Quantitative self-assembly of pure drug cocktails as injectable nanomedicines for synergistic drug delivery and cancer therapy

New strategies to fabricate nanomedicines with high translational capacity are urgently desired. Herein, a new class of self-assembled drug cocktails that addresses the multiple challenges of manufacturing clinically useful cancer nanomedicines was reported. Methods: With the aid of a molecular targeted agent, dasatinib (DAS), cytotoxic cabazitaxel (CTX) forms nanoassemblies (CD NAs) through one-pot process, with nearly quantitative entrapment efficiency and ultrahigh drug loading of up to 100%. Results: Surprisingly, self-assembled CD NAs show aggregation-induced emission, enabling particle trafficking and drug release in living cells. In preclinical models of human cancer, including a patient-derived melanoma xenograft, CD NAs demonstrated striking therapeutic synergy to produce a durable recession in tumor growth. Impressively, CD NAs alleviated the toxicity of the parent CTX agent and showed negligible immunotoxicity in animals. Conclusions: Overall, this approach does not require any carrier matrices, offering a scalable and cost-effective methodology to create a new generation of nanomedicines for the safe and efficient delivery of drug combinations.


Solubility test
To determine the solubility for DAS, 50 mg of DAS was incorporated into the nanoparticles in 5 mL of DI water. The resultant suspension was stirred for 30 min at 25 °C. After centrifugation for 3 min at 5000 rpm, the amount of DAS in the supernatant was measured by UV/vis spectroscopy at a wavelength of 326 nm. S3 CAC was determined using the fluorescence intensity change upon aggregates formation in aqueous solution [1]. Before the aggregates formation, the fluorophores exhibit very weak fluorescence. Once the aggregates were formed, the fluorescence intensity increased significantly.

Determintation of critical aggregation concentration (CAC)
This method was used to determine the CAC of CD NAs and fluorescence intensity at 422 nm was plotted against the DAS concentrations in different media such as DI water, PBS and FBS (10%, v/v) at room temperature.

Molecular dynamics simulations
The structures of CTX and DAS were optimized, and the partial atomic charges were calculated by the restrained electrostatic potential charge from the calculation with the Gaussian09 package at the level of HF/6-31g* [2]. MMFF94x force field parameters were used for these two molecules.
CTX and DAS molecules were mixed at a molar ratio of 1:8.5 (CTX: DAS) and initially packed randomly by PACKMOL in a cubic box with a length of 100 Å [3]. Then, the mixture was neutralized by adding sodium/chlorine counterions and solvated in a cuboid box of TIP3P water molecules with 10 Å solvent layers between the box edges and solute surface.
All MD simulations were performed using AMBER16 [4]. The AMBER GAFF force field was applied, and the SHAKE algorithm was used to restrict all covalent bonds involving hydrogen atoms with a time step of 2 fs. The Particle mesh Ewald method was employed to treat long-range electrostatic interactions. For each solvated system, two minimization steps were performed before the heating step. The first 4000 cycles of minimization were performed with all heavy atoms restrained with 50 kcal/(mol·Å 2 ), whereas solvent molecules and hydrogen atoms were able to move freely.

S4
Then, nonrestrained minimization was carried out with 2,000 cycles of steepest descent minimization and 2,000 cycles of conjugated gradient minimization. Afterwards, the whole system was first heated from 0 K to 300 K in 50 ps using Langevin dynamics at a constant volume and then equilibrated for 400 ps at a constant pressure of 1 atm. A weak constraint of 10 kcal/(mol·Å 2 ) was used to restrain all the heavy atoms during the heating steps. Periodic boundary dynamics simulations were carried out for the whole system at NPT (pressure = 1 atm, and temperature = 300 K) in the production step. In the production phase, a 50 ns simulation was carried out.

Synergistic effect evaluated by the combination index (CI)
The degree of synergy between two drugs can be quantified by calculating the CI using

Cellular uptake of CD NAs
We utilized CLSM to detect the cellular uptake of self-assembled CD NAs in NCI-H1975 cells. The cells were plated in 20 mm glass coverslips at 2.0 ×10 5 cells per well and incubated for 24 h. Then, the solution containing CD NAs (40 µg/mL DAS coencapsulated with 1 µg/mL CTX) was added.
The cells were processed after 15 min, 30 min, 1 h, 2 h and 3 h of incubation with two washes in PBS and then stained with LysoTracker Green (100 × 10 −9 M) for 30 min at 37 °C. After washing, the cells were immediately observed using a confocal microscope (FV3000, Olympus, Japan). The images were further analyzed with ImageJ (NIH, USA).

Colony formation assay
A colony formation assay was conducted to evaluate the long-term Bioscience, UK). Images were taken using a gel imaging system (5200, Tanon, China) and analyzed with ImageJ software (NIH, USA).

Animal study
Male Balb/c athymic nude mice (4-5 weeks) were purchased from Shanghai Experimental Animal Center, Chinese Academy of Science, and housed at the animal facility of the Zhejiang Academy of Medical Sciences.
The studies involving animals were approved by the Ethics Committee of the First Affiliated Hospital, Zhejiang University School of Medicine. All animal experiments were performed in compliance with the guidelines of the Zhejiang University Committee for Animal Use and Care.

In vivo and ex vivo imaging study
To evaluate the tumor-targeting ability and in vivo drug distribution of CD NAs, NCI-H1975 mouse model was established by subcutaneous injection of 1×10 6 NCI-H1975 cells into the right flank of Balb/c nude mice (4-5 weeks old). When the tumor volume reached ~500 mm 3 , the mice were randomly divided into two groups (n = 5 in each group). A near-infrared dye Cy5.5 was coassembled into CD NAs (termed Cy5.5@CD NAs, DAS: CTX: Cy5.5 = 5:1:1, weight ratio) to track the in vivo distribution of CD NAs. The mice were injected with free Cy5.5 or Cy5.5@CD NAs at a Cy5.5 dose of 20 μg per mouse via the tail vein. Whole-body NIR fluorescence imaging was performed using an in vivo imaging system (IVIS ® Lumina LT Series III, PerkinElmer, USA) at predetermined time points. At 48 h, the mice were sacrificed to collect tumors and organs for ex vivo imaging.

In vitro hemolysis
The blood compatibility of the self-assembled nanomedicines was investigated according to a previous report [5]. Blood was collected in heparinized tubes from healthy SPF SD rats, followed by centrifugation at

Immunohistochemical analysis
The tumor-bearing mice were killed at day 12 post-administration. The tumors were excised, fixed in 10% paraformaldehyde and embedded in paraffin. The tissues from NCI-H1975 tumor xenograft-bearing mice were then sectioned for histopathological analysis by H&E staining, Ki67 and TUNEL immunohistochemistry, while the tissues from melanoma PDX tumors were stained with H&E and TUNEL immunofluorescence. The sections were imaged using an Olympus Microscope (IX73, Olympus) or confocal microscope (FV3000, Olympus, Japan).
Immunotoxicity assay S10 ICR mice were divided into five groups (n = 3) and treated with the same administration regimen used in the system toxicity study. At days 0, 1, 8, and 21 after administration, blood samples were allowed to clot for 30 min and then centrifuged at 14,000 rpm for 10 min. Then, the supernatant sera were collected, frozen and stored at -80 °C. Before testing, the samples were further thawed and centrifuged at 13,000 rpm for 10 min at 4 °C. Twenty-five microliters of serum was diluted with assay buffer (25