Study On Multi-functional Nanoparticles Based On Low Molecular Weight Heparin

Cancer has still been a major global challenge to public health,the most common treatment strategy of which is still chemotherapy in clinic.However,the efficacy of traditional chemotherapy has been largely restricted by the following problems of drugs:low aqueous solubility,poor stability,non-specificity and severe side effects to normal tissues.Additionally,the therapeutic efficacy could not be timely evaluated,making it hard to adjust the therapeutic schedule in time.Therefore,it is of urgent importance to develop a novel nano-sized drug delivery system,which could not only kill tumor cells efficiently but also provide a real-time diagnosis of tumor during the treatment process.Accordingly,a novel multi-functional drug delivery system was proposed in this project,which could on the one hand rapidly release chemotherapeutics in response to the reductive environment in tumor cells,thus improving the chemotherapy efficiency;on the other hand,realize the photodynamics therapy(PDT)of tumor,thus combining chemotherapy and PDT in one nanosystem.Additionally,this system could also be utilized for tumor NIR imaging.The work of this thesis could be divided into two parts:(a)Hep-cys-TOS polymer with cystamine as the redox-sensitive linker was synthesized,which was then used to encapsulate hydrophobic drug paclitaxel(PTX).Also,non-redox-sensitive polymer Hep-ADH-TOS was synthesized as a control.The biological performance of both redox-sensitive and non-redox-sensitive nanosystems was compaed,suggesting that the redox-sensitive property could enhance the curative effect.This part could lay the foundation for the development of multi-functional nanoparticles.(b)Based on the preliminary work in Part(a),Ce6 was further conjugated to Hep-cys-TOS polymer and the obtained polymer,Ce6-Hep-TOS(CHT),was utilized to encapsulate PTX.Due to the presence of PTX,this system could be used for cancer chemotherapy;due to the presence of Ce6,this system could be used for PDT and NIR imaging of tumor.Thus,this nanosystem could successfully integrate redox-triggered drug release,tumor diagnosis and tumor therapy(chemotherapy and PDT).The main methods and results are exhibited as following.1.The synthesis and characterization of Hep-cys-TOS and Hep-ADH-TOS polymersHep-cys-TOS and Hep-ADH-TOS polymers were synthesized via amidation using cystamine and ADH as the linker between LWMH and TOS,respectively.The successful synthesis was confirmed by 1H-NMR and FT-IR.By changing the ratio of TOS to LWMH,Hep-cys-TOS polymers with different degree of substitution(DS)were prepared,the DS of which were 1.72,3.48 and 6.95,respectively.2.The preparation of polymeric nanoparticles(NPs)based on Hep-cys-TOS polymerThe critical aggregation concentration(CAC)of different Hep-cys-TOS polymers was investigated by fluorescence spectroscopy with pyrene as the fluorescence probe,suggesting that the CAC values ranged from 0.026 to 0.093 mg/mL.Also,the CAC value of Hep-ADH-TOS polymer was 0.050 mg/mL.The low CAC values of all the polymers indicated that the self-assembled NPs based on these polymers may exhibit good stability.Blank NPs based on different polymers were prepared with a sonication method;the size of these NPs ranged from 127.7±2.2 to 212.6±9.8 nm and the zeta potential ranged from-16.17±2.21 to-8.53±0.99 mV.Hep-cys-TOS 2 was selected as the carrier material to encapsulate PTX and PTX-loaded Hep-cys-TOS NPs(PTX/Hep-cys-TOS NPs)were prepared.The drug/carrier ratio and water/organic phase ratio could influence the drug loading ability of Hep-cys-TOS NPs.An optimized formulation was obtained when the drug/carrier ratio(w/w)was 36:80,water/organic phase ratio(V/V)was 3:0.375.The optimized DL and EE values were 18.99%and 52.93%,respectively.The physicochemical properties of the optimized PTX/Hep-cys-TOS NP were investigated,suggesting that the mean diameter of NPs was 224 nm(PDI=0.264)and the mean zeta potential was-26.2 mV.Also,TEM observation revealed that the NPs were spherical in shape with a uniform size distribution.DSC assay proved that a strong interaction existed between polymer and PTX.Hep-cys-TOS NPs possessed a desirable redox-sensitivity considering the changes of size distribution,morphology of NPs and appearance of NP solution in response to reductive condition.Also,an accelerated PTX release from Hep-cys-TOS NPs was also observed in the presence of 20 mM DTT,which would further confirm the redox-sensitivity of the nanosystem.The uptake of Hep-cys-TOS NPs by MCF-7 cells was observed by using fluorescence microscope and flow cytometry(FCM),suggesting that the NPs could be efficiently internalized by MCF-7 cells.It has also been confirmed that Hep-cys-TOS NPs were taken by MCF-7 cells via energy-dependent,caveolae-mediated and cholesterol-dependent endocytosis.MTT assay was employed to evaluate the cytotoxicity of both blank and PTX-loaded NPs.The results suggested that both Hep-cys-TOS and Hep-ADH-TOS polymers exhibited negligible toxicity against MCF-7 and L929 cells.For MCF-7 cells,the