Construction And In Vitro Anti-tumor Activity Of Carrier Free Nanomedicines Derived From The Assembly Of Indomethacin And Paclitaxel

As a major disease with a high mortality rate,cancer has seriously threatened the health of all human beings.Chemotherapy,surgery,and radiation are common choices for most cancer treatments,however,there are still some inevitable defects in some aspects of these treatments.With the vigorous development of nanomedicine technology in recent years,it has provided new ideas for solving the above problems because of its specificity,targeting,easy absorption,sustained release,increased blood drug concentration,and reduced drug degradation.Despite this,the carrier-assisted nanomedicine has a low drug loading,which severely limits its therapeutic effect.At the same time,the side effects caused by the carrier may also hinder its application.Therefore,the development of a simple,"green"method for the preparation of unsupported nanomedicine without toxic solvents or nanocarriers has become an extremely urgent task in this field.In our previous study,we found a strong intermolecular force between the non-steroidal anti-inflammatory drug indomethacin?IDM?and the traditional chemotherapy drug paclitaxel?PTX?.Based on this,we successfully constructed an indomethacin/paclitaxel carrier-free nano drug delivery system by dialysis without using other inactive carrier materials.Through high-resolution laser confocal microscopy,high-resolution transmission electron microscopy,and computer simulation of its self-assembly process,we were surprised to find that these nanomedicines shared a unique structure similar to?brick-cement?and can be tuned by simply adjusting the proportion of drug ratio.Its micro-morphology allows it to be further loaded with inorganic nanoparticles for a wider range of uses.In addition,indomethacin as a non-steroidal anti-drug can regulate the macrophage phenotype in the tumor area,thereby changing the immune environment of the tumor area,and contributing to the action of chemotherapy drugs,thereby achieving the synergistic effect of immunotherapy and chemotherapy.Methods1.Construction of a carrier-free indomethacin/paclitaxel nano drug delivery systemIndomethacin and paclitaxel solids at different mass ratios were dissolved in 1 mL of dimethyl sulfoxide?DMSO?,and the resulting solution was placed in a dialysis bag?intercepting molecular weight of 1.5 kDa?.Then,the dialysis bag was putted into 2 L of water for dialysis at 25°C,and ultrapure water was replaced every 2 hours.After 24 hours of dialysis,the samples in the dialysis bag were taken out for use.2.Measurement and calculation of drug loading and encapsulation efficiency of carrier-free indomethacin/paclitaxel nano drug delivery system100?L of the dialysis dispersion was taken,and the solvent was removed by a nitrogen gas blower,and the total mass??m?of the nano drug was calculated by a differential method.The sample was dissolved in 100?L of acetonitrile?HPLC grade?,and the contentof indomethacin and paclitaxel in the nano drug was measured by a high-performance liquid chromatography analyzer,and the masses of indomethacin and paclitaxel were m _IDM and m_PTX,respectively.The chromatographic conditions were:C8 reverse phase column,4.6×150 mm;mobile phase A:0.02%aqueous glacial acetic acid,45%;mobile phase B:acetonitrile,55%;flow rate:1 mL/min;detection wavelength:227 nm.3.The determination of diameter distribution and surface potential of nanoparticlesA 1 m L sample was taken and measured by a Zetasizer Nano?Nano ZS?with a temperature set at 25°C,a measurement angle of 90°,and a detection wavelength of 633nm.4.Observation of microscopic morphology by transmission electron microscopeA small amount of sample suspension was taken and added onto the copper mesh membrane.After drying at room temperature,the morphology was observed by transmission electron microscopy?TEM?.The acceleration voltage was 80 kV.5.Observation of crystal structure by high resolution transmission electron microscopeA small amount of sample suspension was added onto the copper mesh membrane and allowed to dry at room temperature.The crystal structure of the sample was observed by high-resolution transmission electron microscopy?HR-TEM?with an accelerating voltage of 200 