Drug-loaded Polymeric Micelles Internalized In Mesenchymal Stem Cells For Tumor Targeting In Glioma Therapy

Tumor targeting is one major realm in development of advanced drug delivery system.Tumor-targeted delivery system is able to enhance the antitumor efficiency of chemotherapeutics by increasing the drug concentration in tumors while reduce the side effects.In addition to the application of tumor-targeted nanocarriers,targeting delivery can also be achieved by cell-based therapies.Mesenchymal stem cells also called multipotent mesenchymal stromal cells which have tumor homing ability could be potential candidates,are widely used in cell therapy,tissue regeneration and immunotherapy.MSCs loaded with chemotherapeutant is a promising vehicle to delivery drugs.In present stduy,hyaluronic acid-b-poly(d,l-lactide-co-glycolide)copolymer(HA-b-PLGA)was synthesized and PTX-HA-PLGA was prepared to target CD44 receptor expressed both on the surface of MSCs and glioma.The MSCs-Micelles biotargeting system(MSCs-Micelles)was constructed by loading micelles into MSCs via endocytosis.MSCs-Micelles not only increased the intracellular concentration of PTX in MSCs through the specific interation between HA and CD44,but also protected MSCs from direct exposure to antimicrotubule agent by encapsulating PTX into HA-PLGA micelles.The migratory capacity of MSCs was preserved.In an orthotopic glioma model,the extensive distribution of MSCs as well as PTX could be observed in the glioma area after the therapeutic injection at contralateral hemisphere.With the sustained and increased accumulative release,enhanced drug delivery into tumor cells and enhanced anti-glioma activity was achieved by MSCs-Micelles.The life span of the tumor-bearing rat was significantly prolonged.Three HA-b-PLGA502H polymers were first synthesized with different HA molecular weights(Mw = 5.8,6.7,9.1 kDa).1,4-butanediamine was linked to the end of HA by reductive amination.Then,the activated HA was conjugated to succinimide ester of PLGA to form HA-b-PLGA copolymers.1H-NMR and GPC were applied to confirm the chemical structure of HA-b-PLGA.Pyrene was employed to determine the critical micelle concentration(CMC)of different HA-b-PLGA copolymers.With an increase in the molecular weight of HA,the CMC value increased.The CMC values to HA5.8k-b-PLGA,HA6.7k-b-PLGA and HA9.1k-b-PLGA were 1.42 mg/L,2.08 mg/L and 2.84 mg/L,respectively.A solvent-dialysis method was used to prepare HA-PLGA micelles.With an increase in the molecular weight of HA,the size of HA-PLGA micelles increased correspondingly.In contrast,the zeta potential of HA-PLGA micelles was not affected and exhibited a negative charge of-15 mV to-16.0 mV,which may contribute to their stability and long circulation in vivo.Finally,HA5.8k-b-PLGA502H micelles,with the smallest size and a suitable CMC were developed for further investigation.In order to obtain a higher drug loading rate and encapsulation efficiency,we optimized the formulation of micelles.The optimal formulation was PTX/polymer ratio of 1:20 with a copolymer concentration of 2 mg/mL.The classical core-shell structure was observed by transmission electron microscopy(TEM).The encapsulation efficiency and drug loading of PTX-HA-PLGA micelles were 53.64 ± 1.08%and 2.57 ± 0.03%,respectively.In vitro release experiments showed that the drug could be sustained released over 50%within 3 days.The cytotoxicity of PTX-HA-PLGA micelles toward MSCs and C6 tumor cells was evaluated by MTT assay.A significant difference in PTX sensitivity was observed between MSCs and C6 cells.The IC50(1226.81 ± 20.53 ng/mL)of HA-PLGA-PTX micelles to MSCs was 300 times higher than that of C6 cells(3.8 ± 0.14 ng/mL).This difference of tolerance is crucial for loading and releasing sufficient amounts of PTX from MSCs to target sensitive glioma cells.Encapsulation of PTX into HA-PLGA micelles enabled MSCs to be protected from direct exposure to the antimicrotubule agent.Then,6-coumarin was used as a fluorescent probe to investigate the uptake of MSCs-Micelles in MSCs.Saturated uptake experiments and endocytosis inhibition experiments showed that the enhanced uptake of HA-PLGA micelles was dependent on the HA-CD44 spetific interation in MSCs.Moreover,the clathrin-mediated endocytic