A Combined Drug Delivery Strategy Of Remodeling Tumor Microenvironment And Targeting Mitochondria For Metastasis Suppression

Breast cancer has become the most frequently occurring cancer and the leading cause of cancer death among women.Majority of breast cancer patients die from metastasis.As the energy and metabolic center of tumor cells,mitochondria can provide energy for the intravasation of tumor cells and help tumor cells survive in the bloodstream.Thus,mitochondria are crucial for maintaining the high metastatic ability of tumor cells.It has been previously confirmed that mitochondrial targeted therapy showed surprisingly anti-metastasis efficacy via activating Bcl-2 related apoptosis pathway and downregulating the expression of pro-metastasis cytokines.Therefore,mitochondria targeting provieds promising way for anti-tumor metastasis treatment.Furthermore,tumor microenvironment（TME）composed of tumor cells,stromal cells,extracellular matrix components and various cytokines is also pivotal in promoting tumor metastasis.The physiological characteristics of TME distinct from normal tissues,such as hypoxia and chronic inflammation,are closely related to the migration,invasion and extravasation of cancer cells.Therefore,it is necessary to inhibit the tumor cells’own metastatic potential and regulate the tumor microenvironment simultaneously for successful metastasis suppression.This study assumes that alleviating hypoxia and inflammation in the tumor microenvironment combined with mitochondria damage might be a two-pronged strategy to inhibit breast cancer metastasis.This has not been reported yet.（1）Targeting tumor-associated fibroblasts and mitochondria for alleviating hypoxic microenvironment and inhibiting breast cancer metastasisHypoxia is a hallmark feature of TME and is emerging as potent factor that promotes metastasis.Cancer-associated fibroblasts（CAFs）are hugely responsible for the lack of oxygen supply,because CAFs hold the largest proportion of stroma cells and produce mounts of extracellular matrix（ECM）components that consequently stiffen the tumor and abnormally squeeze tumor blood vessels.Therefore,a combined drug delivery system for depleting CAFs and damaging mitochondria was constructed to alleviate hypoxia in the tumor microenvironment and inhibit mitochondrial respiration.We developed a precursor peptide denoted as E8R8 that can be specifically cleaved by fibroblast activator protein-α（FAP-α）over-expressed on the surface of CAFs and then the positively charged penetrating sequence R8 can be exposed to mediate enhanced intracellular penetration.E8R8 were modified on the surface of Dox-loaded liposome to obtain CAFs targeted liposomes（E8R8-Lip）.Also,unmodified（Lip）or R8 modified liposomes（R8-Lip）were prepared as controls.All these liposomes displayed similar particle size（100-140 nm）,doxorubicin encapsulation efficiency（～90%）and drug loading（～4.5%）,as well as good plasma stability and slow drug release behavior.Next,Lonidamine（LND）was used to dysfunction mitochodria of cancer cells because it can inhibit tumor cell mitochondrial respiratory chain complexes ⅠandⅡ.Our previous studies found that 3-（guanidinopropyl）methacryloyl（GPMA）,a positive charged monomer,exerted potent mitochondria efficacy due to the high negative potential in inner mitochondrial membrane of cancer cells.N-（2-hydroxypropyl）methacrylamide（HPMA）polymer,a water-soluble vehicle with good biocompatibility,was a promising system for LND delivery.Thus,HPMA polymer-LND conjugates without or with GPMA modification（P-LND and P-GPMA-LND）were synthesized by one-step free radical polymerization.P-LND and P-GPMA-LND displayed well-defined structures with similar molecular weight ranging from 19.1 to 22.2 k Da and LND loading content around 15 wt%.In vitro studies towards CAFs showed that the conditioned medium of CAFs（CM）contained certain levels of CXCL12,TGF-β,IL-6.When incubated with 4T1cells,CM significantly promoted the migration and invasion of 4T1 cells（p<0.01）.Compared with Lip,E8R8-Lip displayed significantly increased cellular uptake（p<0.01）and cytotoxicity in CAFs.Of note,E8R8-Lip greatly weakened the pro-metastasis effect of CAFs by inhibiting the secretion of the above-mentioned pro-metastasis cytokines.Subsequently,the mitochondrial accumulation of P-GPMA-LND-FITC was investigated.P-GPMA-LND significantly increased the mitochondria accumulation by nearly 8 times compared with non-guanidine modified P-LND（p<0.001）.Thus,P-GPMA-LND efficiently damaged mitochondrial function,reduced the synthesis of adenosine triphosphate（ATP）by nearly 40%（p<0.01）and suppressed the metastasis of 4T1 cells.Furthermore,P-GPMA-LND reduced