Preparation Of Drug-loaded Nano-in-microparticles By Supercritical CO₂ Assisted Atomization With An Enhanced Mixer

Drug-loaded nanoparticles used in cancer treatment can effectively improve the physicochemical properties and stability of drugs,facilitate the tumor targeted delivery and cancer cell uptake of drugs.The nano-in-microparticles can be used as dry powder inhalers for lung cancer therapy,which achieves high efficiency pulmonary targeting,prolongs drug residence time in lung,improves lung mucus penetration and tumor permeability;thus,tumor inhibition effect is enhanced and side effects are severely reduced.The structures,morphologies,particle size,size distribution and solid state of drug-loaded nanoparticles and nano-in-microparticles are very important for drug efficacy and storage stability.Therefore,green and efficient preparation technology is particularly desirable for drug-loaded nanoparticles and nano-in-microparticles with excellent properties.Supercritical fluid assisted atomization with an enhanced mixer(SAA-HCM)has many advantages over traditional methods based on non-atomization process and atomization process,including no residual of organic solvents,mild operation conditions,one-step preparation of dry powder particles,wide operation window and flexible control of particle morphologies and size.However,the application of SAA-HCM is limited to the system of homogeneous solution of a solvent,and co-solvent and nanosuspension were rarely refererred to.There are few reports on the application of SAA-HCM technology in drug-loaded nanoparticles,especially in nanoparticles co-loaded with different solubility drugs and nano-in-microparticles suitable for inhalable lung cancer targeted therapy.In this work,SAA-HCM technique was attempted to prepare drug-loaded nanoparticles and nanoparticles co-loaded with two drugs of different solubility.Furthermore,SAA-HCM technology was applied to the preparation of nano-in-microparticles.The disintegration behavior of nano-in-microparticles and redispersibility of nanoparticles in simulated lung environment were studied.This work could provide a reference for preparing drug-loaded nanoparticles and nano-in-microparticles suitable for lung cancer targeted therapy using a green and sustainable technique.First,relation between cavitation number and velocity near the orifice at different recovery pressure and temperature was studied.The results showed that the possibility of hydrodynamic cavitation in liquid CO2,supercritical CO2 and water dissolved with CO2 increased with the increase of velocity of CO2 near the orifice.Doxorubicin hydrochloride(DOX)-loaded chitosan(CS)nanoparticles(DOX@CS)were successfully prepared from aqueous solution by SAA-HCM technique.The effects of the solution concentrations,CO2/solution mass flow ratios,mixer pressures,CS/DOX mass ratios and the molecular weights of CS on the morphologies and size distribution of DOX@CS nanoparticles were studied.The results showed that spherical nanoparticles with well dispersion could be prepared under operating conditions.When the solution concentration was in the range of 0.5～3 g/L,the average size of nanoparticles determined from SEM images was between 119.9±2.2 nm and 232.9±8.2 nm,and almost all nanoparticles were smaller than 300 nm.The percentage of nanoparticles prepared at the solution concentration of 0.5 g/L with diameter smaller than 250 nm was up to 95%,and the volume fraction was up to 55%.An obvious effect of CO2/solution mass flow ratios on the morphologies and size distribution of DOX@CS nanoparticles was also observed.When the concentration of solution was 0.5 g/L,the average particle diameter decreased from 229.7±7.4nm to 119.9±2.2 nm with the increase of CO2/solution mass flow ratio from 1.8 to 4.3.The effects of mixer pressure,CS/DOX mass ratio and CS molecular weight were relatively limited.DOX@CS nanoparticles prepared under the optimal conditions were dispersed in deionized water and the average diameter and polydispersity index of the nanoparticles was 184.8±7.6 nm and 0.21±0.02 respectively,which indicated that the nanoparticles could be stored in dry powders and redispersed to form nanosuspension when needed.In vitro cytotoxicity test showed that DOX activity remained integrated after SAA-HCM process.Based on the preparation of single drug-loaded nanoparticles,water-soluble drug DOX and lipophilic drug paclitaxel(PTX)co-loaded CS nanoparticles(DOX&PTX@CS)were one-step prepared by SAA-HCM technology with water/ethanol mixture as co-solvent.The effects of operation parameters on the morphologies and size distribution of DOX&PTX@CS were investigated.The emphasis was then focused on the effects of the volume ratio of water/ethanol and the concentration of the solution on its stability at the phase of mixing with SC-CO2.At the same time,the influences of the evaporation rate of water/ethanol on the