Preparation And Biological Evaluation Of Antibody Targeted Metal-organic Framework Drug Delivery System In HER2-positive SK-BR-3 Cells

Objective The objective of this study is to devise a delivery system that can accurately transport therapeutic drugs to specific cells.Among various candidates,metal-organic framework(MOF)has shown great potential for application in drug delivery systems.In the biomedical field,a number of promising MOFs have been identified as drug carriers,and the desired therapeutic effect can be achieved with some adjustments in the experimental process.MOF materials exhibit high stability,ultra-high porosity,adjustable pore size,structural diversity,biodegradability,and facile modification of the material itself,all of which make them promising candidates for biomedical applications.Among these materials,Zeolitic Imidazolate Frameworks-8(ZIF-8)have emerged as excellent drug carriers owing to their outstanding stability under aqueous physiological conditions,slow drug release time,and enhanced cellular uptake.While a few studies have reported the targeting of MOFs to cells via small ligands such as folate or RGD peptides and hyaluronic acid,only a limited number of reports have explored the use of MOFs for antibody-targeted drug delivery.To this end,we have designed a multifunctional drug delivery system that involves loading ZIF-8 with adriamycin and coupling it to antibodies via a carboxymethyl-coated interface.This antibody-targeted drug delivery system aims to improve therapeutic efficacy,reduce therapeutic dose,mitigate toxic side effects,and enhance patient survival and quality of life.By precisely delivering therapeutic drugs to the targeted cells or tissues,the negative impact on normal tissues caused by the drug can be minimized,and the therapeutic effect of the drug on the targeted tissues can be enhanced.Methods Trastuzumab@ZIF-8@DOX nanoparticles were synthesized through a one-pot method,involving the in situ synthesis of ZIF-8 and DOX followed by coating the drugloaded particles with carboxymethyl dextran(CMD)and coupling trastuzumab to the coating surface via covalent reaction.The size,crystal structure,and drug loading of the nanoparticles(NPs)were determined using infrared(FTIR)and ultraviolet-visible(UVVis)spectroscopy,Raman spectra,and nanoparticle size testing.Furthermore,the release of DOX from the nanoparticles under varying p H environments was evaluated.The study employed two types of cells,namely,HER2-positive SK-BR-3 cells and HER2-negative Ha Ca T cells.The MTT colorimetric method was utilized to evaluate the performance of different nanoparticle formulations,including ZIF-8@DOX nanoparticles,a mixture of free trastuzumab and ZIF-8@DOX,and Trastuzumab@ZIF-8@DOX nanoparticles,on SK-BR-3 cells in vitro.This allowed for the superior performance of the comparative somatic-targeted drug delivery system to be assessed.Moreover,the selectivity of the antibody-targeted drug delivery system for different cells was evaluated through MTT colorimetric assay,which determined the in vitro killing activity of Trastuzumab@ZIF-8@DOX nanoparticles on both SK-BR-3 cells and Ha Ca T cells.Fluorescence microscopy and high intracellular imaging system were utilized to observe the uptake of nanoparticles by cells.Specifically,the uptake of nanoparticles was analyzed based on the number of bright spots in the cells and the intensity of fluorescence,since DOX has red fluorescenceResults 1.The results from scanning electron microscopy(SEM)and transmission electron microscopy(TEM)analysis revealed that ZIF-8@DOX and CMD-ZIF-8@DOX,as well as Trastuzumab@ZIF-8@DOX nanoparticles,exhibit regular and spherical structures.In contrast,ZIF-8 nanoparticles have an irregular,round-like shape.Moreover,Trastuzumab@ZIF-8@DOX nanoparticles showed good dispersion,although some aggregation was observed.The particle size measurements of ZIF-8@DOX,CMD-ZIF-8@DOX,and Trastuzumab@ZIF-8@DOX were found to be 101.68 nm,120.24 nm,and 131.22 nm,respectively.These findings indicate that the size of the nanoparticles increased with the incorporation of CMD and Trastuzumab.2.EDS spectrum: The EDS spectrum of the element analysis shows that the atomic weights of the elements contained in Trastuzumab@ZIF-8@DOX particles are 70.54% C,14.60% N,12.24% O,1.00% Na,0.14% S,and 1.49% Zn.3.Infrared spectrum: In the CMD-ZIF-8@DOX spectrum,the wide peak in the range of 2500-3300 cm-1 is caused by the stretching vibration of O-H,and the strong band at 1760-1690 cm-1 corresponds to the stretching vibration of C=O.The band at 950-910 cm-1 is caused by the bending vibration of O-H.In the Trastuzumab@ZIF-8@DOX spectrum,the bands at 1575 and 1412 cm-1 are assigned to NH bonds,and the peak at 1654 cm-1 represents the stretching vibration of the amide group’s C=O.4.Raman spectrum: The characteristic absorption peaks of Trastuzumab are also observed in the Raman spectrum of Trastuzumab@ZIF-8@DOX.The peaks at 1373 and 1610 cm-1 are characteristic of the C-N,C-H,and N-H bonds of tryptophan and phenylalanine