the oxygen consumption and resulted in alleviated hypoxia degree of 4T1 cells.In vivo distribution experiments found that E8R8-Lip exerted significantly higher tumor accumulation than Lip and R8-Lip（p<0.05）without increasing the distribution in normal tissues.Mitochondrial accumulation of P-GPMA-LND-FITC after intratumoral injection was 4.2 times higher than that of P-LND（p<0.001）,further revealing the mitochondria targeting capacity of GPMA.Surprisingly,pre-administration of E8R8-Lip greatly depleted CAFs and reduced tumor matrix deposition,and then intratumoral penetration of P-GPMA-LND was greatly promoted.This sequential administration strategy might be beneficial for in vivo efficacy.Having demonstrated that E8R8-Lip and P-GPMA-LND exploited distinct mechanisms to inhibit metastasis of 4T1 cells,we then investigated whether their sequential combination could suppress metastasis more efficiently.Notably,the sequential combination of E8R8-Lip-CM→P-GPMA-LND resulted in significant higher suppression of migration and invasion of 4T1 cells than single drug treatment.Mixture of 4T1 cells and 3T3 cells（1:1）were implanted into one mammary fat pad of female BALB/c mice to establish a stroma-rich orthotopic breast cancer mice model.Then,the effect of E8R8-Lip combined with P-GPMA-LND in regulating tumor hypoxia microenvironment and inhibiting breast cancer metastasis was investigated.After E8R8-Lip treatment,CAFs in the tumor microenvironment were greatly depleted,leading to decreased deposition of collagen fibers and fibronectin.And the oxidized hemoglobin（Hb O₂）content in E8R8-Lip treated group was1.6-fold higher than that of saline treated group（p<0.01）.E8R8-Lip or P-GPMA-LND alone alleviated the degree of hypoxia and downregulated the expression of hypoxia-inducible factor 1α（HIF-1α）.Notably,sequential administration of E8R8-Lip and P-GPMA-LND（E8R8-Lip→P-GPMA-LND）exerted higher Hb O₂content than single drug teratment（p<0.01）,resulting in greatly decreased hypoxia degree and HIF-1αexpression.These results demonstrated that the separate mechanisms of CAFs depletion（increasing oxygen perfusion）and mitochondria disruption（decreasing oxygen consumption）,when combined,were complementary to relieve the tumor hypoxia.Furthermore,after treatment of E8R8-Lip→P-GPMA-LND,the expression of lysyl oxidase（LOX）and metalloproteinase-2（MMP-2）in lung,as well as pulmonary recruitment of bone marrow-derived cells（BMDCs）were significantly decreased（p<0.01）.Thus,the formation of lung PMN was greatly inhibited.The metastasis of luciferase expressed 4T1 cells（4T1-Luc）in lung tissue was also investigated.Consistent with the results of lung PMN formation,E8R8-Lip→P-GPMA-LND inhibited the lung colonization of 4T1-Luc cells“seeds”more efficiently than single drug treated group（p<0.01）.This might be ascribed to that combination of E8R8-Lip and P-GPMA-LND coordinated in inhibiting pulmonary PMN formation by alleviating hypoxia in primary tumor.Finally,in vivo anti-tumor effect was further investigated.Either P-GPMA-LND alone or E8R8-Lip alone was insufficient to suppress the growth of primary tumor and failed to completely eradicate the lung metastasis.By comparison,E8R8-Lip→P-GPMA-LND resulted in considerable suppression of primary tumor with the least tumor burden（72%tumor inhibition rate）and best anti-metastasis effect（the average number of lung metastasis nodules was about 3per lung）among all tested groups.In addition,E8R8-Lip→P-GPMA-LND exhibited favorable safety with no damage in heart,liver,spleen,or kidney observed.These results indicated that this sequential strategy attained the efficient anti-tumor and anti-metastasis capacity with considerable safety.In summary,E8R8-Lip and P-GPMA-LND cooperatively alleviated tumor hypoxia by killing CAFs and inhibited aerobic respiration of cancer cells by damaging mitochondrial function.At the same time,this strategy curbed the promotion effect of CAFs on tumor cell metastasis and cut off the energy supply for the metastasis of tumor cells,therefore inhibiting metastasis potently.These results provided a proof of concept that regulating tumor microenvironment combined with mitochondrial targeting was a feasible way to inhibit breast cancer metastasis.