Mitochondrial Targeting And Responsive Drug Release Mediated Cancer Therapy By Functional Modified Glycolipid-like Drug Delivery System

Mitochondria,crucial regulators of inducing tumor cells apoptosis,can be treated as the prime target for tumor therapy.The selective and responsive release of proapoptotic therapeutics into mitochondria may significantly eliminate severe off-target effect and realize high antitumor efficiency as well as low systemic toxicity In our study,we focus on developing smart nano drug delivery systems for effective mitochondrial targeting and responsive drug release by taking advantage of pathlogical parameters inside tumor mitochondria and external stimulation to improve the efficacy-of this promising therapeutic modality.Cationic chitosan was chosen as the basic framework material and conjugated with hydrophobic stearylamine for polycationic glycolipid polymer(CSOSA).CSOSA was further modified with lipophilic cations(4-Carboxybutyl)triphenylphosphonium bromide(CTPP)for mitochondria-targeted polymer C-P-CSOSA.C-P-CSOSA with 3.88%of CTPP can self-assemble into micelles and exhibited strong proton buffering ability.C-P-CSOSA micelles were more internalized by MCF-7 tumor cells than normal L02 cells.CTPP modification can promote micelles realize lysosomal escape and then target as well as aggregate inside mitochondria effectivelyLoaded with DOX,C-P-CSOSA/DOX micelles were obtained by dialysis method.The size of C-P-CSOSA/DOX was 67.2±2.82 nm and the zeta potential was 18.7±1.78 mV.C-P-CSOSA/DOX exhibited higher tumor cell cytotoxcity by remarkably activating mitochondrial apoptosis signal pathway.In MCF-7 tumor model,C-P-CSOSA had an enhanced accumulation in tumor tissue,compared with CSOSA.Moreover,C-P-CSOSA/DOX demonstrated the highest tumor-inhibition rate(75.0%),comparecd with C SOSA/DOX(44.8%)and DOX·HCl(55.0%).The weak alkaline pH environment in mitochondria was further utilized to design mitochondrial responsive drug release.Loading with weakly acidic drug Celastrol(Cela),C-P-CSOSA/Cela was prepared for weakly alkaline responsive drug release inside mitochondria by dialysis method.The particle size and zeta potential of C-P-CSOSA/Cela was 63.5 ±18.0 nm and 22.1 ±0.3 mV,respectively.C-P-CSOSA/Cela micelles could selectively respond to mitochondrial alkaline pH(pH 8.0)with 80.7%of Cela released in 12 h.However,there was a slow drug release behavior at pH 7.4 and pH 5.0 PBS.The solubility of Cela in pH 8.0 PBS was 21.1±0.38 pg/mL,which was 3.73 fold of that in pH 7.4 PBS.Cela was insoluble in pH 5.0 PBS.The higher solubility of Cela at pH 8.0 could prompt the weaker interaction between hydrophobic core of micelles and Cela,which induced fast drug release.It illustrated that C-P-CSOSA/Cela could realize mitochondrial fast drug release,and decrease drug leakage in the cytoplasm and lysosome.Mitochondria in MCF-7 cells of C-P-CSOSA/Cela group showed swollen,crista fragmentation and degeneration.C-P-CSOSA/Cela highly enhanced ROS levels,which further induced mitochondria membrane potential decreasing and more Cytochrome C releasing into cytoplasm,then promoted tumor cells apoptosis notably.In MCF-7 tumor model,C-P-CSOSA polymer had a significant mitochondrial co-localization.Moreover,the tumor-inhibition rate of C-P-CSOSA/Cela was 80.2%,which was significantly higher than CSOSA/Cela(58.4%)and Cela(54.9%).C-P-CSOSA/Cela micelles with mitochondrial targeting and alkaline pH-responsive release capability could provide a new strategy for tumor therapyExogenous photothermal stimulation was used in the design of responsive drug release inside mitochondria in our study.This strategy relies on mitochondrial-targeted delivery of doxorubicin(DOX)through a photothermal and lipophilic agent IR-780 iodide(IR780)-modified glycolipid conjugates(CSOSA).The resulted IR780-CSOSA showed a strong absorption at 795 nm with 3.12%of IR780.IR780-CSOSA exhibited effective photothermal conversion ability and good photothermal stability by protecting IR780 from degradation.The particle size and zeta potential of IR780-CSOSA/DOX was 119.0± 7.6 am and 37.8 ±0.9 mV,respectively.When IR780-CSOSA/DOX micelles solution was irradiated with a laser,a fast release behavior was observed in the first 10 h with a total release rate of 63.0%.As expected,the total release rate of DOX increased to 82.3%up to a release period of 48 h.Specifically,upon laser irradiation,the photothermal conversion by IR780-CSOSA can not only weaken the hydrophobic interaction between the core of micelles and DOX and trigger unexpected micelle swelling to fast release DOX from micelles by enhancing water solubility of DOX at the hyperthermal conditionIR780-CSOSA/DOX nanoparticles could selectively target into tumor mitochondria and realize photothermal conversion upon NIR-laser irradiation,which not only triggered responsive DOX release inside mitochondria for the amplification of ROS,but also induced mitochondria-specific heat shock to promote the fast evolution of ROS at the same locus.The combined generation of ROS subsequently promoted prompt mitochondria depolarization to facilitate the translocation of Cytochrome C to cytosol,which further activated a cascade of Caspase 9/3 reaction and efficiently induced tumor cells apoptosis.Furthermore,IR780-CSOSA/DOX may accumulate in tumor tissues.The temperature of IR780-CSOSA/DOX micelles in the tumor region rapidly increased under laser irradiation and the hyperthermia promoted more IR780-CSOSA/DOX nanoparticles penetrate into deeper tumor sites.Such multifunctional IR780-CSOSA/DOX with mitochondria-responsive drug release was the most effective in inhibiting tumor growth(tumor inhibition rate:85.3%),compared with DOX·HC1 group(55.0%)and photothermal IR780-CSOSA group(54.7%).HSP70 expression in tumors of IR780-CSOSA/DOX group increased remarkably after laser irradiation,suggesting the local hyperthermia and heat shock in tumor tissues.The expression of Cleaved Caspase-3 and CD8 in tumors of IR780-CSOSA/DOX group under laser irradiation increased sharply,suggesting that tumor cells occurred significant apoptosis and activated the host immune response with enhanced recruit of CD 8 T cells into tumor tissues.The levels of Ki67-positive cells and CD31 in tumors from the mitochondrial chemo-photothermal IR780-CSOSA/DOX was the lowest among all groups,illustrating that tumor growth and the angiogenesis of blood vessel were notably restrained.