| Chemotherapy is one of the most important method treating malignant tumors.Due to the poor water solubility and high toxicity of most chemotherapeutic agents,traditional chemotherapeutic formulations are found with low efficacy and severe side effects.With the development of material sciences and better understanding in tumor biology,the above mentioned problems have been partly solved by the emrgence of nanoscale drug delivery systems(NDDSs).However,the NDDSs are still unsatisfactory due to low drug loading capacity and poor physical stability.Moreover,the therapeutic effect of cationic nanoparticles is limited by the conflict between high cellular uptake and poor tumor penetration to some extent.In this study,concerning the problems of poor drug laoding and conflict between tumor penetration and cellular uptake,a NDDS with high drug loading and high tumor penetration without affecting cellular uptke has been designed.Therefor,this essay is divided into two major parts.In the first part,the role of medium chain triglycerides(MCT)as a crystallization inhibitor against polycaprolactone(PCL)in improving the drug loading of nanoparticles with PCL cores was introduced.Based on this finding,the method controlling the charge reversal profile via changing the structre of polyion complex layer was introduced in the second part.The drug release,cytotoxicity,cellular uptake,pharmacokinetics,biodistribution,intratumoral distribution,in vivo tumor cell uptake and tumor suppression of nanoparticles with different charge reversal profiles were studied.First,the crystallization inhibition effect of MCT on PCL as well as the solid-liquid phase separation between the two components were studied using polarized microscope and differential scanning calorimeter(DSC),together with atomic force microscope(AFM).It was found that the introduction of MCT could effectively decrease the crystallinity of PCL,and the liquid phase separated when MCT content reached to 40%of the weight of PCL.Block copolymer with different lengths in the hydrophobic block(mPEG5k-b-PCL10x,13x,16k)were synthesized.And the particle size and morphology of polyester-lipid mixed nanoparticles prepared with different MCT contents and block copolymer with different lengths in the hydrophobic block were investigated using dynamic light scattering(DLS)and transmission electron microscope(TEM).It was found that the particle size of the mixed nanoparticles was determined mutually by the length of the hydrophobic block and the amount of MCT incorporated;and mixed nanoparticles with high MCT content were found with emulsion-like morphology,while the hydrophobic core of mixed nanoparticles with low MCT content was still a homogeneous phase system.The critical aggregation concentration(CAC),core polarity and core viscosity were characterized using pyrene and 1,3-(1-1’-dipyrenyl)-propane(P3P).And it was found that the core polarity and core viscosity of mixed nanoparticles decreased with amount of MCT incorporated,while the CAC of the mixed nanoparticles showed a first increase then decrease pattern with the increase of MCT content.Three drugs,disulfiram(DSF,low compatibility with PCL),cabazitaxel(CTX,middle compatibility with PCL)and TM-2(high compatibility with PCL)were chosen as model drugs to study the drug loading profiles of mixed nanoparticles with different MCT contents.It was found that loading of all the three drugs was facilitated by reducing core crystallinity:at 25%MCT content,the drug loading contents of DSF,CTX and TM-2 were 3.19,1.61 and 1.34-fold of those in MCT-free micelles,but the amount of drug loading remained to be determined by drug-PCL compatibility.At low MCT content,the particle size of drug-loaded mixed nanoaparticles was primarily determined by drug loading content,while MCT content became the key parameter determing particle size when the MCT content was high.The mixed nanoparticles with high drug loading exhibited good physical stability since the particle size and drug loading content remained nearly unchange during 3-day storage under room temperature.The results of in vitro drug release study indicated that the mixed nanoparticles would release drugs in a more sustained manner when MCT incorporated.The cytotoxicity and cellular uptake in PC-3 and MCF-7 cell lines of MCT-free polymeric micelles and mixed nanoparticles with MCT content of 50%were compared,and not significant differences