pathway and the caveolin-dependent endocytic pathway were involved in the internalization process.Subsequently,the effects of PTX-HA-PLGA micelles on cell cycle and cell migration ability of MSCs were investigated.The optimal PTA-HA-PLGA-micelles-incubation concentration was determined to be 8 ng/mL(equivalent to 1.3 pg/cell intracellular PTX),owing to almost complete restoration of migratory activity and cell-cycle patterns.Next,the release of the MSCs-Micelles was evaluated.The results showed that MSC-Micelles released payloads rapidly in the first 6 hours,and then the intracellular drug was maintained at a stable level,and sustained release within following 5 days,showing an initially fast and then slow excretion process.Approximately 25%and 40%and 62%of intracellular PTX,corresponding to 0.232 pg/cell,0.379 pg/cell and 0.792 pg/cell,was released from MSCs-PTX,MSCs-NPs and MSCs-Micelles,respectively.Compared with MSCs-PTX,MSCs-Micelles can release the drug slowly within three days,prolonging the release time of the drug.Then the transfer of PTX from MSCs-Micelles to C6 cells was confirmed by transwell system.The uptake kinetics of C6 in the transwell system was also shown that the intracellular PTX was significantly enhanced,about 0.03688 ± 0.00034 pg/cell and 0.04825 ± 0.00042 pg/cell,respectively,which was 1.3 times than that in the MSCs-NPs group.Subsequently,efficient PTX transfer was also confirmed by MSCs-Micells cytotoxicity against C6 cells,and that anti-tumor activity was dose-and MSCs number-dependent.At 8 ng/mL and a cell ratio of 1:5,MSCs-Micelles decrease C6 cells survival by 50%.A 3D tumor spheroid model was constructed,and the ability of MSCs and the loaded micelles to penetrate into the tumor spheroids and the tumor sphere viability were also investigated.The results demonstrated that MSCs would migrate to the tumor spheroid,and can further permeate into the 3D tumor spheroid.Due to HA modification,MSCs-Micelles showed deeper penetration.The viability of 3D tumors was evaluated by trypan blue exclusion assay.The results showed that MSCs-Micelles could induce tumor cell apoptosis and the antitumor activity was dose-and cell number-dependent.Finally,the tumor-targeted and anti-tumor activity of the MSCs-Micelles were evaluated in an orthotopic glioma model by means of contralateral tumor administration.Oregon Green(?)488 fluorescent PTX(PTX-Flu)micelles were prepared and loaded into MSCs,while CM-Dil was used as a red fluorescent probe to track the in vivo distribution of MSCs-Micelles in a rat model.Two days after MSCs-Micelles injection,a large number of scattered MSCs were found in the tumor area.Only a small amount of MSCs were clustered at the injection site,which proved the homing ability of MSCs-Micelles to C6 glioma.At the same time,a large amount of drug green fluorescence was observed in the tumor site,and most of the drugs were not co-localized with MSCs,indicating that MSCs-Micelles migrated to the tumor area can effectively unload the drug.This apparent tumor tropism and efficient drug release of the MSCs-Micelles system indicates the effectiveness of this biotargeting system in vivo.The anti-glioma activity of MSCs-Micelles system was subsequently evaluated in vivo.MSC-Micelles were injected contralaterally at a dose of 1 ?g/kg of PTX.Compared with other groups,the rats in the MSC-Micelles group had no significant abnormalities in consciousness or motor response and dramatically prolonged survival time in tumor-bearing rats,with medians of 14.5,13.5,22.5,23.5,38.5 and 60 days for Saline,MSCs,PTX-HA-PLGA micelles,MSCs-PTX,MSCs-NPs and MSCs-Micelles,respectively.After H&E staining,there were no significant differences between the control and MSC groups,both of which exhibited large tracts of tumor tissue in the brain with amplified hyperchromatic nuclei.No necrosis or obvious apoptosis was observed in the tumor mass,and the tumor boundary was obvious.After receiving PTX treatment,both showed significant nuclear contraction and significant tumor cell necrosis or apoptosis.The most significant reduction in the glioma area was observed in the MSCs-Micelles group,and tumors were hardly observed.These results clearly indicate that MSCs-Micelles had an effective anti-tumor effect in vivo.