International Journal of Pharmaceutics 466 (2014) 96–108

Pulmonary sustained release of insulin from microparticles composedof polyelectrolyte layer-by-layer assembly

Kiran Prakash Amancha a,1, Shantanu Balkundi b,2, Yuri Lvov b, Alamdar Hussain a,3,*aDepartment of Basic Pharmaceutical Sciences, College of Pharmacy, University of Louisiana at Monroe, 700 University Ave., Monroe, LA 71209, United StatesbDepartment of Chemistry, Institute for Micromanufacturing, Louisiana Tech University, 911 Hergot Ave., Ruston, LA 71272, United States

A R T I C L E I N F O

Article history:Received 4 October 2013Received in revised form 29 January 2014Accepted 6 February 2014Available online 22 February 2014

Keywords:Pulmonary deliverySustained releaseInsulinLayer-by-layer assemblyMicroparticlesPulmonary toxicity

A B S T R A C T

The present study tests the hypothesis that layer-by-layer (LbL) nanoassembly of thin polyelectrolytefilms on insulin particles provides sustained release of the drug after pulmonary delivery. LbL insulinmicroparticles were formulated using cationic and anionic polyelectrolytes. The microparticles werecharacterized for particle size, particle morphology, zeta potential and in vitro release. Thepharmacokinetics and pharmacodynamics of drug were assessed by measuring serum insulin andglucose levels after intrapulmonary administration in rats. Bronchoalveolar lavage (BAL) and evans blue(EB) extravasation studies were performed to investigate the cellular or biochemical changes in the lungscaused by formulation administration. The mass median aerodynamic diameter (MMAD) of the insulinmicroparticles was 2.7 mm. Confocal image of the formulation particles confirmed the polyelectrolytedeposition around the insulin particles. Zeta potential measurements showed that there was chargereversal after each layering. Pulmonary administered LbL insulin formulation resulted in sustained seruminsulin levels and concomitant decrease in serum glucose levels. The BAL and EB extravasation studiesshowed that the LbL insulin formulation did not elicit significant increase in marker enzymes activitiescompared to control group. These results demonstrate that the sustained release of insulin could beachieved using LbL nanoassembly around the insulin particles.

ã 2014 Elsevier B.V. All rights reserved.

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International Journal of Pharmaceutics

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1. Introduction

Protein and peptide drugs are frequently administered throughintravenous and subcutaneous injections and are rapidly clearedfrom the body. These routes of drug administration are painful and,as many of the protein drugs are indicated to treat chronicconditions such as diabetes, chronic administration results inpatient non-compliance (Todo et al., 2001). Systemic delivery ofprotein and peptide drugs through pulmonary route has distinctadvantages compared to other routes, such as low proteaseactivity, large absorptive surface area, thin alveolar epithelium,high perfusion capacities, no first-pass metabolism and highbioavailability (Adjei and Gupta, 1994; Komada et al., 1994).Pulmonary delivery of sustained release formulations of a protein

* Corresponding author. Tel.: +1 405 271 6593x47472; fax: +1 405 271 7505.E-mail address: alamdar-hussain@ouhsc.edu (A. Hussain).

1 Current Position: Insys Therapeutics, Inc., 444 S. Ellis St., Chandler, AZ 85224.2 Current Position: College of Medicine, University of Nebraska Medical Center,

42nd and Emile, Omaha, NE 68198.3 Current Position: College of Pharmacy, The University of Oklahoma, 1110 N.

Stonewall Ave., Oklahoma City, OK 73117.

http://dx.doi.org/10.1016/j.ijpharm.2014.02.0060378-5173/ã 2014 Elsevier B.V. All rights reserved.

drug could protect the protein over a prolonged period fromdegradation or elimination, reduce the frequency of administra-tion, increase the systemic availability, and improve patientcompliance. However, until now, there is no pulmonary insulindosage form available in the market.

The types of current insulin delivery systems used in themanagement of diabetes include: 1. Short acting insulin formu-lations deliver insulin at a rate that reaches into the bloodstream in30 min after injection and stays effective till 3–6 h (Humulin1).2. Rapid acting insulin formulations deliver insulin at a rate thatbegins to work after about 5 min and work for 2–4 h (Novolog1).3. Intermediate acting insulin formulations reaches bloodstream in2–4 h and stay effective for 12–18 h (Novolin N1, NPH1). 4. Long-acting insulin formulations deliver the drug at a rate that reachesbloodstream in 6–10 h and stays effective for 20–26 h (Lantus1).All of these dosage forms are administered via injections. Thechronic use of these dosage forms for the treatment of diabetescould decrease the patient compliance.

