Preparation, Statistical Optimization and In-vitro Characterization of a Dry Powder Inhaler (DPI) Containing Solid Lipid Nanoparticles Encapsulating Amphotericin B: Ion Paired Complexes with Distearoyl Phosphatidylglycerol

Document Type : Research article


Department of Pharmaceutics, School of Pharmacy, Hamadan University of Medical Sciences, Hamadan, Iran.


The aim of this study was to prepare dry powder inhalers (DPIs) containing amphotericin B-loaded solid lipid nanoparticles (AMB-SLNs) as an alternative approach for prevention of pulmonary aspergillosis. For solubilizing AMB in small amounts of organic solvents ion paired complexes were firstly formed by establishing electrostatic interaction between AMB and distearoyl phosphatidylglycerol (DSPG). The SLN formulations containing AMB-DSPG complexes were prepared using glycerol monostearate (GMS) as the lipid matrix and soybean lecithin and tween 80 as the surfactants by solvent emulsification-evaporation technique. The nanoparticles were optimized through a fractional factorial design. DPIs were prepared by lyophilization technique using lactose as the inhalational carrier and then after, the formulations were evaluated in terms of aerodynamic particle size distribution using an Andersen cascade impactor. The morphology of the particles was examined using scanning electron microscopy (SEM) and in-vitro drug release profiles were evaluated. Following the statistical results, the particle size, Poly dispersity index (PdI), zeta potential, entrapment efficiency (EE%), and drug loading (DL%) of the optimized SLNs were 187.04 ± 11.97 nm, 0.188 ± 0.028, -30.16 ± 1.6 mV, 89.3 ± 3.47 % and 2.76 ± 0.32 %, respectively. Formulation containing 10% w/v of lactose with the calculated fine particle fraction value as 72.57 ± 4.33% exhibited the appropriate aerodynamic characteristics for pulmonary drug delivery. SEM images revealed de-agglomerated particles. In-vitro release studies showed sustained release of AMB from the carriers and the release kinetics were best fitted to the first order kinetic model.

Graphical Abstract

Preparation, Statistical Optimization and In-vitro Characterization of a Dry Powder Inhaler (DPI) Containing Solid Lipid Nanoparticles Encapsulating Amphotericin B: Ion Paired Complexes with Distearoyl Phosphatidylglycerol