were observed between the two nanoparticles.The pharmacokinetic profiles of MCT-free polymeric micelles and mixed nanoparticles with MCT content of 50%in Sprague-Dawley rats were also studied,the nearly superimposed concentration-time curve and similar AUC0-t(1317.7 ± 107.5 and 1325.8 ± 153.4 mg/L h)indicated that the biological performances of mixed nanoparticles were not affected by MCT incorporation.Then,PLys-b-PCL was synthesized starting from PCL-OH,and cationic mixed nanoparticles were prepared by dialysis.The nanoparticles were coated with PGlu-g-mPEG after shell crosslinking with glutaraldehyde.The prepared coated mixed nanoparticles were found with diameters around 130 nm.Manipulation of charge reversal profiles was realized by varying the shell crosslinking degree,and non-crosslinked nanoparticles were unable to reverse surfacr charge in pH 6.5 PBS;and with higher crosslinking degree,the charge reversal rate increased.According to gel permeation chromatograph(GPC)analysis it was found that charge reversal was realized by shedding of the coating materials.The prepared nanoparticles with different charge reversal profiles(CTCL30 and CTCL70)showed good colloidal stability in FBS with good capability in reducing drug leakage.Also,the nanoparticles showed good sustained release behavior,and the release profile was pH dependent with faster drug release in low pH medium.The cytotoxicity of coated nanoparticles with different charge reversal profiles under media with different pH was investigated in human non-small cell lung cancer cell(A549)and mouse hepatoma cell(H22)by MTT assay,and the cellular uptake in A549 cell line of the nanoparticles under different pH and incubation time was also studied using coumarin 6 as fluorescent marker.Due to the sustained drug release profile,the cytotoxicity of nanoparticles was lower than that of the CTX DMSO solution.In pH 6.5 medium,the cytotoxicity of coated mixed nanoparticles were higher than that in pH 7.4 medium.The results of cellular uptake study indicated that the prepared nanoparticles were more readily internalized by cells after decoating.The pharmacokinetic profiles of CTX solution and the two coated mixed were compared in Sprague-Dawley rats.Encapsulation of CTX into nanoparticles was found helpful in prolonging the blood retention time.The AUC0-t of CTCL30 and CTCL70 were 3271.7 ±244.1 and 2916.0 ± 209.8 μg/L*h,respectively,and the t1/2 of the two nanoparticles were 3.89± 1.84 and 3.54 ± 1.63 h,respectively,which were all higher than the AUC0-t and ti/2 of CTX solution(1217.1 ± 427.3 μg/L*h and 0.562 ± 0.129 h).While negligible differences in the pharmacokinetic parameters between the two nanoparticles were observed.The biodistribution of CTX solution,CTCL30 and CTCL70 was investigated using kunming mice bearing mouse hepatoma H22 tumor xenograft.The two nanoparticles showed improved CTX accumulation in tumor tissue than that of the CTX solution group.And the intratumoral CTX content in the nanoparticle groups remained nearly constant.PLys-b-PCL was labeled with rhodamine B isothiocyanate(RBIC)before the preparation of coated mixed nanoparticles with different charge reversal profiles,and the RBIC-labeled nanoaprticles were employed in the investigation of tumor penetration after intravenous injection.The results turned out that CTCL30 with delayed charge reversal profile could distribute wider in tumor tissue and better tumor cell uptake.The in vivo tumor growth surpression effect of CTX solution,CTCL30 and CTCL70 was studied in kunming mice bearing mouse hepatoma H22 tumor xenograft with normal saline used as negative control.It was found that CTX solution showed limited tumor suppression effect with a tumor inhibition rate(TIR)of 39.3%,and the body weight of mice flunctuated dramatically.CTCL30 was found with a higher TIR than that of the CTCL70(72.3%v.s.60.2%).And the mice body weights in the two nanoparticle groups grew steadily during experiment,indicating relative lower side effects of nanoparticles.Both the tumor weights and tumor sizes in CTCL30 group were significantly lower than those in the CTCL70 group(P<0.05),indicating that delayed charge reversal nanoparticles could improve the percentage of tumor cells exposed to drug-containing environment through better tumor penetration,and thus improve the therapeutic effect of NDDSs. |
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