Development Of Nanoparticles Embedded Porous Microspheres Co-loaded With SiRNA And Doxorubicin By Compressed CO₂ Antisolvent Process

Multidrug resistance is the major chemotherapeutic burden in the lung cancer therapy.In the current situation,the shortcoming in the use of traditional methods to produce porous microspheres(PMs)for pulmonary drug delivery is evidenced.To address these issues,we attempt to employ the supercritical fluid technology to prepare the nanoparticles embedded PMs for co-delivery therapeutics.The chitosan nanoparticles(CS NPs)containing siRNA are prepared by complex coacervation technique,and further,these were encapsulated in the doxorubicin(DOX)-imbedded PMs by supercritical fluid anti-solvent process to protect the activity of siRNA.Firstly,the preparation parameters of porous microspheres were optimized,and the physical and chemical properties of siRNA and DOX co-loaded microspheres were also characterized.The biocompatibility of the microspheres was evaluated systematically by molecular,and animal models.The work is summarized as follows:Firstly,the chitosan nanoparticles loaded with siRNA were prepared by complex coacervation technique.The physical characterization evidenced smooth surfaced spherical particles with a particle size distribution between 50-200 nm.Other characterization techniques of chitosan nanoparticles include FTIR and TGA for confirming the chemical modification and degradation profiles,respectively.Further,the construction process of PMs and processing parameters of(supercritical fluid)SCF technology such as temperature,pressure,flow rate of emulsion,carbon dioxide flow rate and single factor investigation are optimized to yield revealed the better geometric size and aerodynamic diameter.The optimized conditions were as follows,temperature-30 °C,pressure-80 bar,emulsion flow rate-4 mL/min,CO2 flow rate-40 g/min.Blank PMs have good sphericity with an average particle size of 16.32 μm,aerodynamic diameter of 4.05 μm and an excellent aerodynamic performance(FPF-58.28%).Next,the biocompatibility of the microspheres was evaluated comprehensively using both cellular and animal models.The cytotoxicity degree in L929 cells was 0 and 1,demonstrating that the microspheres are highly biocompatible and biologically safe.In vitro experiments showed that the PM-induced cellular ROS levels are concentration dependent however they are significantly lower than the positive control and in a safe range.The hemolysis rate was lower than 5%,which are in agreement with the ROS measurements.We observed a certain degree of genetic toxicity in CHO-K1 cells and is concentration dependent.However,it is significantly lower than the positive control group.Acute systemic toxicity test showed that no significant effect of porous microspheres on mice.Further,the pathological analysis of liver tissue revealed that the microspheres had no significant effect on hepatocytes.The lungs of rats may result in a certain degree of damage due to PMs,in turn,releases intracellular substances such as LDH,ALP,and others,which resulted in slight immune responses.HE staining of lung tissue have shown a slight damage,but still in an acceptable range.In addition,Masson staining also showed that the microspheres had little effect on pulmonary fibrosis.Finally,the drug loading and other properties of the microspheres were investigated.The results showed that the particle size distribution was uniform,and the surface roughness was porous.The particle size(16.90 μm)and aerodynamic diameter(< 4.39 μm)of the microspheres with a significant FPF value(57.92%)is suitable for pulmonary drug delivery.The actual drug loading(3.7%,5.2%,6.8%)also showed an increasing trend,but the encapsulation efficiency(80%,78%,70%)had a certain reduction.In vitro,DOX release studies showed that the release rate was 75% for 60 h.Medicinal properties showed that the atomization effect was 0.16 s,and the atomization efficiency was 90.4±5.2%.The in vivo distribution of PMs showed that they were deposited in lungs and the action might execute within 24 h.Anticancer activity studies on H69 AR cells revealed that the effect of drug loaded microspheres is significantly higher compared to the groups treated with the pure drug and single drug group in 72 h.AO/EB staining also showed that the cells underwent apoptosis and anti-cancerous efficacy is quite effective to overcome the resistance in cancer.In summary,we have employed the supercritical fluid technology to prepare the DOX immobilized PMs enclosing siRNA carrying chitosan nanoparticles as a core-shell strategy.The designed combinatorial strategy of multifaceted nanocontainer is most promising and can emerge as an efficient delivery system for anti-MDR applications.