Research On Multi-targeted Core-shell Nano-delivery System Based On Polylysin

Cancer has surpassed cardiovascular disease and become the leading cause of threat to human health in the world.The commonly used methods of cancer treatment in clinic include surgical intervention,chemotherapy and radiotherapy,among which chemotherapy is widely used because of its simple operation,but it is toxic and easy to cause drug resistance.make some new treatments such as photothermal therapy and immunotherapy become the focus of research.However,chemotherapy,photothermal therapy and immunotherapy are faced with limitations that are difficult to target and difficult to deliver,and these limitations can be broken through the construction of drug delivery system.The carrier material is the key to construct the nano-drug delivery system.At present,a variety of polymers are used in the field of delivery,among which polyethylene glycol peptides have been widely used because of their excellent biodegradability and biosafety.However,due to the multi-step chemical synthesis of amphiphilic peptides,there are differences between batches and hidden dangers in biological safety.Based on the previous research results,we found that single hydrophilic carriers can also load hydrophobic drugs through electrostatic interaction,so we consider simplifying the carrier,using hydrophilic peptide polylysine(PLL)as the carrier and Methotrexate(MTX)as the model drug to construct nanoparticles(NPs).Compared with amphiphilic carrier materials such as polyethylene glycol-polylysine(PEG-PLL),which are commonly used in gene delivery system,α-Polylysine(α-PLL),a monohydrophilic material without PEG modification,was selected as the object of study.Drug-loaded nanoparticles PEG-PLL/MTX NPs and αPLL/MTX NPs were prepared by medium grinding combined with high pressure homogenization method.The particle size,morphology,drug release behavior and antitumor effect of the two kinds of drug-loaded nanoparticles were systematically compared.it was found that the average particle size of α-PLL/MTX NPs was 163.90 ± 3.01 nm,the polydispersity index(PDI)was 0.19 ± 0.01,and the zeta potential was 41.90 ± 2.77 mV.The average particle size of PEG-PLL/MTX NPs was 204.40± 2.71 nm,the PDI was 0.20±0.02 and the zeta potential was 37.70±1.31 mV.α-PLL/MTX NPs were agglomerated and PEG-PLL/MTX NPs were crystalline.Both the two nanoparticles remained stable within 14 days,both could be stable in 5%glucose and plasma,and both had sustained release function,and the release behaviors were in accordance with Higuchi equation.The anti-tumor effects of the two nanoparticles were also similar in vivo and in vitro,indicating that α-PLL can be used as a simplified carrier of PEG-PLL.After confirming that α-PLL can be used as a simplified carrier for drug loading,in order to improve the efficacy of the nano-drug delivery system,it is necessary to further investigate the effect of carrier secondary structure on the efficacy of the drug delivery system.PLL has two secondary structures,α-PLL and ε-Polylysine(ε-PLL),according to the difference of peptide bonding position.According to the literature,the secondary structure of peptides can affect the efficacy of drug-loaded nanoparticles,so α-PLL and εPLL are used as carriers and MTX as model drug carriers to study the specific effects of different secondary structures on nanoparticles.After the two kinds of polylysine and MTX were constructed into nanoparticles,it was found that α-PLL/MTX NPs and ε-PLL/MTX NPs had similar stability and sustained release behavior,but there were great differences in morphology and antitumor effect in vivo and in vitro.The average particle size of αPLL/MTX NPs was 181.80 ± 2.74 nm,the PDI was 0.15 ±0.01,the zeta potential was 40.10 ± 3.95 mV;the average particle size of ε-PLL/MTX NPs was 98.17± 1.03 nm,the PDI was 0.14± 0.02,the zeta potential was 33.3±1.37 mV;ε-PLL/MTX NPs were smaller and more uniform than α-PLL/MTX NPs.α-PLL/MTX NPs is agglomerated spherical andε-PLL/MTX NPs is spiral rod.The anti-tumor effect of ε-PLL/MTX NPs in vivo and in vitro was 1.3-fold and 2.6-fold higher than that of α-PLL/MTX NPs,respectively.The possible mechanism of these phenomena was that the secondary structure affects the morphology of nanoparticles,and different morphology affects the transport or internalization rate of nanoparticles,resulting in different anti-tumor effects in vivo and in vitro.These results also showed that ε-PLL was a more ideal carrier material than α-PLL.We imagine to construct core-shell nanoparticles with double targeting across membrane and mitochondria,and at the same time,chemotherapeutic drugs and immunotherapeutic agents can be delivered to the tumor site.in response to the stimulation of the weakly acidic environment of the tumor site,the immune drugs are released to the immune cells of the tumor site,and the chemotherapeutic drugs are delivered to the tumor cells to realize the synergistic treatment of chemotherapy and immunity.Therefore,based on the determination that ε-PLL is an ideal carrier material,it is optimized and coupled with triphenylphosphine(TPP)which was the mitochondrial targeting material to form TPP-PLL to target tumor cells.On the other hand,the active tumor targeting material cRGDfk was selected as the targeting ligand and γ-polyglutamic acid(y-PGA)was used as the main chain to synthesize cRGDfk-polyglutamic acid(RGD-PGA).RGD-PGA and TPP-PLL have opposite charge,and core-shell nanoparticles can be constructed by layerby-layer encapsulation method.The final result showed that the average particle size of TPP-PLL/MTX NPs was 107.80 ± 1.84 nm,the PDI was 0.15 ± 0.02,the zeta potential was 26.10± 1.05 mV.The particle size of RGD-PGA/TPP-PLL/MTX NPs was 167.30± 1.99 nm,the PDI was 0.17 ± 0.01,and the zeta potential is-13.80 ± 0.53 mV.The potential has been reversed.It could be seen by electron microscope that there is no damage to the core,the morphology of the core does not change obviously after the shell material is loaded,and the halo layer appears around it,indicating that the core-shell structure has been constructed successfully.In this study,it is confirmed that ε-PLL which was a monohydrophilic peptide carrier,could be loaded with MTX which was hydrophobic chemotherapeutic drug to form rodshaped nanoparticles through electrostatic interaction,and after coupling TPP,core-shell nanoparticles can be prepared by electrostatic interaction with negatively charged RGDPGA by layer-by-layer coating method to achieve multiple targeting and cooperative delivery.