Study On The Antitumor Efficacy And Tumor Targeting Of Doxorubicin Loaded Bletilla Striata Polysaccharides-based Micelles In Vivo

Bletilla striata polysaccharides(BSPs),a kind of natural biomaterial,could be easily modified for drug delivery due to the presence of abundant hydroxyl groups on its molecular chains.The folate(FA)mediated and stearic acid(SA)modified BSPs polymers were obtained by covalent attachment of FA and SA to BSPs backbone.The substituted degree values of SA and FA conjugated to the monosaccharide residue of BSPs were 12.94%and 5.6%,respectively,which were confirmed by ~1H Nuclear Magnetic Resonance and Ultraviolet spectra.The results of gel permeation chromatography indicated that the average molecular mass of BSPs-SA and FA-BSPs-SA were 22361 Da and 24448 Da,respectively.Besides,the critical aggregation concentration values of BSPs-SA and FA-BSPs-SA slightly increased with the decrease of pH values.The critical aggregation concentration of FA-BSPs-SA was lower than that of BSPs-SA.Three different kinds of bile salt and doxorubicin hydrophpbic complex were prepared via electrostatic interaction.The effects of types,concentrations and amounts of bile salts on drug-loaded micelles were screen.The results indicated that the complex between doxorubicin and sodium cholate could be an alternative to be directly incorporated into micelles with high drug loading content and encapsulation efficiency when the concentration of the complex and the drug versus vehicles mass ratio were kept at 2.0 mg/mL and 1:6,respectively.Dox/FA-BSPs-SA and Dox/BSPs-SA micelles displayed pH-sensitive property.Besides,the particle size and zeta potential slightly decreased with the increase of pH values.The results of differential scanning calorimeter and thermogravimetric analysis suggested that doxorubicin might be existed in an amorphous state in FA-BSPs-SA micelles.The doxorubicin release from and Dox/BSPs-SA and Dox/FA-BSPs-SA micelles exhibited two stages.In the first stage,the drug release might be the combination of diffusion and erosion control meachnisms.At the second stage,the drug release of Dox from micelles might be controlled by the cooperative function of degradation,diffusion and erosion meachnisms.More over,release rates of doxorubicin from drug-loaded micelles were accelerated at low pH values.Dox/BSPs-SA and Dox/FA-BSPs-SA micelles presented better stability in RPMI 1640 medium containing 10%of fetal bovine serum at 37°C for 48 h.The results of hemolysis revealed that BSPs-SA and FA-BSPs-SA micelles had better blood compatibility.The cytotoxicity exposed to Dox/BSPs-SA micelles or Dox/FA-BSPs-SA micelles was distinctly higher than that of free doxorubicin on HepG2 cells and 4T1 cells,which were confirmed by MTT and migration assays.Besides,Dox/FA-BSPs-SA micelles presented greater inhibitory effects on 4T1 cells in comparison to Dox/BSPs-SA micelles at corresponding drug concentration.Nevertheless,no remarkable difference for the cytotoxicity of Dox/FA-BSPs-SA and Dox/BSPs-SA micelles on HepG2 cells was found.The cellular uptake amounts of Dox/FA-BSPs-SA micelles were greater than those of Dox/BSPs-SA micelles and free doxorubicin,which displayed time-dependent and dose-dependent.Moreover,the escape ability of Dox/FA-BSPs-SA micelles from lysosomes was stronger than Dox/BSPs-SA micelles,which enhanced the distribution of Dox in the cytoplasm and allowed the drug to fastly reach the nuclei.The results indicated that the internalized pathways of Dox/FA-BSPs-SA micelles and Dox/BSPs-SA micelles into 4T1 cells were mainly controlled by the clathrin-mediated endocytosis.Besides,the internalization of Dox/FA-BSPs-SA micelles and Dox/BSPs-SA micelles also involved caveolae-induced endocytosis and macropinocytosis.Folate receptor-mediated endocytosis played an important role in the uptake of Dox/FA-BSPs-SA micelles.The cellular uptake of micelles was an energy-dependent process.Compared with free doxorubicin,Dox/BSPs-SA micelles and Dox/BSPs-SA micelles presented lower clearance rates,longer mean residues time and biological halftime.The absolute bioavailability was also enhanced after encapsulating the doxorubicin into the hydrophobic core of micelles.It could be observed from Maestro in vivo imaging system that DiR/BSPs-SA micelles and FA-BSPs-SA micelles presented weaker florescence intensities in the heart and the greater florescence intensities in the tumor tissues.Besides,Dox/FA-BSPs-SA micelles exhibited more superior tumor inhibition compared to free doxorubicin and Dox/BSPs-SA micelles,which might chiefly be attributed to the folate receptor-induced endocytosis and enhancement of permeation and retention effect of micelles.Chemo-photodynamic therapy was a promising method for cancer treatments.1-(4-carboxyphenoxy)zinc phthalocyanine conjugated with doxorubicin(ZnPc-C-Dox)were gained and characterized by mass spectrometry,~1H Nuclear Magnetic Resonance and Fourier transform infrared spectrum.ZnPc-C-Dox loaded FA-BSPs-SA micelles were prepared by a dialysis method with high encapsulation efficiency and loading capabicity,which also showed a pH-sensitive property.The fluorescence quantum yields and singlet oxygen generation were also maintained after the ZnPc-C-Dox were incorporated into the hydrophobic core of FA-BSPs-SA micelles.For 4T1 cells,the anticancer efficacy of ZnPc-C-Dox loaded FA-BSPs-SA micelles was improved by the chemo-photodynamic synergistic effect.Bovine serum albumin(BSA)was often selected as model protein to explore the interactions with BSPs-SA and FA-BSPs-SA micelles via the fluorescence emission spectrum,ultraviolet spectrum,circular dichroism and molecular modeling.The results suggested that the interaction process was spontaneous and exothermic reaction.The negative value of entropy change(ΔS)and enthalpy change(ΔH)suggested van der Waals forces and hydrogen bonds played important roles in their interactions.Moreover,BSPs-SA micelles or FA-BSPs-SA micelles were mainly bound to the subdomain IIA(site I)around Trp-213 residue in BSA along with a non-radiative energy transfer,which caused the quenching of fluorescence.Furthermore,the BSPs-SA micelles or FA-BSPs-SA micelles interacted with BSA resulted in decreasing theα-helical contents and causing a slightly conformational alteration in second structure of BSA.To further confirm the results and acquire more information about the interaction between BSPs-SA/FA-BSPs-SA micelles and BSA,molecular modeling was applied to illuminate the binding process.Based above resuts,the tumor targeting and anticancer effects of doxorubicin were enhanced after encapsulating into the FA-BSPs-SA micelles,which might be releated to the accumulation caused the folate receptor-induced endocytosis.Besides,the conformation of BSA was slightly altered after interacting with micelles.Furthermore,chemo-photodynamic therapy provides a new approach for the cancer treatments.