'-methylenebis (N-phenylcarbamate) was shown to induce inhibitory effects on tubulin polymerization in the Chinese hamster cell lines (CHL and V79) and a human cancer cell line (HeLa S3), causing mitotic arrest even greater than that of PTX and colchicine and eliciting chromosome aberrations .
To examine possible DNA fragmentation, the comet assay was exploited with results showing significant DNA refraction in the treated cells with the PTX loaded nanomicelles (Figure 9). We speculate that the fragmentation of DNA occurs indirectly as a result of activation of apoptosis pathways and/or perhaps direct interaction of these nanomicelles with subcellular elements such as tubulin and DNA itself.
Metastasis in cancerous cells appears to be a crucial problem for cancer therapy. Thus, the impact of nanoformulations should be examined regarding their possible potential for induction of inadvertent intrinsic metastasis. To pursue this concept, we looked at the expression pattern of the CTTN gene (cortactin) which is an important gene for promoting lamellipodia and invadopodia formation as well as cell migration [46, 47]. We found downregulation of CTTN gene in the treated cells with the PTX loaded nanomicelles (Figure 8), which may be considered as further indication for the safety of the PTX loaded nanomicelles from this point of view.