OBJECTIVE： To prepare Baicalin-loaded Polyethylene glycol-derivatized phosphatidylethanolamine （BAI@PEG-PE） nanomicelles， and to characterize it and study its cytotoxicity. METHODS： BAI@PEG-PE nanomicelles were prepared by film hydration method and their appearance characteristics were observed. The particle size， polydispersity index， Zeta potential， drug-loading amount and encapsulation efficiency of the nanomicelles were detected. Drug release of BAI raw material and BAI@PEG-PE nanomicelles in pH 7.4 phosphate buffer were compared within 1-84 h. Using coumarin 6 as fluorescent probe， the distribution of PEG-PE nanomicelles in H9c2 cardiomyocytes were observed. H9c2 cardiomyocytes were divided into model group， BAI raw material group and BAI@PEG-PE nanomicelles group. After treated with serum-free DMEM medium containing no or corresponding drugs for 0.5 h， isoproterenol was used to induce cardiomyocyte apoptosis. Nuclear morphology， cell apoptosis rate and protein expression of Bcl-2 and Bax were compared with among 3 groups. RESULTS： Prepared BAI@PEG-PE nanomicelles were uniform globular shape. The particle size was （16.7±0.8） nm， PDI was 0.11±0.01 and Zeta-potential was （－18.4±0.6） mV； drug-loading amount was （7.84±0.65）％， encapsulation efficiency was （85.7±4.9）％ （n＝3）. Accumulative release rate was 76.5％ within 84 h. BAI raw material was released completely within 24 h. PEG-PE nanomicelles could strengthen the intake of coumarin 6 in H9c2 cardiomyocytes， mainly gathering around mitochondria. Compared with model group， the apoptosis morphology of cardiomyocytes were improved significantly in BAI raw material group and BAI@PEG-PE nanomicelles group； apoptosis rate was decreased significantly； protein expression of Bcl-2 was increased significantly； protein expression of Bax was decreased significantly with statistical significance （P＜0.05 or P＜0.01）. Above effects of BAI@PEG-PE nanomicelles group were more significant （P＜0.05 or P＜0.01）. CONCLUSIONS： BAI@PEG-PE nanomicelles are prepared successfully， and show significant sustained-release effect and myocardial targeting， and can prevent cardiomyocyte apoptosis.

Objective:To effectively express the receptor binding domain (RBD) of SARS-CoV-2 spike protein in Pichia pastoris and to evaluate its immunogenicity. Methods:The gene encoding the RBD protein was synthesized and cloned into the pPICZαA plasmid. After linearization, the plasmid was transferred and integrated into the genome of Pichia pastoris. The expressed RBD protein in culture supernatant was analyzed by Western blot and Biolayer interferometry. After screening, a single clone expressing the RBD protein was selected. The high-level expression of RBD protein was achieved by optimizing the fermentation process, including the salt concentration adjusting of the medium and induction condition optimization (pH, temperature and duration). The immunogenicity of the expressed RBD protein was evaluated in a mouse model. Results:A single clone with a high expression level of RBD protein was obtained and named RBD-X33. The expression level of RBD protein in the fermentation supernatant reached up to 240 mg/L after optimization of the induction condition (HBSM medium, pH=6.5±0.3, 22℃ and 120 h). In the mouse experiment, the recombinant RBD protein was formulated with Alum+ CpG dual adjuvant and injected into mice. The binding IgG antibody levels were up to 2.7×10 6 tested by ELISA and the neutralizing antibody levels were up to 726.8 tested by live virus neutralizing antibody assay (prototype). Conclusions:The RBD protein could be efficiently expressed in Pichia pastoris and induce stronger immune response in animals. This study suggested that the recombinant SARS-CoV-2 RBD protein expressed in Pichia pastoris could serve as a candidate antigen in the development of SARS-CoV-2 vaccine.