kV,the dot resolution is 0.24 nm.6.Microscopic morphology of nanoparticles observed by scanning electron microscope?SEM?A small amount of the sample suspension was taken and applied evenly onto the mica plate.The sample was dry out at room temperature and sprayed with gold.The microscopic morphology was observed with scanning electron microscopy?SEM?.The accelerating voltage is 15 kV.7.The fluorescence properties of nanoparticles were studied by laser confocal microscopy.A small amount of sample suspension was dropped onto glass slide.After standing for5 min,select the corresponding excitation and emission wavelength according to the fluorescence properties of the sample,and observe the fluorescence properties by Confocal laser scanning microscopy?CLSM?.8.Differential Scanning Calorimeter?DSC?thermograms of nanoparticlesThe appropriate amount of indomethacin,paclitaxel,mixture of indomethacin and paclitaxel,nanoparticles at different feed ratios were accurately weighed.After that,all of samples were placed in the sample tray and the thermograms were determined by DSC under nitrogen protection?20?to 250?,10?/min?.9.The X-ray diffraction?XRD?patternsThe X-ray diffraction?XRD?patterns were collected on a PANalytical X'Pert Powder instrument under the following conditions:scanned from 3 to 60°?2?value?at a scanningspeed of 5°/min.X-ray scattering measurements were performed using a SAXS MC2 high flux small-angle X-ray scattering instrument?Anton Paar,Austria,Cu K?,?=0.154 nm?,equipped with a Kratky slits system in transmission mode.10.The determination of intermolecular forces by Ultraviolet spectrophotometer,fluorescence spectrometer,infrared spectrometerThe appropriate amount of indomethacin,paclitaxel,and non-feeding ratio nanoparticle samples were placed in an ultraviolet spectrophotometer,a fluorescence spectrometer,or infrared spectrometer to measure spectral information,respectively.Observation conditions:UV spectrophotometer,200 nm?800 nm;fluorescence spectrometer,3D scanning mode,250 nm?750 nm;infrared spectrometer,400?4000 cm^-1.11.The study of in vitro release profiles of nanoparticlesThe in vitro release experiments were carried out via dialysis.1 mL of IDM/PTX assembly suspension was put into a dialysis membrane?MWCO 1000 Da?.A mixture suspension of PTX and IDM was taken as control.The dialysis membranes were immersed into vials containing 25 mL of PBS in a shaking bed at 37°C.Tween 80?0.1%v/v?was added into the release medium to improve the solubility of PTX and IDM in PBS solution?pH 7.4,0.01 M?.The amount of drug released was detected by HPLC.12.Computational Simulations.The multiscale simulation methodology adopted here synergizes both molecular dock and DPD techniques.Interaction energy was theoretically assessed by molecular modeling using the AutoDock software package?AutoDock 4.2,the Scripps Research Institute?.To further validate this point,DPD was used to simulate the formation of IDM/PTX assemblies.PTX,IDM,and water molecules were firstly coarse-grained to six,three,and one bead,respectively.With a fully atomistic detail,the Blends module incorporated in the Materials Studio 2017 software?Accelrys Inc.,kindly provided by Analytical&Testing Center ofSichuan University?was employed to calculate the Flory-Huggins interaction parametersbetween binary components.On this basis,DPD simulations were applied at a coarse-grained level to investigate the assembly process of IDM/PTX assemblies with various morphologies.Because the morphologies and aggregate sizes are almost the same in the boxes of 100×100×100r_c³ and200×200×200 r_c³,a box of 100×100×100 r_c³ was employed to simulate the morphology of IDM/PTX assemblies and a box of 200×200×200 r_c³ was employed to simulate the dynamic formation process of IDM/PTX assemblies.13.Intracellular Uptake.For the cellular uptake study,MDA-MB-231 cells?human mammary cancer cells?were seeded in a 24 well plate at a density of 1.0×10⁵ cells per well in 500?L of growthmedium.IDM/PTX assemblies or raw PTX at 8.5?g/m L was added to the dishes.The cellswere then incubated for 2,4,and 8 h.Before observation,the cells were washed three times with PBS and then lysed.The drug content in cells was measured by HPLC,while the content of total proteins was quantified by the BCA method.The content of cellular drug was normalized to the protein content.Results1.Autodock results showed that there was a strong intermolecular force between IDMand PTX,mainly composed of?