 (1) Zhou QT, Leung SS, Tang P, ParumasivamT, Loh ZH and Chan HK. Inhaledformulations and pulmonary drug deliverysystems for respiratory infections. Adv.
Drug. Deliv. Rev. (2015) 85: 83-99.
 (2) Bulpa P, Dive A and Sibille Y. Invasivepulmonary aspergillosis in patients withchronic obstructive pulmonary disease. 60Mehrabani Yaganeh E et al. / IJPR (2020), 19 (3): 45-62Eur. Respir. J. (2007) 30: 782-800.
(3) Salama A, Burger M and Mueller EckhardtC. Acute immune hemolysis induced by adegradation product of Amphotericin B.Blut. (1989) 58: 59-61.
(4) Wang LH, Smith PC and Andersen KL.High- performance liquid chromatographicanalysis of amphotericin B in plasma, bloodurine and tissues for pharmacokinetic andtissue distribution studies. J. Chromatogr.(1992) 579: 259-68.
 (5) Fanos V and Cataldi L. AmphotericinB-induced nephrotoxicity: A review. J.Chemother. (2000) 12: 463-70.
(6) Scheuch G, Kohlhaeufl MJ, Brand P andSiekmeier R. Clinical perspectives onpulmonary systemic and macromoleculardelivery. Adv. Drug Deliv. Rev. (2006) 58:996-1008.
(7) Traini D and Young PM. Delivery ofantibiotics to the respiratory tract: anupdate. Expert. Opin. Drug Deliv. (2009)6: 897-905.
(8) Telko MJ and Hickey AJ. Dry powderinhaler formulation. Respir. Care (2005)50: 1209-27.
(9) Walsh TJ, Anaissie EJ, Denning DW,Herbrecht R, Kontoyiannis DP, Marr KA,Morrison VA, Segal BH, Steinbach WJ,Stevens DA and van Burik JA. Treatment ofaspergillosis: clinical practice guidelines ofthe infectious diseases society of America.Clin. Infect. Dis. (2008) 46: 327-60.
(10) Garbati MA, Alasmari FA, Al-Tannir MAand Tleyjeh IM. The role of combinationantifungal therapy in the treatment ofinvasive aspergillosis: a systematic review.Int. J. Infect. Dis. (2012)16: 76-81.
(11) Espuelas MS, Legrand P, Irache JM,Gamazo C, Orecchioni AM, DevissaguetJP and Ygartua P. Poly (ε-caprolacton)nanospheres as an alternative way to reduce
amphotericin B toxicity. Int. J. Pharm.(1997) 158: 19-27.
(12) Saravolatz LD, Ostrosky Zeichner L, MarrKA, Rex JH and Cohen SH. AmphotericinB: time for a new “gold standard”. Clin.Infect. Dis. (2003) 37: 415-25.
(13) Ekambaram P, Sathali AA and PriyankaK. Solid lipid nanoparticles: a review. Sci.Rev. Chem. Commun. (2012) 2: 80–102.
(14) Naseri N, Valizadeh H and ZakeriMilani P. Solid lipid nanoparticles andnanostructured lipid carriers: structure,preparation and application. Adv. Pharm.Bull. (2015) 5: 305-13.
(15) Xia D, Sun WK, Tan MM, Zhang M, DingY, Liu ZC, Su X and Shi Y. Aerosolizedamphotericin B as prophylaxis for invasivepulmonary aspergillosis: a meta-analysis.Int. J. Infect. Dis. (2015) 30: 78-84.
(16) Sternal K, Czub J and Baginski M.Molecular aspects of the interactionbetween amphotericin B and a phospholipidbilayer: molecular dynamics studies. J.Mol. Model. (2004) 10: 223-32.
(17) Lemke A, Kiderlen AF and KayserO. Amphotericin b. Appl. Microbiol.Biotechnol. (2005) 68: 151-62.
(18) Rahman Z, Zidan AS and Khan MA. Nondestructive methods of characterization ofrisperidone solid lipid nanoparticles. Eur.J. Pharm. Biopharm. (2010) 76: 127-37.
(19) Patel S, Chavhan S, Soni H, Babbar AK,Mathur R, Mishra AK and Sawant K. Braintargeting of risperidone-loaded solid lipidnanoparticles by intranasal route. J. Drug.Target. (2011) 19: 468-74.
(20) Espada R, Josa JM, Valdespina S, DeaMA, Ballesteros MP, Alunda JM andTorrado JJ. HPLC assay for determinationof amphotericin B in biological samples.Biomed. Chromatogr. (2008) 22: 402-7.
(21) Barman RK, Iwao Y, Funakoshi Y,Ranneh AH, Noguchi S, Wahed MI and ItaiS. Development of highly stable nifedipinesolid–lipid nanoparticles. Chem. Pharm.Bull. (2014) 62: 399-406.
(22) Shahsavari Sh, Bagheri G, Mahjub R,Bagheri R, Radmehr M, Rafiee Tehrani Mand Dorkoosh FA. Application of artificialneural networks for optimization of insulinnanoparticles composed of quaternizedaromatic derivatives of chitosan. Drug Res.(2014) 64: 151-8.
(23) Joshi MR and Misra A. Liposomalbudesonide for dry powder inhaler:preparation and stabilization. AAPS Pharm.Sci. Tech. (2001) 2: 44-53.
(24) Bai S, Gupta V and Ahsan F. Inhalablelactose-based dry powder formulations oflow molecular weight heparin. J. Aerosol.Med. Pulm. Drug Deliv. (2010) 23: 97-104.
(25) Van de Ven H, Paulussen C, Feijens PB, 61Dry Powder Inhaler Containing Solid Lipid Nanoparticles Encapsulating Amphotericin BMatheeussen A, Rombaut P, Kayaert P, Vande MooterG, Weyenberg W, Cos P, MaesL and Ludwing A. PLGA nanoparticlesand nanosuspensions with amphotericinB: Potent in-vitro and in-vivo alternativesto Fungizone and AmBisome. J. Control.Release. (2012) 161: 795-803.
(26) Zu Y, Sun W, Zhao X, Wang W, Li Y,Ge Y, Liu Y and Wang K. Preparationand characterization of amorphousamphotericin B nanoparticles for oraladministration through liquid antisolventprecipitation. Eur. J. Pharm.Sci. (2014) 53:109-17.
(27) Shah R, Eldridge D, Palombo E andHarding I. Optimisation and stabilityassessment of solid lipid nanoparticlesusing particle size and zeta potential. J.Phys. Sci. (2014) 25: 59-75.
