Combinatorial nanococktails via self-assembling lipid prodrugs for synergistically overcoming drug resistance and effective cancer therapy

Background Combinatorial systemic chemotherapy is a powerful treatment paradigm against cancer, but it is fraught with problems due to the emergence of chemoresistance and additive systemic toxicity. In addition, coadministration of individual drugs suffers from uncontrollable pharmacokinetics and biodistribution, resulting in suboptimal combination synergy. Methods Toward the goal of addressing these unmet medical issues, we describe a unique strategy to integrate multiple structurally disparate drugs into a self-assembling nanococktail platform. Conjugation of a polyunsaturated fatty acid (e.g., linoleic acid) with two chemotherapies generated prodrug entities that were miscible with tunable drug ratios for aqueous self-assembly. In vitro and in vivo assays were performed to investigate the mechanism of combinatorial nanococktails in mitigating chemoresistance and the efficacy of nanotherapy. Results The coassembled nanoparticle cocktails were feasibly fabricated and further refined with an amphiphilic matrix to form a systemically injectable and PEGylated nanomedicine with minimal excipients. The drug ratio incorporated into the nanococktails was optimized and carefully examined in lung cancer cells to maximize therapeutic synergy. Mechanistically, subjugated resistance by nanococktail therapy was achieved through the altered cellular uptake pathway and compromised DNA repair via the ATM/Chk2/p53 cascade. In mice harboring cisplatin-resistant lung tumor xenografts, administration of the nanococktail outperformed free drug combinations in terms of antitumor efficacy and drug tolerability. Conclusion Overall, our study provides a facile and cost-effective approach for the generation of cytotoxic nanoparticles to synergistically treat chemoresistant cancers. Supplementary Information The online version contains supplementary material available at 10.1186/s40824-022-00249-7.


Synthesis of Cisplatin-Derived Prodrug
. The synthetic procedure of the Pt-LA2 conjugate.
The prodrug of Pt-LA2 was synthesized according to our previous works [1]. In brief, cis, cis, trans-Pt(NH3)2Cl2(OH)2 was first synthesized through an oxidation reaction. Cisplatin (1.0 g, 3.3 mmol) was added to a solution of 30% hydrogen peroxide (H2O2, 30 mL) in a round-bottomed flask and continuously stirred at 80°C for 8 h in dark. The reaction solution was then stored at 4°C overnight to obtain precipitate, followed by washing with deionized water, ethanol, and ethyl ether. The resulting residue was dried under vacuum to afford pale-yellow solid (0.91 g, 82%).

Size Characterization
The hydrodynamic diameters (DH), ζ potentials, and polydispersity indexes (PDI) of prodrug-loaded nanoparticles were obtained from dynamic light scattering (DLS) measurement on a Malvern Nano-ZS90 instrument (Malvern, UK) at 37 °C, with each sample repeated thrice.

Transmission Electron Microscopy Analysis
Transmission electron microscopy (TEM) images were captured by using TECNAL 10 (Philips). NPs at a concentration of 0.5 mg/mL (cisplatin and SN38 equivalence) were dripped on a 300-mesh copper grid. After a 2-minute deposition, the surface water was removed with filter paper and then air-dried. The surface of each grid was further positively stained with an aqueous solution of 2 wt % uranyl acetate.

Cell viability assay and analysis of synergistic effects
A total number of 3 × 10 3 cells of A549 cells and A549 cisR cells were seeded in 96well plates and cultured for 24 h for cell adherence. Next, each cell line was treated with a variety of concentrations of Pt (IV) -NP, SN38-NP, or the nanococktails (NCs) at constant molar ratios of 10:1, 5:1, 2:1, 1:1, 1:2, 1:5, or 1:10 for an additional 72 h at 37 °C. Finally, cell viability was measured by a CCK-8 kit according to the manufacturer's protocol. Half-maximal inhibition values were extrapolated from inhibition curves by using Graphpad Prism software (8.0). A combination analysis was performed using the method described by Chou and a Calcusyn software program for automated analysis [2]. The synergy of Pt (IV) -NP in combination with SN38-NP was evaluated by calculating the combination index at IC50 (termed CI50) using the following indexes were classified as synergistic when CI was < 0.9 and antagonistic when it was > 1.1, with CI among 0.9-1.1 indicating additive.

