Design Of Targeting Drug Loading Peptide And Analysis Of Peptide-doxorubicin Microsphere On Anti-tumor Activity

Cross-multidisciplinary is becoming more and more important in drug development.It is of great significance to design polypeptide sequences reasonably and make them as macromolecule nanomaterials and endow them with certain abilities of targeting and recognizing cancer cells,carrying drugs and penetrating cell membranes.As drug carriers,amphiphilic polypeptide have many advantages:(1)T he solubility of poorly soluble drugs is improved.The hydrophobic core of micelles can encapsulate poorly soluble drugs in the form of covalent bonds,hydrophobic interactions and van der Waals force.Because of the high partition coefficient of these drugs in the hydrophobic core of micelles,they can achieve a higher drug loading.(2)The drug resistance to the environment is improved.By encapsulating of amphiphilic polypeptide chains,free drugs can enter the human body,avoiding the influence of some tissue microenvironment factors(for example,some enzymes can degrade protein drugs,phagocytosis of vesicles to capture and deplete small molecules of drugs).(3)The human body's drug absorption rate is improved.The drug is coated with a "protective shell"by the hydrophilic segment of amphiphilic polypeptide chain,which is not easy to be recognized by phagocytosis vesicles or endophagic vesicles,prolongs the retention time of drugs in metabolism and improves the absorption and utilization rate of drugs in human body.(4)Targeting effect.By leading into some polypeptide sequences with specific recognition of cell surface receptors,drug-loaded micelles can actively identify the location of tumors,improve the efficacy,reduce unnecessary drug waste and reduce toxicity.Based on solid-phase synthesis technology,amphiphilic polypeptide P10(DGRGAAAA)with targeting and drug-loading functions was designed and prepared.On this basis,doxorubicin(DOX)was used as drug carrier to prepare targeted drug-loaded nanomicrospheres.The P10 was characterized by infrared spectroscopy and high resolution mass spectrometry to determine the synthesis of the target product and to meet the purity requirements of cell experiments in vitro.The morphology,matrix structure,critical micelle concentration(CMC)and in vitro drug release properties of P10-DOX nanoparticles were systematically investigated to provide theoretical basis for their further application.The main research contents are as follows:(1)Amphiphilic targeting polypeptide P10 was synthesized by solid-phase synthesis method,and the solid-phase synthesis conditions were optimized.The optimum synthesis conditions were determined as follows:2-cl resin as carrier,DIEA as activation reagent,TBTU as catalyst,the mass ratio of amino acid to resin was 0.5:1,reaction time was 1.5 h.Under these conditions,the yield of crude peptide was 87.06%and the purity was 95.55%.The molecular weight of P10 was characterized by liquid chromatography-mass spectrometry(LC-MS).After purification by high performance liquid chromatography(HPLC),the purity of P10 was 96.08%.The CMC of P10 was analyzed by ultraviolet spectroscopy and fluorescence spectroscopy.The results showed that the CMC value of P10 was about 0.45 mg/L.The interaction force type between P10 and DOX was analyzed by fluorescence spectroscopy.The results showed that DOX had a good static quenching effect on P10 fluorescence,and the interaction force was mainly hydrogen bond or van der Waals force.(2)DOX was loaded by dialysis method.P10 and DOX were mixed at a mass ratio of 6:1 to form P10-DOX drug-loaded nanomicrospheres.The standard curve and linear equation of DOX were constructed by ultraviolet spectrophotometry.The drug loading rate(DLE)and encapsulation efficiency(EE)of P10-DOX nanospheres were calculated.The EE and DLE values of P10 for DOX were 23.011%±2.88%and 10.125%±2.62%respectively,which indicated that P10 had good encapsulation ability for DOX.The structures of P10,DOX and 110-DOX were analyzed by infrared spectroscopy.The comparison of the characteristic peaks of the three substances proved that P10 successfully encapsulated DOX.The morphology and size of nanospheres were studied by scanning electron microscopy(SEM)and Zeta particle size analyzer respectively.The results showed that the P10-DOX composite was spherical micelles with uniform size,and the particle size ranged from 390 to 530 nm.(3)By simulating the conditions of gastric juice(pH 1.2),intestinal juice(pH 6.8)and tumor cell environment(pH 4.5),we studied whether P10-DOX could release effectively in gastric juice,intestinal juice and tumor environment.The results showed that the drug would release rapidly to a certain concentration within the first 24 hours,and then release slowly and steadily within the next 72 hours to achieve the effect of sustained drug release.Six mathematical models were used to analyze the drug release characteristics of P10-DOX nanoparticles in vitro.The results showed that the drug release characteristics of P10-DOX nanoparticles in vitro conformed to Zero-order and Ritger-Peppas models.The cytotoxicity of P10 on murine breast cancer(4T1)cells was investigated by MTT assay.The results showed that P10 was not toxic to 4T1 cells.The anti-tumor activity of P10-DOX in vitro was studied with Hela cells as the research object.The results showed that the inhibition rate of P10-DOX on HaLa cells was higher than that of DOX.When the concentration of P10-DOX was 20?g/mL,the inhibition rate of tumor cells was 44.17%.(4)P13(DGRHHLLLAAAA)peptide was synthesized by solid-phase synthesis.The acid-base buffering ability of P13 was investigated by acid-base titration.FT-IR spectroscopy was used to analyze the connection between the components and the surface morphology was studied by SEM.The results of ultraviolet spectroscopy,fluorescence spectroscopy,infrared spectroscopy,Zeta particle size analyzer and drug release in vitro showed that P13 had good acid-base buffering ability.The CMC value was about 0.21 mg/L.The particle size distribution of P13-DOX nanoparticles was concentrated in 122-164 n..The EE and DLE values of P13 to DOX were 20.40%±1.21%and 21.25%+±2.79%respectively.Under different pH conditions,the drug release characteristics in vitro conformed to Weibull model.Finally,the antitumor activity of 4T1 cells in vitro was investigated.The results showed that when the concentration of P13-DOX was 50 ?g/mL,the inhibition rate was 26.65%.Compared with P10-DOX,the inhibition rate of P13-DOX on cancer cells was higher.When P13-DOX enters the cell,it first locates in lysosome,then enters the nucleus 2 hours later,and induces apoptosis in the nucleus.