Fabrication Of Rapeseed Protein Based Nanoparticles And Investigations Of Their Biological Potency

In recent years,nanoparticles composed of various forms of nanomaterials have received great attention in the treatment of breast cancer due to their potential capability of inhibiting tumor growth and metastasis.Among various developed materials,proteins were a kind of unique functional properties and natural macromolecules with great potential applications in biology and materials.For example,the antitumor drug loaded albumin based nanoparticles is a commonly used protein-based nanoparticles for treating breast cancer and its metastatic cancer,but the expensive price of albumin based nanoparticles,the limited resources of albumin,and the poor controllability of albumin based nanoparticles severely limited their applications.Therefore,there is an urgent necessity to develop a novel protein-based nanoparticle.Rapeseed protein is a superior vegetable protein derived from rich and inexpensive rapeseed meal.It has excellent amino acid composition and contains a variety of active peptide sequences.Herein,in this thesis,one intends to use rapeseed protein as bioactive nanomaterial to construct protein-based nanoparticles and load antitumor drugs to treat breast cancer.This rationally designed rapeseed protein based nanoparticles may provide a more effective approach for breast cancer treatment.Firstly,the rapeseed protein was modified by high pressure,acylation and enzymatic hydrolysis.The rapeseed protein based microparticles were prepared by spray drying method,and the effects of different modifications on rapeseed protein and its microparticles were explored.Alkaline protease hydrolysis of rapeseed protein resulted in a large loss of stable secondary structure(?-helical and ?-sheet)and increasing of lots of random structures(rose by 8%)of rapeseed protein compared to the acylation,high pressure treated and unmodified rapeseed protein of spatial structure.The succinic anhydride acylation of rapeseed protein maintained the stable secondary structure of the protein,as evidenced by little change of the ?-helical structure and the unordered structure of the acylated rapeseed protein structure and the partial transformation of ?-turned structure into ?-sheet,resulting in acylated rapeseed protein having the most stable secondary structure in modified rapeseed protein.The nano-indentation experiment further demonstrated that a large number of stable secondary structures were beneficial for improving the mechanical properties of rapeseed protein microparticles.Compared with unmodified rapeseed protein microparticles(405 MPa),enzymatic modification of protein significantly reduced the Young's modulus of rapeseed protein microparticles(245 MPa),whereas high pressure and acylation modifications,especially acylation rendered the Young's modulus of the rapeseed protein microparticles reaching 450 MPa.After loading the rapeseed peptide,the surface of the microparticles of the enzymatically modified rapeseed protein was observed to be ruptured,and the acylation and high pressure modified rapeseed protein were not only microscopically intact,but also the particle size of microparticles were similar to that of the unmodified rapeseed protein based microparticles.Compared with unmodified rapeseed protein,the encapsulation rate of enzymatically modified rapeseed protein on active rapeseed peptide decreased by 7.9%-25.6%,and it was positively correlated with the degree of hydrolysis.The acylation and high pressure treatment significantly increased the encapsulated ability of rapeseed protein,and the encapsulation rate of rapeseed protein microparticles with 47% acylation degree and 400 MPa treatments was 99% and 94%,respectively,increased by 9.0% and 3.2%.In vitro simulated release revealed that the release rate of rapeseed peptide from the acylation treated rapeseed protein microparticles was significantly slower than that of the unmodified and other modified rapeseed protein microparticles as well.These properties may be related to the the hard polymer structure induced by modification.The above results indicate that acylation modification is more advantageous for the particles of rapeseed protein than other methods.Secondly,based on the acylated rapeseed protein,stable rapeseed protein-based nanogel was prepared by heat-induced gelation method.The rapeseed protein based nanogel efficiently loaded the hydrophobic curcumin and improved its antitumor activity.Through systematic investigations on the main factors affecting the formation of protein nanogel: pH,protein concentration,heating