Induction Of Enhanced Immunogenic Cell Death Through Ultrasound-Controlled Release Of Doxorubicin By Liposome-Microbubble Complexes

Objective: Exploring ultrasound-controlled release of doxorubicin in liposome-microbubble complexes to expand immunogenic cell death for the treatment of tumors.Methods: Firstly,the liposome-microbubble-doxorubicin complex(Mb Dox)was prepared to understand its biological properties.Then,the LL/2 and CT26 cell lines were dealt with the Ultrasound-Controlled release of doxorubicin(Mb Dox+US)in liposomemicrobubble complexes and the corresponding control.Flow cytometry was used to detect the number of apoptotic cells,calreticulin(CRT)and endoplasmic reticulum associated protein disulfide isomerase ERp57;Western Blot was used to detect the translation initiation factor(e IF2-?)phosphorylation(p-e IF2-?),HMGB1;The chemiluminescence ATP assay kit was used to detect ATP content,the purpose of these test is to explore whether doxorubicin in ultrasound-controlled liposomemicrobubble complexes(Mb Dox+US)could cause immunogenic cell death(ICD).Moreover the LL/2 and CT26 cell lines were treated with ultrasound-controlled release of doxorubicin in liposome-microbubble complexes(Mb Dox+US)and the corresponding control,and the supernatant was collected,Co-culture with dendritic cells(DCs),flow cytometry was used to detect dendritic cells(DCs)mature markers(CD80 and CD86)and activation markers(INF-g).The distribution of fluorescencelabeled doxorubicin in the cells and tissues of tumor-bearing mice was observed by confocal microscopy.At the same time,tumor cells(105)treated with different forms were injected to homologous C57BL/6 mice or BCLB/c mice to model as preventive tumor vaccine,then the same tumor was re-inoculated,Spleen mononuclear cells were subjected to cytotoxic T lymphocyte(CTLs)reaction experiments;serum was taken for Western Blot assay to detect CT26 tumor lysate related antibodies;splenic monocyte-derived B lymphocytes antibodies induced by LL/2 or CT26 cleavage products were detected by ELISPOT assay;anti-CD8 antibodies and anti-INFantibodies of spleen mononuclear cells,INF-g expression in CD8+ T cells and CD25+ FOXP3+ regulatory T cell(Tregs)cells were detected by flow cytometry.the purpose of these test is to explore whether doxorubicin in ultrasound-controlled release of liposome-microvesicle complexes can induce immunogenic cell death-related immune responses.Finally,tumor vaccine treatment models were established to observe the growth of LL/2 and CT26 tumor in immune-competent(homologous C57BL/6 and BALB/c)and immunodeficient(BALB/c Nu/Nu)mice,the purpose of these test was to investigate whether doxorubicin in ultrasound-controlled release of liposomemicrovesicle complexes can mediate anti-tumor immune responses.Results: We prepared a doxorubicin-liposome-microbubble complex(Mb Dox),and then the Mb Dox was characterized and tested for US-controlled release of Dox(Mb Dox+US treatment)to enhance the induction of ICD in LL/2 and CT26 cancer cells and in syngeneic murine models.We found that Mb Dox+US treatment caused more cellular uptake and nuclear accumulation of Dox in tumor cells,and more accumulation of Dox in tumor tissues.Enhanced induction of ICD occurred both in vitro and in vivo.Mb Dox+US treatment induced more apoptosis,stronger membrane exposure and the release of ER stress proteins and DAMPs in tumor cells,and increased DC maturation in vitro.In addition,Mb Dox+US treatment also resulted in stronger therapeutic effects in immunocompetent mice than in immunodeficient mice.Moreover,Mb Dox+US enhancement of ICD was also evidenced by a higher proportion of activated CD8+ T-lymphocytes but lower Tregs in tumor tissues.Conclusions: US-controlled release of ICD inducers into nuclei using liposome-microbubble complexes may be an effective approach to enhance the induction of ICD for tumor treatment.