Cellular uptake and intracellular distribution of NCs
For the colocalization assay, A549 cisR cells were seeded in glass-bottom dishes at a density of 3 × 10 4 cells and incubated overnight. The cells were then treated with DiI labeled NC for predetermined time intervals, followed by the incubation with Hoechst the cells were observed under confocal laser-scanning microscope (CLSM).
To verify that cellular uptake of the NCs was due to the energy-dependent endocytosis, the incubation temperature was decreased to 4°C, and cellular uptake of the NCs was examined. A549 cisR cells were seeded in 6-well plates at a density of 2 × 10 5 cells/well and incubated overnight, and then treated with DiI-loaded NCs at 37°C or 4°C for 4 h. For incubation at 4°C, the cells in the 6-well plate were placed into a sealing bag that was prefilled with 5% CO2, and then the bag with the cells was transferred to a 4°C incubator for a 4-hour incubation. The cells were harvested and rinsed with PBS, and fluorescence intensities were analyzed by flow cytometry.
To investigate the exact endocytotic routine for the NCs, A549 cisR cells were preincubated with specific endocytosis inhibitors at 37°C, with chlorpromazine at 10 µg/mL, cytochalasin D at 40 µM, and filipin III at 5 µg/mL. Following 60 min of incubation, DiIlabeled NCs were added to the cells and incubated for another 4 h. The cells were harvested and washed with PBS three times. Finally, the cellular uptake was analyzed with flow cytometry.

Cell apoptosis
After being seeded in 6-well plates overnight, cells were treated with Pt (IV) -NP, SN38-NP, or the NCs for 24 h. Then cells were harvested by trypsinization and rinsed with PBS. Cells were resuspended with a binding buffer containing Annexin V-FITC/PI double staining. After being incubated at room temperate under the dark condition for 20 minutes, cells were subsequently analyzed by florescence-activated cell sorting (FACS).

Cell cycle distribution
3 × 10 5 A549 cisR cells were seeded in 6-well plates and incubated for 24 h to allow attachment. Next, cells were treated with Pt (IV) -NP (5 μM), SN38-NP (1 μM), or their combination for another 24 h. Followed by trypsin digestion and twice washes with PBS, cells were then fixed with 75% cold ethanol for 30 minutes at 4 °C. Lately, cells of different groups were stained with propidium iodide (PI) solution at room temperate for 30 minutes. Cell cycle distributions were determined on a flow cytometer, and a total number of 10 4 events were recorded for further analysis.

Western blotting analysis
Whole-cell lysates were prepared by RIPA Lysis Buffer (Beyotime) containing the protease and phosphatase inhibitor cocktails on ice for 20 minutes. The protein concentration of each sample was first quantified by a BCA protein determination reagent and subsequently diluted with sample loading buffer to give a concentration of 2 μg/μL. After boiled on a dry heat block, equal amounts of proteins were electrophoresed on an SDS-10% polyacrylamide gel and transferred onto polyvinylidene fluoride membranes. Membranes were then blocked with 5% skimmed milk in 1 × Tris-buffered saline containing 0.1% Tween 20 for 1 h at room temperature, followed by incubation with primary antibodies at 4 °C overnight and visualization of horseradish peroxidase (HRP) -conjugated secondary antibodies by Bio-Rad ChemiDoc analysis system with enhanced chemiluminescence. Primary and secondary antibodies used for immunoblotting were listed in Table S1.

Immunofluorescence Staining Assay
A suspension of 5 × 10 4 A549 cisR cells was seeded in a glass-bottom dish and cultured for 24 h. The cells were treated with Pt (IV) -NP, SN38-NP, or the NCs for 24 h.
Cells without treatment were used as a control. After a 24 h incubation, the cells were washed three times with PBS and fixed with 4% paraformaldehyde for 30 min at room temperature. The cells were then permeated with 0.5% Triton X-100 in PBS for 15 minutes and blocked with goat serum for 1 h at room temperature. Subsequently, the cells were immunostained with γH2AX antibody (Cell Signaling Technology) at 4 °C overnight. After brief washes, the cells were stained with Alexa Fluor 555-labeled secondary antibody (Thermo Fisher Scientific) for 40 minutes at room temperature, followed by nuclei staining with DAPI for 15 minutes. Finally, the cells were imaged by a Nikon fluorescence microscope (Nikon, Japan).

qPCR analysis
Total RNA was extracted from cell lysates according to the protocol of the manufacturer (YiShan Biotech, Shanghai, China). After that, 1 μg RNA of each sample was converted into cDNA by a fast all-in-one reverse transcription kit (YiShan Biotech, Shanghai, China). The cDNA was then treated as templates for further quantification of mRNA transcription by using a CFX96 Real-Time PCR Detection System (Bio-Rad).
All primers were provided by Tsingke Biological Technology (Hangzhou, China) and S11 their sequences were summarized in Table S2. All real-time qPCR analyses were performed in triplicate. Fold change was calculated using the Ct method. The results of the target genes were normalized to β-2 Microglobulin.