Co-delivery of Docetaxel and Disulfonate Tetraphenyl Chlorin in One Nanoparticle Produces Strong Synergism between Chemo- and Photodynamic Therapy in Drug-Sensitive and -Resistant Cancer Cells

Abstract

Cancer therapies based on the combinations of different drugs and/or treatment modalities are emerging as important strategies for increasing efficacy and cure, decreasing unwanted toxicity, and overcoming drug resistance, provided that optimized drug concentration ratios are delivered into the target tissue. To these purposes, delivery systems such as nanoparticles (NPs) offer the unique opportunity to finely tune the drug loading and the release rate of drug combinations in the target tissues. Here, we propose double-layered polymeric NPs for the delivery of the chemotherapeutic docetaxel (DTX) and the photosensitizer disulfonate tetraphenyl chlorin (TPCS2a) coated with hyaluronic acid (HA), which allows cell targeting via CD44 receptors. The simultaneous delivery of the two drugs aims at killing DTX-sensitive (HeLa-P, MDA-MB-231) and DTX-resistant (HeLa-R) cancer cells by combining chemotherapy and photodynamic therapy (PDT). Using the Chou and Talalay method that analyses drug interactions and calculates combination index (CI) using the median-effect principle, we compared the efficiency of DTX chemotherapy combined with TPCS2a-PDT for drugs delivered in the standard solvents, coloaded in the same NP (DTX/TPCS2a-NP) or loaded in separate NPs (DTX-NPs + TPCS2a-NPs). Along with the drug interaction studies, we gained insight into cell death mechanisms after combo-therapy and into the extent of TPCS2a intracellular uptake and localization. In all cell lines considered, the analysis of the viability data revealed synergistic drug/treatment interaction especially when DTX and TPCS2a were delivered to cells coloaded in the same NPs despite the reduced PS uptake measured in the presence of the delivery systems. In fact, while the combinations of the free drugs or drugs in separate NPs gave slight synergism (CI < 1) only at doses killing more than 50% of the cells, the combination of drugs in one NPs gave high synergism also at doses killing 10-20% of the cells. Furthermore, the DTX dose in the combination DTX/TPCS2a-NPs could be reduced by ∼2.6- and 10.7-fold in HeLa-P and MDA-MB-231, respectively. Importantly, drug codelivery in NPs was very efficient in inducing cell mortality also in DTX resistant HeLa-R cells overexpressing P-glycoprotein 1 in which the dose of the chemotherapeutic can be reduced by more than 100 times using DTX/TPCS2a-NPs. Overall, our data demonstrate that the protocol for the preparation of HA-targeted double layer polymeric NPs allows to control the concentration ratio of coloaded drugs and the delivery of the transported drugs for obtaining a highly synergistic interaction combining DTX-chemotherapy and TPCS2a-PDT.

CD44 Targeting Mediated by Polymeric Nanoparticles and Combination of Chlorine TPCS2a-PDT and Docetaxel-Chemotherapy for Efficient Killing of Breast Differentiated and Stem Cancer Cells In Vitro

“…The release of the drugs from NPs was checked in cell culture medium enriched with 10% serum in order to reproduce the experimental conditions of in vitro cell studies. As demonstrated for HA@DTX/TPCS 2a -NPs with a drug ratio 1:35 [16], in this novel formulation the release of the PS was slow, thus highlighting its strong association with NP. Concerning the chemotherapeutic, the 70% of entrapped DTX was released in 72 h, thus showing a fast burst effect in the first 6 h of incubation ( Figure S1).…”

“…In a previous work [16], we reported that the combination of DTX-chemotherapy and TPCS2a-PDT produced synergistic killing of differentiated CD44 over-expressing HeLa and MDA-MB-231 cells. Here we found that the analysis, according to the Chou and Talalay method [25], of the viability of MCF-7 cells (Figure 2a) treated with the HA-NPs co-loaded with the 1:35 DTX/TPCS2a molar ratio revealed antagonism (Figure 2b, blue line, CI > 1: antagonism) instead of synergism.…”

“…In preliminary experiments we observed that MCF-7 cells are less sensitive to treatment with free DTX (IC 50 0.134 µg/mL) compared to MDA-MB-231 cells (IC 50 0.016 µg/mL) (Tables S1 and S2, and [16]). In addition, it is well known that in tridimensional cell cultures and aggregates (e.g., mammospheres) the drug accumulation inside the cells is generally reduced in comparison to monolayers [24].…”

“…On these premises, the 1:35 DTX/TPCS 2a ratio inside NPs, that had been optimized for MDA-MB-231 cells, was modified by keeping un-altered the loading of TPCS 2a and increasing that of DTX. Using the method already described in [16], we were able to prepare NPs containing DTX/TPCS 2a with the 1:5 ratio. The properties of the new nanoformulation are reported in Table 1 and compared with those of NPs loaded with a single drug (HA@DTX-NPs and HA@TPCS 2a -NPs) used as controls.…”

“…Properties of NP formulations a . HA@DTX/TPCS 2a -NPs contain DTX and TPCS 2a in the molar ratio 1:5; for HA@DTX/TPCS 2a -NPs loaded with the drugs in the ratio 1:35 refer to our previous work [16]. All the formulations showed drug entrapment efficiencies higher than 95%, sizes comprised in the 160-210 nm range, a polydispersity index (PI) of 0.2 and a high negative zeta potential, due to the presence of the negatively charged HA external layer.…”