NIR-dye bridged human serum albumin reassemblies for effective photothermal therapy of tumor

Human serum albumin (HSA) based drug delivery platforms that feature desirable biocompatibility and pharmacokinetic property are rapidly developed for tumor-targeted drug delivery. Even though various HSA-based platforms have been established, it is still of great significance to develop more efficient preparation technology to broaden the therapeutic applications of HSA-based nano-carriers. Here we report a bridging strategy that unfastens HSA to polypeptide chains and subsequently crosslinks these chains by a bridge-like molecule (BPY-Mal2) to afford the HSA reassemblies formulation (BPY@HSA) with enhanced loading capacity, endowing the BPY@HSA with uniformed size, high photothermal efficacy, and favorable therapeutic features. Both in vitro and in vivo studies demonstrate that the BPY@HSA presents higher delivery efficacy and more prominent photothermal therapeutic performance than that of the conventionally prepared formulation. The feasibility in preparation, stability, high photothermal conversion efficacy, and biocompatibility of BPY@HSA may facilitate it as an efficient photothermal agents (PTAs) for tumor photothermal therapy (PTT). This work provides a facile strategy to enhance the loading capacity of HSA-based crosslinking platforms in order to improve delivery efficacy and therapeutic effect.


Characterizations
All chemical structures were confirmed by 1 H and 13 C NMR spectra and high-resolution mass spectra (HRMS) spectrometry. 1H and 13 C nuclear magnetic resonance (NMR) spectra were measured on a Bruker BBFO 400/600 spectrometer using deuterated chloroform (CDCl3) as the solvents.High-resolution mass spectrometry (HRMS) was performed on a Q-tof Premier MS spectrometer (Waters) and a MAT95XP mass spectrometer (Thermo Fisher Scientific).The in vivo and ex vivo living images were obtained by IVIS Lumina II (PerkinElmer).The photothermal conversion efficiency (PCE) was determined by analyzing the heating and cooling curves according to the following equations 1 .
where η is PCE, h represents heat transfer coefficient, s represents surface area, Qs represents the heat-related to the light absorbance of the deionized water, I represents the power of the laser, A808 represents the absorbance of sample in 808 nm.The hs could be calculated by equation (2), where m represents the mass of the sample, C represents the heat capacity of water, and τ could be calculated by the cooling curves according to equation ( 3) and ( 4).one (1 eq, 1.42 g, 6.46 mmol) were dissolved in absolute ethanol (10 mL).Aqueous sodium hydroxide solution (3 eq, 0.76 g, 19.38 mmol, 10 mL) was added.Resulting mixture was stirred at room temperature for 24 h, during which the product precipitated.The solution was poured into 1 M HCl (10 mL), and further acidified with concentrated HCl.The crude product was washed with water to obtain a white solid used for further synthesis without purification (2.2 g, 84.6%). 1

Compound 2
Compound 2 was synthesized according to the method reported in previous literature. 2Briefly, a S5 solution of compound 1 (1 eq, 2.2 g, 5.47 mmol), nitromethane (20 eq, 5.87 mL, 109.3 mmol) and KOH (1.2 eq, 0.37 g, 6.54 mmol) in EtOH (10 mL) was heated at 60 ºC under reflux for 12 h.After cooling down to room temperature, the solvent was removed and residue obtained was acidified with 4 M HCl, and the crude product was extracted using EtOAc and washed with deionized water.
The organic layers were collected and dried against Na2SO4.The crude product was purified by silica gel chromatography to obtain the final product (2.4 g, 94.5%).The obtained product (1 eq, 2.4 g, 5.18 mmol) and ammonium acetate (35 eq, 13.97 g, 181.2 mmol) were dissolved in butanol (50 mL).The mixture was heated at 130 ºC under reflux for 24 h.After cooling down, the residue was diluted with EtOAc followed by filtration.The crude product was extracted with EtOAc, and then washed with brine and ethanol.The organic layers were collected and dried against Na2SO4.
The solvent was removed to give the product used for further synthesis without purification (0.92 g, 21.2%).The last product (1 eq, 0.32 g, 0.38 mmol) was dissolved in dry DCM (50 mL), treated with triethylamine (4 mL) and BF3•Et2O (4 mL), and stirred under the nitrogen atmosphere for 48 h, followed by washing against water.The organic layers were collected and dried against Na2SO4.

BPY-Mal2
Compound 2 (1 eq, 100 mg, 0.113 mmol) and DBCO-maleimide (2.4 eq, 115 mg, 0.27 mmol) were dissolved in dry DCM (30 mL), and the mixture was stirred at room temperature for 12 h.The solvent was removed, and the crude product was purified by silica gel chromatography using methanol/acetone/DCM (v/v/v, 1:6.5:50) as eluent to remove the impurity, and then using acetone/DCM (v/v, 1:2) as eluent to give the final product as dark blue metallic solid (0. 18.3% a Covalent binding formulated HSA-platforms are based on covalent bonds between drugs and HSA, thus covalent binding strategy requires the drugs to have reactive groups with sulfhydryl groups or amino groups that are naturally existed on HSA protein, and the drugs containing sulfhydryl groups, maleimide groups, and NHS esters could be loaded to HSA by covalent binding.
b Noncovalent binding formulating strategies are based on hydrophobic interaction, electrostatic attraction, aromatic stack, and some weak bonds, which is not requiring the drugs to own covalent S38 binding groups.Such formulating strategies could be employed to load hydrophobic small molecular drugs, charged molecular drugs, and some biomolecules.
c HSA-based self-assembled nanoparticles are prepared by noncovalent interactions of HSA and drug units, which own hydrophilic parts and hydrophobic tails that could provide larger hydrophobic space for efficient drug loading.
d HSA-based polymers are kinds of HSA polypeptide chain derivates, which are prepared by modifying residues on denatured HSA polypeptide chains.Such polymers enlarged the molecular binding sites for loading more drugs, however, the carrier skeleton would gain the weight as well.
e HSA crosslinked nanoparticles require crosslink agents to serve as "glues" to bind the drugs and HSA proteins to form nanoparticles, which relate both covalent and noncovalent interactions among the agents, drugs, and HSA proteins.

Table 2 .
IC50 values calculated from CCK-8 experiments of each treatment.