(28) Shah M and Pathak K. Developmentand statistical optimization of solid lipidnanoparticles of simvastatin by using 23full-factorial design. AAPS PharmSci.Tech. (2010) 11: 489-96.
(29) Mehnert W and Mäder K. Solid lipidnanoparticles: production, characterizationand applications. Adv. Drug Deliv. Rev.(2001) 47: 165-96.
(30) Trotta M, Debernardi F and Caputo O.Preparation of solid lipid nanoparticles by asolvent emulsification–diffusion technique.Int. J. Pharm. (2003) 257: 153-60.
(31) Turk CT, Oz UC, Serim TM and HascicekC. Formulation and optimization ofnonionic surfactants emulsified nimesulideloaded PLGA-based nanoparticles bydesign of experiments. AAPSPharm. Sci.Tech. (2014) 15: 161-76.
(32) Tiyaboonchai W, Tungpradit W andPlianbangchang P. Formulation andcharacterization of curcuminoids loadedsolid lipid nanoparticles. Int. J. Pharm.(2007) 337: 299-306.
(33) Liu D, Jiang S, Shen H, Qin S, Liu J,Zhang Q, Li R and Xu Q. Diclofenacsodium-loaded solid lipid nanoparticlesprepared by emulsion/solvent evaporationmethod. J. Nanopart. Res. (2011) 13:2375-86.
(34) Fang YP, Wu PC, Huang YB, Tzeng CC,Chen YL, Hung YH, Tsai MJ and Tsai YH.Modification of polyethylene glycol ontosolid lipid nanoparticles encapsulating anovel chemotherapeutic agent (PK-L4) toenhance solubility for injection delivery.Int. J. Nanomed. (2012) 7: 4995-5005.
(35) Mahjub R, Dorkoosh FA, Amini M,Khoshayand MR and Rafiee TehraniM. Preparation, statistical optimization,and in-vitro characterization of insulinnanoparticles composed ofquaternizedaromatic derivatives of chitosan. AAPSPharm. Sci. Tech. (2011) 12: 1407-19.
(36) MuÈller RH, MaÈder K and GohlaS. Solid lipid nanoparticles (SLN) forcontrolled drug delivery–a review of thestate of the art. Eur. J. Pharm. Biopharm.(2000) 50: 161-77.
(37) Severino P, Santana MH and SoutoEB. Optimizing SLN and NLC by 22 fullfactorial design: effect of homogenizationtechnique. Mater. Sci. Eng. (2012) 32:1375-9.
(38) Schwarz C. Solid lipid nanoparticles(SLN) for controlled drug delivery II.Drug incorporation and physicochemicalcharacterization. J. Microencapsul. (1999)16: 205-13.
(39) Zimmermann E, Müller RH and MäderK. Influence of different parameters onreconstitution of lyophilized SLN. Int. J.Pharm. (2000) 196: 211-3.
(40) Mahjub R, Radmehr M, DorkooshFA, Ostad SN and Rafiee Tehrani M.Lyophilized insulin nanoparticles preparedfrom quaternized N-aryl derivatives ofchitosan as a new strategy fororaldeliveryof insulin: in-vitro , ex-vivo and in-vivocharacterizations. Drug Dev. Ind. Pharm.(2014) 40: 1645-59.
(41) Howard MD, Lu X, Jay M and DziublaTD. Optimization of the lyophilization
process for long-term stability of solid–lipid nanoparticles. Drug Dev. Ind. Pharm.(2012) 38: 1270-9
(42) Abdelwahed W, Degobert G and FessiH. A pilot study of freeze drying of poly(epsilon-caprolactone) nanocapsulesstabilized by poly (vinyl alcohol):formulation and process optimization. Int.J. Pharm. (2006) 309: 178-88.
(43) Abdelwahed W, Degobert G, StainmesseS and Fessi H. Freeze-drying ofnanoparticles: formulation, process and 
62Mehrabani Yaganeh E et al. / IJPR (2020), 19 (3): 45-62storage considerations. Adv. Drug Deliv.Rev. (2006) 58: 1688-713.
(44) Al-Qushawi A, Rassouli A, Atyabi F,Peighambari SM, Esfandyari-Manesh M,Shams GR and Yazdani A. Preparation andcharacterization of three tilmicosin-loadedlipid nanoparticles: physicochemical properties and in-vitro antibacterialactivities. Iran. J. Pharm. Res. (2016) 15:663-76.
(45) De Chasteigner S, Cavé G, Fessi H,Devissaguet JP and Puisieux F. Freezedrying of itraconazole-loaded nanospheresuspensions: a feasibility study. Drug Dev.Res. (1996) 38: 116-24.
(46) Pisponen A, Mootse H, Poikalainen V,Kaart T, Maran U and Karus A. Effects oftemperature and concentration on particlesize in a lactose solution using dynamiclight scattering analysis. Int. Dairy J.(2016) 61: 205-10.
(47) Daniher DI and Zhu J. Dry powderplatform for pulmonary drug delivery.Particuology (2008) 6: 225-38.
(48) Vivek K, Reddy H and Murthy RS.Investigations of the effect of the lipidmatrix on drug entrapment, in-vitro release,and physical stability of olanzapine-loadedsolid lipid nanoparticles. AAPS Pharm. Sci.Tech. (2007) 8: 16-24.
(49) Venkateswarlu V and Manjunath K.Preparation, characterization and in-vitrorelease kinetics of clozapine solid lipidnanoparticles. J. Control. Release (2004)95: 627-38.
(50) Kushwaha AK, Vuddanda PR,Karunanidhi P, Singh SK and Singh S.Development and evaluation of solid lipidnanoparticles of raloxifene hydrochloridefor enhanced bioavailability. Biomed. Res.Int. (2013) 2013: 584549.
 (51) Priyanka K and Hasan SA. Preparationand evaluation of montelukast sodiumloaded solid lipid nanoparticles. J. YoungPharm. (2012) 4: 129-37.
(52) Kakkar V, Singh S, Singla D and KaurIP. Exploring solid lipid nanoparticlesto enhance the oral bioavailability ofcurcumin. Mol. Nutr. Food Res. (2011) 55:495-503.