Study Of Self-assemble Nanoparticles Based On Biological Macromolecules

Nanotechnology could change the form of drugs in the body.For example,nanocarriers can sustainably release the drugs,and promote the absorption of drugs,and change the metabolism of drugs,so as to increase the drug efficacy and reduce the toxicity.Existing nanocarriers include liposomes,polymers,and inorganic materials.However,these nanomaterials can cause several problems in vivo,such as difficulty in metabolism and immune response.Biological macromolecules(such as human serum albumin(HSA),human hemoglobin(Hb),Transferrin(Tf)and erythrocyte membrane(RBC M),etc.)can overcome general problems caused by the nanocarriers(eg:immune response,it is difficult metabolism).More importantly,due to the special structures of biological macromolecules,they can form stable nano-assembly with small molecule ligands via a variety of forces(hydrogen bonding,ionic bonding or hydrophobic interaction,etc.).Nano-assembly also realizes both the passive targeting(EPR effect)and active targeting(receptor binding biological macromolecules).To this end,we chose hemoglobin,transferrin and red cell membrane as biological macromolecules,and took either "acid induced self-assembly" or "one-step synthesis"technology to prepare hemoglobin nanoparticles(Hb-CCM NPs,PEG-Hb-PTX NPs),transferrin nanoparticles(Tf-PTX-NPs,Tf-IR780 NPs),and erythrocyte membrane nanoparticles(IR780@RBCM NPs).We also investigated their formation mechanisms and drug efficacy,as follows.1)Hemoglobin nanoparticles(Hb-CCM NPs)There are a lot of hemoglobins in the body,and hemoglobin probably targets CD 163 overexpressed at the tumor surface.To this end,this research used hemoglobin as a drug carrier and the "acid induced self-assembly" technology to prepare hemoglobin drug nanoparticles(Hb-CCM NPs),and investigated the formation mechanism of nanoparticles.Through the optimization of "acid self-assembly"technique,such as pH,and concentration,we obtained Hb-CCM NPs nanoparticles with particle size of 90nm and drug loading up to 20.9%.Our experiments showed that the"acid self-assembly" technique,can be used to prepared nanoparticles hemoglobin.2)PEG-hemoglobin nanoparticles(PEG-Hb-PTX NPs)Hemoglobin nanoparticles prepared by "acid induced self-assembly" technology could encapsulate more than 20%of drug,however,the stability was poor.Therefore,PEG conjugated hemoglobin was used to increase the stability of nanoparticles.PEG-Hb-PTX nanoparticles is 182 nm diameter and more than 10%drug loading.The stability of PEG-Hb-PTX NPs was significantly enhanced.Cell experiments showed that the cellular uptake of PEG-Hb-PTX NPs increased,as well as the anti-tumor activity,circulation time in vivo and tumor accumulation.3)Transferrin-paclitaxel nanoparticles(Tf-PTX-NPs)It was reported that transferrin had better tumor targeting properties compared to hemoglobin.Compared to the CD 163 receptor,transferrin receptors have wider distribution in the body,for example,the capillary endothelium of the blood-brain barrier system expresses a large number of transferrin receptors.Most tumor cell lines have high expression of transferrin receptors at the surface.Therefore,transferrin as a drug carrier have better application prospect.Currently nanoparticles using transferrin as a carrier mainly self-assembled through covalent synthesis.The paper used "one-step synthesis" to prepare transferrin-paclitaxel-nanoparticles.The characterization experiments of nanoparticles indicated their uniform particle size,high drug loading and stability in the serum.Paclitaxel inside the nanoparticles was in the amorphous state,in favor of the dissolution of the drug.During the formation of nanoparticles,the secondary structure of transferrin remained unchanged,indicating that the preparation conditions were relatively mild.The synchronous changes of fluorescence in the tryptophan and tyrosine position of transferrin proved that paclitaxel may bind to the site of the two amino acids.The particle size of nanoparticles kept changing with the amount of paclitaxel.This study can be used to control the particle size.Cell experiment demonstrated that transferrin nanoparticles entered the cell by transferrin receptor-mediated pathway.In vivo pharmacodynamics experiments also showed that compared with commercially available paclitaxel injection,nanoparticles achieved better drug efficacy of paclitaxel.4)Transferrin-IR780 nanoparticles(Tf-IR780 NPs)At present,tumor treatment includes surgery,radiation therapy,chemotherapy,heat treatment and photodynamic therapy.None of them are perfect.Compared to chemotherapy,near-infrared photothermal and photodynamic therapy have stronger effect.This part used the "one-step synthesis" technology to prepare transferrin-IR780 nanoparticles.IR780 was a near-infrared dye.Experiments demonstrated that transferrin increased the solubility of IR780.In vitro experiments also demonstrated its excellent photothermal and photodynamic effect.At the cellular level,this nanoparticle has tumor-targeting and better cellular uptake.Either photodynamic or photothermal effect achieved high cytotoxicity of a variety of tumor cells.In vivo near-infrared imaging experiments also proved its ability to target tumors.The pharmacokinetic data indicated that nanoparticles achieved a long half-time in vivo.Pharmacodynamic results demonstrated its strong anti-tumor effect.This section proveed that transferrin-based nanocarriers also has application prospects in the field of photothermal and photodynamic therapy.5)Erythrocyte membrane loading IR780 nanoparticles(IR780@RBC M NPs)Erythrocyte membrane as composite biological macromolecules have more advantages that a single macromolecule.For example,CD47 receptor expressed on the surface of erythrocyte can help immune escape and hence achieve long circulation time.In this part,the cell membrane and near-infrared dye IR780 were self-assembled into nanoparticles,wherein IR780 was adsorbed on the cell membrane to increase its stability.This nanoparticle has higher cellular uptake and good photodynamic and photothermal effect.It also achieved better in vivo tumor imaging and targeting thanfree IR780.In summary,this study uses hemoglobin,transferrin and erythrocyte membrane as carriers to prepare drug-loaded nanoparticles.A series of experiments showed the advantages of using biological macromolecules as carriers.By investigating the interactions of biological macromolecules and hydrophobic drugs,the formation mechanism of nanoparticles was studied.Tumor therapeutic effects of nanoparticles including chemical treatment,photothermal and photodynamic therapy had also been investigated.