Lactide glycolide

Branched polyethylenimine (PEI, M_w 1800 Da) and succinic anhydride (SA) were purchased from Innochem Technology Co., Ltd. (Beijing, China). 2,3-Dimethylmaleic anhydride (DMMA), PLGA (lactide : glycolide = 65 : 35, M_w 40000-75000 Da), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), cytochalasin D (Cyt. D), methyl-β-cyclodextrin (MBCD), sucrose, collagenase I, and collagenase IV were purchased from Sigma-Aldrich (Shanghai, Trading Co., Ltd). Polyethylene glycol monostearate (C₁₈-PEG₂₀₀₀-OH, CPO) was obtained from Tokyo Chemical Industry Co., Ltd. C₁₈-PEG₂₀₀₀-TPP (CPT) was synthesized as our previously reported 38 (link). Doxorubicin hydrochloride (DOX·HCl) was provided by Aladdin Reagent (Shanghai, China). N,N-dimethyl formamide (DMF), chloroform (CHCl₃), and isopropanol were purchased from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). Lysotracker Green (Yeasen), Mitotracker Green (Beyotime), BCA protein assay kit (Beyotime), mitochondrial membrane potential assay kit with JC-1 (Beyotime), mitochondrial isolation kit (Beyotime), nuclei extraction kit (ExKine), XF24 extracellular flux analyzer kit (Seahorse Bioscience), and matrigel matrix (BD Biosciences) were all obtained from domestic suppliers. All other reagents and solvents were of analytical grade and used as received.

PDMAEMA-PLGA NPs were synthesized following our previous publications with modifications using a single emulsion/solvent evaporation method [9 (link),10 (link),11 (link)]. One hundred milligrams of poly(lactic-co-glycolic acid) with lactide:glycolide of 50:50 (Sigma, St. Louis, MO, USA) were dissolved in 10 mL of dichloromethane (RCI Labscan, Bangkok, Thailand). The aqueous solution was prepared by dissolving 10 mg of PDMAEMA (MW 10 kDa) that was synthesized following our previous publication [9 (link)] and 120 mg of polyvinyl alcohol (PVA, MW 9–10 kDa, 80% hydrolyzed; Sigma) in 30 mL of endotoxin-free water. The organic solution of PLGA was then homogenized in the prepared aqueous solution using a handheld homogenizer (ErgoMixx, Bosch, Gerlingen, Germany) for 2 min. Subsequently, the organic solvent was evaporated at 25 °C under magnetic stirring for 2 h. The obtained particles were centrifuged at 500 rcf to remove large particles with the size greater than 1000 nm. The supernatant was then centrifuged at 2000 rcf to collect NPs with 500 nm in size. PLGA-PDMAEMA NPs were lyophilized and stored at 4 °C until use.