amino acid residues.A weak but distinct Raman spectrum was detected in the low-frequency region of 400-700 cm-1 due to the vibration of the disulfide bond.5.UV absorption spectrum: The characteristic absorption peaks of DOX,Trastuzumab,and Trastuzumab@ZIF-8@DOX were detected by UV-Vis.DOX has two absorption peaks at 254 nm and 495 nm,while there is no significant DOX absorption band in Trastuzumab@ZIF-8@DOX.6.Results of DOX release from nanoparticles in different p H environments: The aim of this study was to investigate the p H-dependent release behavior of Trastuzumab@ZIF-8@DOX nanoparticles.The drug loading capacity of Trastuzumab@ZIF-8@DOX was determined to be 12.53%,indicating a high drugloading efficiency.The cumulative drug release profile showed that Trastuzumab@ZIF-8@DOX exhibited a significant p H-dependent sustained release behavior that increased with decreasing p H change.Specifically,the release of DOX was observed to be p Hdependent,with a gradual increase in release as p H decreased from 7.4 to 5.0.At p H 7.4,only 16.09% of DOX was released from the drug carriers,while the majority of the drug was encapsulated in ZIF-8 nanoparticles.This can be attributed to the stable backbone of ZIF-8 under neutral conditions,which inhibits drug release.However,as the p H decreased,the backbone of ZIF-8 became unstable,resulting in an accelerated collapse and subsequent release of DOX.At p H 6.0,the release of DOX increased significantly to 65.22%,and at p H 5.0,almost complete release of DOX was achieved(99.75%)7.Cell toxicity test results: The cytotoxicity of Trastuzumab@ZIF-8@DOX on SKBR-3 and Ha Ca T cells was assessed via MTT assay.Our findings indicate that ZIF-8 nanoparticles have a limited effect on the proliferation of SK-BR-3 cell line.Specifically,when SK-BR-3 cells were treated with different concentrations of ZIF-8 nanoparticles for 24 hours at concentrations below 50 μg/m L,there was minimal toxicity.However,we found that ZIF-8@DOX,free Trastuzumab + ZIF-8@DOX mixture,and Trastuzumab@ZIF-8@DOX exhibited varying degrees of toxicity on SK-BR-3 cells,depending on the concentration.The concentrations of all samples were determined based on DOX content.Our results suggest that the cytotoxicity of ZIF-8@DOX was lowest in the concentration range of 0.5-50 μg/m L.Interestingly,Trastuzumab@ZIF-8@DOX exhibited higher cytotoxicity than ZIF-8@DOX with free Trastuzumab and ZIF-8@DOX at the same DOX dose.Specifically,at a DOX dose of 5 μg/m L,the survival rates of Trastuzumab@ZIF-8@DOX and ZIF-8@DOX on SK-BR-3 cells were 47.75% and 94.71%,respectively.Notably,Trastuzumab@ZIF-8@DOX nanoparticles demonstrated higher cytotoxicity against SK-BR-3 cells than Ha Ca T cells across the range of nanoparticle concentrations studied.8.Cell uptake test results: Cellular uptake was evaluated by fluorescence microscopy with DAPI staining for nuclei localization.Co-culturing SK-BR-3 and Ha Ca T cells with ZIF-8@DOX nanoparticles did not induce any apoptotic phenomena such as chromatin condensation and nuclear sequestration.After 2 hours,DOX red fluorescence was observed in both cell types,and the number of emitted red dots increased with incubation time.Subsequently,the cells were co-incubated with the same concentration of ZIF-8@DOX and Trastuzumab@ZIF-8@DOX nanoparticles,and highresolution intracellular imaging was performed.The uptake of Trastuzumab@ZIF-8@DOX nanoparticles and ZIF-8@DOX particles by SK-BR-3 cells was distinguishable,with a higher number of red dots observed in SK-BR-3 cells co-cultured with Trastuzumab@ZIF-8@DOX nanoparticles.Ha Ca T cells co-cultured with ZIF-8@DOX nanoparticles exhibited a higher number of red dots than SK-BR-3 cells.Subsequently,the cytoskeleton was stained using Actin-Tracker Green-488 to delineate the cell range,and bright spots with red fluorescence were observed.The number of red highlights was higher in SK-BR-3 cells than in Ha Ca T cells under the same incubation conditions.Moreover,with increasing incubation time,the uptake of Trastuzumab@ZIF-8@DOX nanoparticles by SK-BR-3 cells was higher than that by Ha Ca T cells.Conclusions1.A novel multifunctional drug delivery system was successfully constructed for p H-responsive release and specific targeting of cells for imaging.2.The Trastuzumab@ZIF-8@DOX nanoparticles possess a regular orbicular structure,high drug loading capacity(up to 12.53% of DOX),low cytotoxicity,and high stability at p H-neutral conditions,thus effectively protecting DOX from loss before drug delivery to tumor cells.3.The drug delivery system demonstrated p H-responsive DOX release behavior,enabling controlled release of DOX to HER2 overexpressing SK-BR-3 cells,and achieved targeted drug delivery.4.Cellular uptake experiments confirmed that HER2 mediated endocytosis facilitated the internalization of Trastuzumab@ZIF-8@DOX nanoparticles and promoted drug accumulation in cancer cells.Specifically,the nanoparticles bound specifically to HER2 expressed on the surface of SK-BR-3 cells,allowing for effective and targeted drug delivery.