（2）A combined strategy of remodeling inflammatory microenvironment and targeting mitochondria for breast cancer metastasis suppressionChronic inflammation is another crucial pro-metastasis feature of TME.COX-2,a protease expressed in a variety of inflammatory cells in tumor tissues,plays important roles in promoting metastasis by up-regulating the expression of multiple cytokines（VEGF,EGFR,CXCL12,MMP,etc.）and matrix components.As a non-steroidal anti-inflammatory drug,celecoxib（Cel）is capable of selectively down-regulating the expression of COX-2 in these inflammatory cells.To further explore the anti-metastasis effect of regulating tumor microenvironment combined with mitochondrial targeting,we then established a combined system consisted of Cel loaded liposome（Lip-Cel）to alleviate tumor inflammation and camptothecin（CPT）loaded nanocomposite to target mitochondria of cancer cells.Though P-GPMA-LND could inhibit metastasis,its wider application might be limited by the weak cytotoxicity of LND and the non-specific distribution in the body due to the positive charge of GPMA.We then tried to construct a more potent mitochondria targeting system.For this purpose,CPT was covalently tethered by a ROS-responsive thioketal linker（TK）in the side chain of HPMA polymers to realize mitochondria-specific drug release.Water soluble 2-（Dimethylamino）ethyl methacrylate（DEA）instead of those hydrophobic mitochondrial targeting ligand（triphenylphosphine and glycyrrhetinic acid）was used as the mitochondrial ligand.Besides,the negatively charged HA was used to cross-link with DEA modified HPMA polymer-CPT conjugates（P-DT-CPT）to form nanocomposites（PDT-NCs）via coulombic interaction.By this way,HA can shield the positive charge of DEA and promote the cellular uptake of P-DT-CPT by binding with the CD44 receptor over-expressed in tumor cells.Firstly,Lip-Cel was prepared by film dispersion method and exhibited spherical shape with the diameter of about 130 nm and encapsulation efficiency of Cel over 90%.Afterwards,DEA unmodified or modified HPMA polymer-CPT conjugates with reactive oxygen species（ROS）responsive TK as the linker（P-TK-CPT and P-DT-CPT）were synthesized.Also,The DEA modified HPMA polymer-CPT conjugate with ROS non-responsive azelaic acid（LA）as linker（P-DL-CPT）was prepared as control.The three polymers displayed similar characteristics with molecular weight ranging from 22 to 24 k Da and CPT loading content about 12%.Next,by screening the ratio of HA to P-DT-CPT,PDT-NCs（150 nm,PDI<0.2）with smaller particle size and uniform dispersion were obtained.The zeta potential of PDT-NCs was-16.15±0.35 m V,indicating that HA successfully shielded the positive charge of DEA.To investigate the ROS responsive drug release behavior,different concentrations of hydrogen peroxide（H₂O₂）were used to simulate in vivo ROS levels.P-DT-CPT,P-TK-CPT and PDT-NCs with TK as linker can achieve accelerated release under higher concentrations of H₂O₂.On the contrary,the P-DL-CPT with azelaic acid as the linker couldn’t realize ROS responsive CPT release.With the aid of the positive charge of DEA,the cellular uptake of P-DT-CPT was nearly 7 times higher than that of P-TK-CPT in 4T1 cells（p<0.01）.Surprisingly,the internalization of PDT-NCs was 2.3 times higher than that of P-DT-CPT（p<0.01）due to the ability of HA to bind with CD44 receptor over-expressed in the surface of 4T1 cells.Furthermore,PDT-NCs could efficiently accumulate in tumor cell mitochondria and damage mitochondrial function.Thus,it potently decreased intracellular ATP level and inhibited the proliferation of 4T1cells,resulting in greatly curbed 4T1 cell metastasis.Of note,despite of the similar cellular uptake and mitochondrial targeting behavior,P-DT-CPT damaged mitochondrial more efficiently than P-DL-CPT（p<0.05）.This might be ascribed to the mitochondrial specific drug release of P-DT-CPT.Western Blot and immunohistochemistry assay indicated that Lip-Cel could downregulate the expression of COX-2 and secretion of PGE₂,resulting in decreased expression of various inflammatory factors in tumor tissue including tumour necrosis factor（TNF-α）,IL-6,MMP-2 and vascular endothelial growth factor（VEGF）.Finally,4T1 cells were implanted into one mammary fat pad of female BALB/c mice to establish orthotopic breast cancer mice model.And the anti-tumor and anti-metastasis effect of Lip-Cel combined with PDT-NCs（Lip-Cel/PDT-NCs）was investigated in orthotopic breast cancer mice model.Lip-Cel/PDT-NCs displayed significantly higher tumor inhibition rate（80%）and anti-metastasis efficacy than single treatment（p<0.05）.These results further confirmed that remodeling tumor inflammatory combined with mitochondrial dysfunction can inhibit breast cancer metastasis potently.In summary,the strategy of regulating tumor microenvironment（hypoxia,chronic inflammation）combined with mitochondrial dysfunction was a two-pronged approach to inhibit the pro-metastasis effect of tumor microenvironment and suppress the metastatic ability of tumor cells,resulting in synergetic anti-metastasis effect.This study provides a new strategy for anti-metastasis therapy.