formation of DOX&PTX@CS nanoparticles were also disscussed.When the water/ethanol volume ratio was 1:2 and 1:1,well defined spherical DOX&PTX@CS nanoparticles were successfully prepared.While at the volume ratio of water/ethanol of 2:1,the stability of solution decreased when mixing with SC-CO2,leading to precipitating of PTX prior to form rod like particles in DOX&PTX@CS nanoparticles.The solution concentration also had obvious influence on the stability.Well defined spherical DOX&PTX@CS nanoparticles could be fabricated at the solution concentrations of 1 g/L and 2 g/L.Homogeneous solution could not be obtained anymore after mixing of CS solution,DOX solution and PTX solution with the increase of concentration beyond 3 g/L.The limited solubility of PTX in this co-solvent was the main reason for the poor stability of the system at high concentration.Considering the phase of droplets drying,the DOX&PTX@CS nanoparticles with smooth surfaces were prepared at precipitator temperatures of 50℃,90℃ and 100℃.However,there were small bulges on the surfaces of the nanoparticles when the precipitator temperatures were 60℃,70℃ and 80℃,respectively.The results of in vitro cytotoxicity showed that DOX&PTX@CS had the synergistic inhibitory effect on A549 cancer cells.CS-based nano-in-microparticles were prepared by SAA-HCM technique with mannitol as the excipient.The structures of nano-in-microparticles were analyzed.The mechanisms of nano-in-microparticles formation,nanoparticles aggregation and redispersibility were revealed based on exploring the mass transfer behavior of nanoparticles and excipient in the phase of droplets drying.SEM images showed that CS nanoparticles were embedded in the mannitol which was continuously distributed in the nano-in-microparticles.While the TEM images revealed that the CS nanoparticles tended to distribute at the outer of nano-in-microparticles.Under the optimal conditions,the theoretical aerodynamic diameter of the nano-in-microparticles was between 1～2μm,and the angle of repose was less than 40°,which could meet the requirements of inhalable lung drug delivery.When the mass ratio of CS nanoparticles to mannitol was 10:90,the in vitro redispersed fraction of CS nanoparticles was 38.51±12.03%.The CS nanoparticles appeared slight deformation and size increase during atomization.In addition,the enriching of CS nanoparticles at the outer of nano-in-microparticles during droplet drying and the capillary force between nanoparticles made the CS nanoparticles partially aggregate.The preliminary study on the micronization process of nano-in-microparticles and the mechanism of nanoparticle aggregation and redispersion provided the guidance for the subsequent preparation of nano-in-microparticles with good redispersibility of nanoparticles.Furthermore,PTX nanorods were used to prepare drug-loaded nano-in-microparticles with the presence of different excipients.The effects of PTX/excipient mass ratios and specific excipients on the redispersibility of PTX nanoparticles were investigated.When the mass ratios of PTX/excipient were 1:0,1:2 and 1:4,a small amount of drug-loaded nano-in-microparticles were spherical,and most of them were seriously collapsed.When the mass ratios of PTX/excipient were 1:16 and 1:32,well defined spherical drug-loaded nano-in-microparticles were successfully prepared.The redispersibility of PTX nanoparticles was poor when mannitol,lactose and trehalose were used as excipients respectively.The redispersibility of nanoparticles was improved effectively when polyvinylpyrrolidone,polyvinyl alcohol,gum arabic and sodium alginate were used as co-excipients.The redispersibility fraction of PTX was 97.2±4.9%with the mass ratio of PTX/gum arabic/mannitol of 1:10:16.Thus,an efficient strategy was established to improve the redispersibility of nanoparticles,and it was proposed for the first time by adding the materials with the surface enrichment effect in nano-in-microparticles as the co-excipient.The disintegration behavior of drug-loaded nano-in-microparticles and redispersibility of PTX nanoparticles in simulated lung environment were studied.The results showed that the drug-loaded nano-in-microparticles rapidly disintegrated and dispersed back into PTX nanoparticles in 30 min under high humidity simulating the lung environment.PTX nanoparticles in the nano-in-microparticles with co-excipients had good redispersibility and relatively high penetration efficiency in the simulated lung mucus,phagocytic escape ability of macrophages and inhibitory effect of A549 cancer cells.In conclusion,SAA-HCM technology has been successfully extended to the preparation of drug-loaded nanoparticles and nano-in-microparticles suitable for inhalation pulmonary drug delivery,which indicated the prospect of SAA-HCM in the preparation of drug particles with elaborate structures and diverse functions for cancer targeted therapy.