redox-sensitive PTX/Hep-cys-TOS NPs exhibited a lower IC50 value(0.79μg/mL)compared with that of non-redox-sensitive PTX/Hep-ADH-TOS NPs(1.11μg/mL),implying that the redox-triggered release of PTX could enhance the anti-cancer effect of the nanosystem.3.Development of PTX/CHT NPs and characterizations of the physicochemical propertiesEDC/NHS chemistry was utilized to chemically conjugate the carboxyl groups of Ce6 to the unreacted amide groups of Hep-cys-TOS and the carrier material Ce6-Hep-TOS(CHT)was synthesized.By altering the ratio of Ce6,TOS to LWMH,a library of CHT polymers(CHT 1-6)could be obtained.The chemical structure of CHT polymer was characterized by 1H-NMR and UV spectrophotometry;the Ce6 content in different CHT polymers was analyzed via UV spectrophotometry,indicating that the Ce6 content ranged from 1.88%to 22.60%.A sonication method was adopted to prepare drug-free NPs based on different CHT polymers and the size,zeta potential were measured using a DLS method;the morphology was observed using TEM.A modified dialysis method was employed to prepare PTX/CHT NPs and the drug-loading ability of different CHT polymers was evaluated by measuring the DL and EE values of different formulations under different drug/carrier ratio.The CHT NPs were incubated under a reductive environment;the change of morphology,recovery of NIR florescence and ROS generation were recorded to illustrate the redox-sensitivity of the CHT NPs.Additionally,it was found that the release of PTX from PTX/CHT NPs could be accelerated in the release medium containing 20 mM DTT;however,20 μM DTT could not trigger the release of PTX.The results suggested that PTX/CHT NPs could keep stable in blood,while being destroyed by the reductive substance in tumor cells and rapidly release the loaded drug to kill tumor cells.4.In vitro and in vivo biological evaluation of PTX/CHT NPsThe cellular uptake of CHT NPs and Ce6 solution was observed by confocal laser scanning microscope(CLSM),suggesting that the uptake of Ce6 via MCF-7 cells could be dramatically enhanced when it is delivered by CHT NPs compared with Ce6 solution.The flow cytometry(FCM)analysis was conducted to further quantitatively describe the Ce6 internalization,suggesting the same trend with the CLSM observation results:after 1,2,and 4 h of incubation,the mean fluorescence intensity values of CHT NP-treated MCF-7 cells were respectively 4.25,4.96 and 5.12 times higher than those of Ce6-treated ones.The results demonstrated that CHT NPs could effectively enhance the Ce6 internalization.The dark and light cytotoxicity of CHT NPs against MCF-7 cells were evaluated via MTT assay.It was found that CHT NPs(0.5-5 μg/mL of Ce6 equivalents)exhibited negligible dark toxicity against MCF-7 cells,suggesting the good biocompatibility of CHT NPs.When cells were irradiated with NIR laser(660 nm),the survival rate decreased along with the increase of CHT concentration;besides,the viabilities of MCF-7 cells treated with CHT NPs plus NIR irradiation were always lower than those treated with Ce6 solution plus NIR irradiation.The combinational chemotherapy/PDT effect was evaluated via MTT assay.When MCF-7 cells were exposed to PTX/CHT NPs at a low Ce6 concentration(0.000175-0.175 μg/mL),no obvious difference in cell viability was found between the NIR-irradiated and non-NIR-irradiated groups,suggesting that the Ce6 concentration was too low to exert PDT effect;however,when Ce6 concentration increased to 1.75 μg/mL,there was a significant difference in cell viability between these two groups(p=1.9×e8<0.01),suggesting a combinational PDT/chemotherapy effect could be obtained at a high Ce6 concentration.An apoptosis assay was the conducted to evaluate the anti-cancer effect of different treatment strategies,indicating that PTX/CHT NPs exhibited superior cell-killing effect compared with mono-photodynamic or mono-chemotherapy.A wound healing assay was designed to investigate the anti-metastasis activity of CHT NPs.It was found that the CHT NPs could preserve the anti-metastasis activity of LMWH,making CHT NP promising as carrier material for cancer management.The in vivo NIR imaging study was performed by recording the NIR fluorescence intensity at designed time points post injection of CHT NPs or Ce6 solution.It was found that CHT NPs could effectively enhance the Ce6 signal intensity and increase the retention time of Ce6 in tumor foci.According to the results of in vivo combinational therapy evaluation,PTX/CHT NPs could effectively inhibit the tumor growth via a combinational therapy mode compared with the mono-PDT or mono-chemotherapy.Also,according to the results of safety evaluation,the ’PTX/CHT NP plus NIR irritation’ treatment would not cause any severe damage to the major organs or obvious change of body weight,indicating the developed nanosystem exhibited desirable safety.Taken together,the developed smart multifunctional nanosystem successfully integrated NIR imaging capacity,redox-sensitivity,chemotherapy and PDT efficiency,exhibiting great promise for effective cancer treatment with little side effects.