Synthesis and characterization of TPT polymer, d-LND, and HA-DOX

The detailed protocol

1. Synthesis of Mitochondria-Targeting Poly (L-lactic acid) (TPT polymer)

Mitochondria-targeting poly (L-lactic acid) (TPT polymer) was synthesized via ring opening polymerization and conventional coupling reaction. 

Firstly, poly (L-lactic acid) (PLLA diol) polymer was synthesized via ring-opening polymerization of LLA with 1,6-hexanediol (HI) as initiatior in the presence of Sn(Oct)₂ as catalyst. For example, 1, 6-hexanediol (1 mol), L-lactic acid (6 equivalent moles of HI), and then Sn(Oct)₂ (ca. 0.1% of reactants in mass amount) in anhydrous toluene were mixed in a dry polymerization tube and purged with nitrogen for 3 times. After degassed by a vacuum pump for 8 hours, the tube was sealed and put into an oil bath at 120℃ for 48 hours. The mixture was cooled at room temperature and dissolved into dichloromethane. The disolved solution was precipitated in large amount of cold diethyl ether for 2 times. The product (PLLA diol) was obtained after filtered and dried under a vacuum at room temperature. The structure of PLLA diol was characterized by ¹HNMR spectrum (AV-400, Bruker, USA) using CDCl₃ as the solvent, and 0.5% tetramethylsilane was used as the internal standard.

Subsequently, TPT polymer was synthesized via a conventional coupling reaction between a PLLA diol and 4-carboxybutyltriphenylphosphonium bromide (TPP⁺COOH•Br⁻). In brief, PLLA diol (1 mol) was first dissovled in dichloromethane. TPP⁺COOH•Br⁻ (3 equivalent moles of PLLA diol), EDC (1 equivalent mole of TPP⁺COOH•Br⁻), DMAP (1 equivalent mole of TPP⁺COOH•Br⁻) and TEA (1 equivalent mole of TPP⁺COOH•Br) were all dissolved in dichloromethane and stirred for 3 hours to activiate the carboxylic acid of TPP⁺COOH•Br⁻. Afterwards, the PLLA diol in dichloromethane was added into the activated reaction mixture and then stirred for 48 hours under nitrogen atmosphere. The resulting reaction solution was evaporated and concentrated to remove dichloromethane, and then DMSO was added to dissolve the product. Finally, the mixed solution was dialyzed against DMSO and deionized water for 48 h (1000 MWCO) with every 6 hours change of the dialysat, and then dried under a vacuum to obtain the TPT polymer. The structure of TPT polymer was characterized by ¹HNMR spectrum (AV-400, Bruker, USA) using (CD₃)₂SO as the solvent, and 0.5% tetramethylsilane was used as the internal standard.

2. Synthesis of Lonidamine Drug Dimer (d-LND)  

d-LND dimer was synthesized via a conventional coupling reaction between LND and cystamine. Firstly, cystamine (1 mol) was dissolved in DMSO. LND (2.5 equivalent moles of cystamine), EDC (2.5 equivalent moles of LND) and NHS (2.5 equivalent moles of LND) were all dissolved in DMSO and stirred for 3 hours to activiate the carboxylic acid of LND. Afterwards, cystamine in DMSO was added dropwise into the activated reaction mixture and stirred for 36 h at 40℃ under nitrogen atmosphere. Then, the mixture was evaporated and concentrated to remove DMSO, and then extrated with a large amount of denonized water for 4 times. The product was obtained after filtered and dried under a vuccum. The structure of d-LND dimer was characterized by ¹HNMR spectrum (AV-400, Bruker, USA) using CDCl₃ as the solvent, and 0.5% tetramethylsilane was used as the internal standard. High-resolution mass spectrometer (LCMS-IT-TOF, Shimadzu, Japan) were employed for the structural verification.

3. Synthesis of Cellular Active-Targeting HA-DOX

HA-DOX was synthesized via a conventional coupling reaction between HA and DOX. DOX (1 mol) was dissolved in a mixture of DMSO/H₂O (v/v=1:1). EDC (5 equivalent moles of DOX), NHS (5 equivalent moles of DOX) and HA (0.03 equivalent moles of DOX) were dissolved in a mixture of DMSO/H₂O (v/v=1:1) and stirred for 3 hours to activated the carboxylic acid of HA. Subsequently, DOX in the mixture of DMSO/H₂O (v/v=1:1) was added into the activated reaction mixture and stirred for 48 hours at room temperature. After that, the reaction mixture was dialyzed agaisnt with water and DMSO for 72 hours with every 6 hours change of the dialysat (3500 MWCO) to remove the unreacted DOX, EDC and NHS. Then, the HA-DOX was freeze-dried and stored in the absence of light. The structure of HA-DOX was characterized by ¹HNMR spectrum (AV-400, Bruker, USA) using (CD₃)₂SO as the solvent, and 0.5% tetramethylsilane was used as the internal standard.

Category