Insulin dosage forms forpulmonaryadministration areyet to reachthe market. Sustained release formulations for pulmonary delivery inthe form of liposomes, solid-lipid nanoparticles, nanospheres,microcapsules and other types of dosage forms have been under

K.P. Amancha et al. / International Journal of Pharmaceutics 466 (2014) 96–108 97

investigation for many years (Chono et al., 2009; Kawashima et al.,1999; Takenaga et al., 2002). Liposomes provide the stability andprevent the degradation of encapsulatedprotein drugs. These deliveryvehicles are well established and extensively investigated particulatecarrier systems that have been successfully employed for the sitespecific drug delivery (Wang, 1999). However, drug leakage andbinding of proteins with the phospholipid molecules are thedisadvantages of these systems. Proteins are hydrophilic moleculesand encapsulation of these drugs in the core of solid-lipid nano-particles and nanospheres could be difficult due to the partition ofthese molecules into the aqueous compartment (Antonio and Eliana,2007). Protein drugs can be loaded into microcapsules in order toprovide the stability of the encapsulated drugs. However, drug leakageis the main problem associated with this type of drug delivery system(Qi et al., 2008). In addition, a more recent layer-by-layer (LBL) self-assembly technique for preparing ultra-thin films over the drugparticles has begun to receive much attention in sustained releasedelivery systems.

LbL approach relies on the electrostatic attraction betweenoppositely charged polyions and this approach can be used toprepare nanoshell assembly around the drug particles (Qiu et al.,2001). In this methodology, cationic and anionic polyelectrolytesare alternatively adsorbed on a charged drug particle, resulting inmulti-layer assemblies on the solid substrate (Ibarz et al., 2001).Unlike liposomes and micelles, the multilayer assembly over thedrug particles is usually tough and homogeneous, and canprecisely control the release of the drug. The prerequisites to coatsubstrate particles with these multilayers include the presence ofcharge on the substrate molecule and that the substrate must notdissolve completely in the coating solution. The rate of drug releasegenerally depends on the thickness of the nanoassembly aroundthe drug particles which in turn depends on the type, concentra-tion, molecular weight and adsorption capacity of polyelectrolyteused for the coating. Varying the number of layers on drug particlescould result in a formulation with optimal release characteristics(Suzuki et al., 2002). In one study, fluorescein dye microcrystalswere used as core material and coated with anionic polystyrenesulfonate (PSS) and cationic poly (allylamine) hydrochloride (PAH).This process resulted in the sustained release of the dye. Theauthors found that increasing the number of layers in the shellaround the drug core resulted in decreased shell permeability andprolonged core dissolution (Sui et al., 2003). In another study,multi-layer assembly on furosemide drug microcrystals resulted incontrolled release of drug depending on the number of layers,thickness of LbL assembly, and type of polyions used in the processof coating. In this study, it was shown that gelatin/PDDA/PSScoating over the furosemide crystals reduced the drug release up to300 times compared to uncoated drug microcrystals (Ai et al.,2003). Dexamethasone, a potent synthetic member of steroidhormones, was formulated using LbL technique. In this study, drugparticles coated with gelatin A/PDDA/PSS resulted in controlledrelease of the drug (Pargaonkar et al., 2005).

More recently, a glucose sensitive multilayer film wasfabricated on to tablet core by the LbL assembly method (Chenet al., 2011). In this method positively charged poly [2-(dimethylamino) ethyl methacrylate] polymer (star PDMAEMApolymer) and negatively charged insulin and glucose oxidasewere used. The authors found that, in response to stepwise in vitroglucose challenge, the multilayer film showed an on–off regulationof insulin release. Furthermore, the multilayer film continuouslyreleased insulin after being subcutaneously implanted in strepto-zocin induced diabetic rats and reduced the blood glucose level forat least two weeks. In a separate study, the arrangement sequenceof the LbL components was altered. Subcutaneous administrationof this altered LbL system prolonged the hypoglycemic effect from17 days to 36 days (Chen et al., 2012). Moreover, Luo et al. (2012)

prepared supramolecular assembly of porcine insulin (P-SIA) andthen fabricated a glucose sensitive LbL film using a star polymer,glucose oxidase, catalase and P-SIA. A single subcutaneous dose ofthis LbL formulation resulted in effective glycemic control indiabetic rats for up to 295 days without hypoglycemia (Luo et al.,2012). Based on these studies, the LbL encapsulation method canbe used to formulate protein drugs and control the release of thesemacromolecules following pulmonary delivery. Also, to the best ofour knowledge there are no published reports available onpulmonary efficacy and toxicity evaluation of LbL drug micro-particles.