-?stacking,hydrogen bonding and hydrophobicinteraction.2.Through the co-dialysis of IDM and PTX?the toss ratio of IDM/PTX:2/1,dialysis concentration of PTX:5 mg/m L?,nanospheres with an average diameter of 461.6±57.72nm could be successfully prepared.3.The morphology of IDM/PTX assemblies could be effectively controlled by the ratio of dialysis concentration and the toss ratio of IDM/PTX,so as to obtain linear,reticular,honeycomb,spherical,vesicle and membrane structures with different morphology.4.The results of transmission electron microscopy and laser confocal microscopy showed that the vesicular IDM/PTX assemblies?the toss ratio of IDM/PTX:2/1,dialysis concentration of PTX:10 mg/m L?could further load quantum dots.5.The results of differential scanning calorimetry,X-ray diffraction,high-resolution transmission electron microscopy and small angle X-ray scattering,high-resolution projection electron microscopy and fluorescence spectrum scanning showed that IDM and PTX exist in the form of amorphous and nanocrystalline respectively in IDM/PTX assemblies.6.The spectral properties of IDM/PTX assemblies were measured by UV and FTIR,which suggest that the?-?stacking between paclitaxel and indomethacin was the main intermolecular force in IDM/PTX assemblies.7.The assembly process of IDM/PTX assemblies was simulated by dissipative particle dynamics simulation,which showed that in the early stage of the assembly of indomethacin and paclitaxel,paclitaxel crystallized first because of its strong hydrophobicity,while indomethacin was still in the dissolution state.With the further decrease of DMSO ratio during dialysis,indomethacin molecules gradually adsorbed to the surface of the crystal nucleus of paclitaxel,which prevented the growth of paclitaxel crystal,and gradually grew to a variety of nanostructures.8.Indomethacin in IDM/PTX nanocomposites was rapidly released due to its amorphous morphology,while the release rate of paclitaxel molecule was faster than that of paclitaxel crystal powder,but the release was still relatively slow.9.IDM/PTX assemblies could break down rapidly in serum and release nanoparticles with a particle size of about 100 nm.10.The uptake efficiency of MDA-MB-231 cells to paclitaxel in IDM/PTX assemblies was much higher than that of the single drug group.11.Compared with paclitaxel alone,IDM/PTX assemblies showed a stronger anti proliferation effect on MDA-MB-231 and MCF-7 tumor cell lines in vitro.12.Indomethacin can effectively inhibit the up-regulation of cd206 expression of M1macrophages caused by tumor cells.In the co culture system of MDA-MB-231 cells and M1 macrophages from THP-1 cells,the anti-proliferation effect of IDM/PTX assemblies was significantly higher than that of indomethacin or paclitaxel alone.ConclusionWe firstly demonstrated herein the formation of nanocrystal aggregates of PTX with diverse morphologies over multiple length scales by a one-pot strategy because of the special intermolecular interactions between IDM and PTX.In addition,the molecular mechanism driving the formation of IDM/PTX assemblies was proposed.With tunable morphology,perfect drug loading,great clinical transform prospect,and serving as nanovehicles for other nanoparticles,the IDM/PTX assemblies showed great potential to be a brand-new kind of nanotherapeutic.After IDM/PTX assemblies were administered,the assemblies rapidly collapsed into smaller nano-particles and brought PTX nanocrystals retained in them to the tumor tissue effectively due to the EPR effect.Coupled with the immunomodulatory effect of IDM,the IDM/PTX assemblies showed a potential improved in vitro antitumor activity.Of great importance is that IDM/PTX assemblies obtained without any nonactive excipients are far safer in terms of safety and more conducive to clinical transformation than other materials-based nanotherapeutics despite plenty of challenges in the aspects of functionalization,theranostics,and target delivery.In addition,the construction of nanocrystal aggregates of drugs induced by other active agents also provides a new idea for the design of carrier-free nano-medicines.