temperature,heating time,etc.,it was found that the optimized rapeseed protein based nanogel was spherical,a particle size of 170 nm and good dispersion with a typical core-shell structure of nanogels.Compared with unmodified rapeseed protein,acylation and heat treatment could change the secondary and tertiary structure of the rapeseed protein,improving the surface hydrophobicity of the protein,and reducing the content of free sulfhydryl groups in the protein.These changes resulted in the acylated rapeseed protein having amphiphilic properties and self-aggregation ability,forming a nanogel of hydrophobic core which are cross-linked by disulfide bonds and a shell composed of a hydrophilic anhydride group.Such structures enabled rapeseed protein based nanogel to withstand different pH and ionic strengths and good water redispersibility.In vitro antitumor experiments showed that compared with free curcumin,the curcumin loaded rapeseed protein based nanogel significantly enhanced the cytotoxicity of curcumin in cancer cells,such as the human breast cancer cell line MDA-MB-231 and MCF-7,and increased the bioavailability of curcumin.Thirdly,based on the acylated rapeseed protein,the rapeseed protein/chitosan polymer nanoparticle(APRI/CSNP)was prepared by a mild polyelectrolyte self-assembly method.The nanoparticle delivery system was better than the rapeseed protein based nanogel.It has a more efficient ability to treat breast cancer.Since cathepsin B(CathB)is overexpressed in many metastatic cancer cells,such as breast cancer MDA-MB-231 cells,we designed high efficiency nanoparticle delivery system based on CathB degradable acylated rapeseed protein and mild polyelectrolyte self-assembly method.Polyanionic acylated rapeseed protein and polycationic chitosan were self-assembled to form nanoparticles by electrostatic interaction under room temperature,physically embedding doxrubicin(DOX),finally forming rapeseed protein-based polymer nanoparticles(DOX-ARPI/CSNP).It was found that the optimized DOX-ARPI/CSNP is characterized of a spherical structure with a particle size of 188 nm,good stability,dual acidic pH and CathB response ability.DOX-ARPI/CSNP entered breast cancer cells through the clathrin pathway and then were transported to lysosomes.Due to its unique dual response properties,the DOX was rapidly released from DOX-ARPI/CSNP,meanwhile the protonation of the acylated rapeseed protein resulted in the increase of the nanoparticles volume induced by CathB digestion and acidic pH,leading to the lysosome to swell and rupture.As a result,DOX-ARPI/CSNP overcomes the sequestration of lysosomes and prompted the massive transfer of DOX to its nuclear site.When the lysosomal protease CathB hydrolyzed the acylated rapeseed protein,pro-apoptotic peptides(PAS,AGS,YT)were also produced,which could regulate the mitochondrial-related protein apoptosis pathway(p53,Bax,Bcl-2,pro-Caspase-3)and coordinately enhanced the cytotoxicity of DOX,ultimately leading to extremely significant cytotoxicity especially against CathB overexpressing breast cancer MDA-MB-231 cells.Notably,no significant enhancement of the antitumor effect of DOX-ARPI/CSNP was observed in CathB low expression breast cancer MCF-7 cells.These results indicated that the use of rapeseed protein as nanoparticle matrix could specifically enhance the cytotoxicity of antitumor drugs.Finally,orthotopic breast cancer tumor model was constructed in the right mammary gland of NOD SCID mice using breast cancer MDA-MB-231 cells,and then the antitumor effect of the DOX-ARPI/CSNP vector system was studied.The study found that DOX-ARPI/CSNP appeared at the tumor site 2 h post intravenous injection,and reach the maximum concentration at the tumor after 8 h injection.Compared with the saline group and the free DOX group,DOX-ARPI/CSNP significantly inhibited tumor growth and prolonged the survival cycle of mice.The blank ARPI/CSNP and DOX-ARPI/CSNP treatments did not cause any organ damage in mice compared to the obvious cardiac and liver damage of free DOX treatment,indicating that the rapeseed protein based nanoparticle had good biocompatibility and tumor targeting.Pathological section of breast tumor studies further confirmed the ability of DOX-ARPI/CSNP to significantly induce tumor cell necrosis.Immunohistochemical staining showed that there was a significant amount of activated caspase-3 cells in the blank ARPI/CSNP and DOX-ARPI/CSNP treated breast tumor tissues compared with the saline group,suggesting APRI based nanoparticles system can be hydrolyzed by CathB in tumor and cancer cells for enhanced apoptosis through mitochondrial pathway.