Poly-lysine Dry Powder Inhalations For The Effective Delivery And Accurate Treatment Of Pulmonary Infection With Pseudomonas Aeruginosa

The treatment of pulmonary infection with pseudomonas aeruginosa(PA)is of great concern by the adverse factors of high mortality,resisting treatment and expensive cost.The low efficacy of traditional therapy was caused by a rise in antibiotic resistant and failure delivery to the pulmonary lesion site via inappropriate drug delivery systems.To address these problems,developing a precise drug delivery system loaded with the new type of antimicrobial drug,which was equipped with unique antibacterial mechanism,has become the crux of the efficient treatment of pulmonary infection with PA.Poly-lysine(PLL),a new antimicrobial peptide with fine biocompatibility,offers a promising strategy against pulmonary infection with PA for its different antimicrobial mechanism from the traditional antibiotic that it is capable to kill bacteria via physical disruption of the bacterial cell membrane.Dry powder inhalations(DPIs)is the first choice for PLL to treat lung infections,which can directly deliver drug into the lung.The combination of PLL and DPIs provide promising approach to achieve efficient delivery and accurate treatment of pulmonary infection with PA.However,the hygroscopicity of PLL and the hygro-instability of DPIs pose giant challenges to the development of PLL DPIs.In the present study,PLL DPIs with the superiority of aerodynamic property and satisfactory anti-hygroscopicity were designed by introducing optimized carrier material with low hygroscopicity,high stability and good biocompatibility,for achieving the fix spot deposition in the lower respiratory tract and enhanced therapeutic effect of pulmonary infection with PA.Spray-drying,a versatile technique for particle processing in pharmaceutical industry,was employed to fabricate the PLL DPIs with different ration of PLL and carrier material.Further,the formation mechanism of the PLL DPIs was explored detailly,and its enhancement mechanism and anti-hygroscopicity mechanism were elucidated.The PLL DPIs were fabricated with PLL and leucine(LL)from the aqueous solutions by spray-drying.The ratio of PLL and LL was set as 100:0,90:10,85:15,80:20,75:25 and 70:30,and the corresponding formulation were termed as P0,P1,P2,P3,P4 and P5,respectively.The physicochemical characteristics of PLL DPIs were conducted with particle size,density,surface morphology,crystal form,surface area,and hygroscopicity.The particle sizes of the PLL DPIs were determined with laser diffraction,and their surface morphology and surface area were analyzed by SEM and Brunauer-Emmett-Teller.The dynamic vapor sorption,circular dichroism and X-ray diffraction were adopted to detect the hygroscopicity,secondary structure and crystal form of PLL DPIs,respectively.Three bacteria strains,including PA,Escherichia coli and Methicillin-Resistant Staphylococcus aureus,were used to test the minimum inhibitory concentration(MIC)and minimum bactericidal concentration(MBC)of the PLL DPIs,evaluating the in vitro antimicrobial activity of PLL DPIs.Moreover,the bacteria integrity,membrane permeabilization and protein leakage were analyzed to explore the antimicrobial mechanism of PLL DPIs.The in vitro aerosolization performance of PLL DPIs was determined by the next generation impactor,and the enhancement mechanism of pulmonary drug delivery efficacy was elaborated.Furthermore,fourier transform infrared spectrum(FT-IR),contact angle(CA),surface tension(ST)and solid state nuclear magnetic resonance(SS-NMR)were employed in order to unambiguously figure out the structure of PLL DPIs and expounded its formation mechanism.Afterwards,P0 and P2 were stored in 25? and 60%RH in 10 days.And the samples were analyzed with physicochemical property,powder property and in vitro aerosolization performance,intended to illuminate the anti-hygroscopicity of PLL DPIs.And the pharmacodynamics tests of P0 and P2 were conducted with Wister-rats to evaluate the in vivo antimicrobial activity.Moreover,the comprehensive safety evaluation of PLL DPIs including cytotoxicity,hemolysis,ciliotoxicity,pulmonary function measurement,histological assessment and BALF analysis were performed.In addition,the stress,accelerated and long-term stability tests of PLL DPIs were conducted to determine the storage condition.The d0.5 and bulk density of PLL DPIs increased firstly but then dropped with the increase of LL content.The optimal formulation of PLL DPIs(P2)exhibited the lowest d0.5 and bulk density.Besides,the moisture uptake of PLL DPIs was restrained by the increased content of LL.And the pronounced corrugated surface was observed in PLL DPIs with addition of LL,and there was no secondary structure change of PLL DPIs.Desirable broad-spectrum antimicrobial activity was witnessed in PLL DPIs,and the antimicrobial mechanism was related to the rupture of bacterial membrane and then outbreak leakage of protein by PLL DPIs.The physicochemical property and in vitro aerosolization performance of P2 did not alter significantly(p>0.05)after 10-day storage in 60%RH,yet a pronounced increment was witnessed in P0.A hypothesis about formation mechanism of PLL DPIs was supposed by the analysis of SEM,FT-IR,CA,SS-NMR and ST.The surface tension gradients and the migrated of solute produced the PLL DPIs with a LL enriched "shell" and a PLL concentrated "core"structure.Significantly higher FPF and FPD were witnessed in P2 than any other PLL DPIs(p<0.05),which were 3.26-fold and 3.32-fold against P0,respectively,clarifying the growing in vitro aerosolization performance of P2.The enhanced pulmonary drug delivery efficiency was further confirmed by the pharmacodynamics research since the P2 exhibited superior in vivo antibacterial effect than P0 in Wister rats with pulmonary PA infection.The cell viability higher than 95%and hemolysis lower than 5%verified the beneficial cytocompatibility and blood biocompatibility of PLL DPIs.Besides,the scarcely ciliotoxicity,unaffected lung function,inconspicuous inflammatory factor and negligible lung injury in histopathology confirmed the safely of PLL DPIs in pulmonary delivery system.Modified PLL DPIs were successfully established in this research with the purpose of improving the anti-hygroscopicity stability and enhancing the pulmonary deposition efficiency.The formation mechanism of PLL DPIs could be rationally clarified by the surface-enrichment theory of LL.Enhanced pulmonary drug delivery efficiency and anti-hygroscopicity were confirmed in the optimized formulation P2.It was considered that the PLL DPIs with a LL enriched "shell" and a PLL concentrated "core" structure could account for anti-hygroscopicity,while the satisfactory aerodynamics property contributed to aerosolization performance enhancement.As an effective and accurate delivery system to lung,PLL DPIs was expected to be a promising treatment for the pulmonary infection with PA.