Construction And In Vitro Evaluation Of Enzyme Nanoreactors Based On Carboxymethyl Chitosan For Arginine Deprivation In Cancer Therapy

Tumor metabolism as an approach to treat certain malignancies recently triggered increased interest.Deprivation by ADI of arginine which is nonessential for normal human cells but is essential for rapidly proliferating tumor cells may be a selective approach for targeting malignancies.In enzyme replacement therapy,polymeric nanoreactors composed of vesicles that encapsulate an enzyme or a combination of enzymes in their cavities represent a novel approach in therapeutic applications.In this study,a pH-induced morphological transition from micelles to vesicles for an amphiphilic photo cross-linkable biocopolymer(Az-NaCMCS),synthesized by mixing azidobenzaldehyde(Az)and an aqueous solution of carboxymethyl chitosan sodium salt(NaCMCS),was used for encapsulation of l-arginine deiminase(ADI).The aqueous solution of enzyme-loaded vesicles obtained was followed by UV-irradiation to result in shell cross-linked enzyme nanoreactors.The nanoreactors exhibit the potential for arginine deprivation in cancer therapy.The main research work and results are as follows:I.An amphiphilic photo cross-linkable biocopolymer(Az-NaCMCS)were prepared,and its related properties were studied.We prepare micellar solution by self-assembly in aqueous medium of an amphiphilic biopolymer(Az-NaCMCS).Micelles shade into nanovesicles by a pH-induced morphological transition.Then the aqueous solution of evesicles obtained was followed by UV-irradiation to result in shell cross-linked nanoreactors.Its structure was determined,and its related properties were studied.TEM results indicated that micelles and nanovesicles both had obvious core-shell structure,neat and clear morphology,equal distribution,and uniform particle size.Compared with the micelles,the nanovesicles had a tighter shell structurethe,and photo cross-linking could lead to a more tightly packed shell.II.By adding a water-soluble enzyme into aqueous solution before self-assembly,enzyme-encapsulated nanovesicles would be obtained.The aqueous solution of enzyme-loaded vesicles obtained was followed by UV-irradiation to result in shell cross-linked enzyme nanoreactors.The structure of the nanoreactors was determined.Its related properties were studied.(1)The morphological structure of nanoreactors were characterized by TEM.After photo cross-linking,the enzyme-encapsulated sample showed a compact core-shell structure with smaller mean diameter;(2)The particle diameter and Zeta potential of nanoreactors were characterized by DLS.The particle diameter of the samples decreased significantly when the DS of Az increased.There were no significant effects of DS of Az on the Zeta potential.The nanovesicle diameter before shell cross-linking is significantly larger than that after shell cross-linking,which is consistent with that obtained by TEM observation,demonstrating that the photo cross-linking could lead to a more tightly packed shell.There were no significant effects of cross-linking on the Zeta potential;(3)Analysis of encapsulation efficiency,loading capacity and leakage rate showed that the increase in DS of Az resulted in an increase in the encapsulation efficiency and loading capacity for the samples;the shell cross-linking can avoid leakage of the free ADI molecules in their aqueous core through the shells toward the external aqueous environment,thus enhancing encapsulation efficiency and loading capacity.III.The encapsulated enzyme activity,the stability in blood plasma and in vitro inhibitory activity against tumor cells were investigated.The main conclusions were as follows:(1)Assay of enzyme activity of the ADI-loaded nanoreactors indicate that the encapsulation process has no adverse effects on the enzyme activity.The activity of the encapsulated enzymes is nearly identical to that of native ADI.And the cross-linked shell of vesicle is not a barrier for the influx of L-arginine and the outflow of L-citrulline;(2)Semi-permeability of the shell of nanovesicle:The encapsulated show almost the same activities as native ADI.The results indicate that the cross-linked shell has good semi-permeability;(3)Stability of the ADI-loaded nanoreactors in plasma: The result indicated that the stability of ADI-encapsulated sample in plasma could be controlled by the shell cross-linking;(4)Assay of in vitro growth-inhibitory activity: To evaluate the effectiveness of ADI-loaded nanovesicles as an anti-cancer agent for tumor therapy,the in vitro growth-inhibitory activity of native enzyme and encapsulated enzyme against mouse heptoma cells(MH134)was investigated.A strong cell inhibition was observed when the MH134 cells were incubated with native and encapsulated ADI,and the growth inhibitory activity of the encapsulated enzyme was very similar to that of the native enzyme under the same enzyme concentration.The nanoreactors exhibit the potential for arginine deprivation in cancer therapy.