The objective of the present study was to formulate insulin,utilizing LbL nanoassembly technique and to test the in vivoefficacy of the formulation after pulmonary administration in rats.The effect of number of layers on drug release profiles was studied.Pulmonary toxicity due to short-term and chronic administrationof LbL insulin formulation was also investigated.

2. Materials and methods

2.1. Materials

Recombinant human insulin (28.8 U/mg), poly (dimethyldiallylammonium chloride) (PDDA: MW 100,000; cationic polymer),sodium (polystyrenesulfonate) (PSS: MW 70,000; anionic poly-mer), N-Acetylglucosaminidase (NAG) assay kit, sodium mono-dodecyl sulfate (SDS) and evans blue (EB) were purchased fromSigma–Aldrich (St. Louis, MO, USA). Reagents for lactate dehydro-genase (LDH) and alkaline phosphatase (ALP) determination wereobtained from Pointe Scientific Inc. (Canton, MI, USA). Bicincho-ninic acid (BCA) protein assay kit was purchased from ThermoScientific (Rockford, IL, USA). Fluorescein-5-isothiocyanate (FITC;Sigma–Aldrich) was used for labeling LbL insulin microparticles.Human insulin-specific radioimmunoassay (RIA) kit and glucose(Hexokinase) assay kit were purchased from Linco Research Inc.(St. Charles, MO, USA) and Pointe Scientific Inc. (Canton, MI, USA),respectively. Anesthetics, ketamine and xylazine were purchasedfrom Henry Schein, Inc. (Melville, NY, USA).

2.2. Preparation of pulmonary LbL insulin formulations

The procedure used to introduce LbL assembly on insulinparticles was described in previous reports (Pargaonkar et al.,2005). Briefly, based on the negative charge of the suspendedinsulin particles at pH above 5.3 (isoelectric point of insulin), 5 mgof insulin was suspended in 1 ml of PDDA solution (2 mg/ml in PBS,pH 7.4). The suspension was sonicated for 10 s, stirred for 10 minand centrifuged (5700 � g at 4 �C) (Eppendorf, Model 5804 R, VWR,NY, USA). The supernatant polyelectrolyte solution was discardedand the separated drug particles were washed two times with PBS(pH 7.4) to remove unadsorbed polyelectrolyte (PDDA). Thisresulted in the formation of first layer of polyions around insulinparticles. These particles were resuspended in a 1 ml of PSSsolution (2 mg/ml in PBS, pH 5.8) and stirred for 10 min to ensurecoating. The suspension was centrifuged (5700 � g at 4 �C), andseparated particles were washed 2 times to remove unadsorbedPSS. Alternate layers of PDDA and PSS were subsequently depositeduntil the desired number of layers was achieved. In addition,insulin solution for subcutaneous administration was prepared bydissolving the drug in acidic PBS (pH 2). The pH of the solution wasadjusted to a physiological level (pH 7.2–7.4) with NaOH. Theconcentration of insulin in the final formulations was measured byBCA protein assay kit. For this, few drops of 0.1 N HCl was added toformulation while mixing until all the particles are dissolved. ThepH of the solution was adjusted to 7.4 with the addition of 0.1 NNaOH and the insulin concentration was determined.

98 K.P. Amancha et al. / International Journal of Pharmaceutics 466 (2014) 96–108

2.3. Particle size and morphology

Scanning electron microscopic images of micronized insulinparticles were taken using a Philips XL30 scanning electronmicroscope (Philips, Eindhoven, Netherlands). The applied voltagewas 50 kV and the magnification was 3380�. Morphology of LbLinsulin microparticles was studied using confocal laser scanningmicroscopy (Leica, Allendale, NJ, USA). For fluorescence, FITC-labeled PDDA was used as the outermost layer on LbL formulationparticles. Particle size analysis was performed using eight-stagenon-viable cascade impactor (Gonda and Khalik, 1988). Onemililiter of 6-LbL insulin formulation was dispensed three timeswith five-second interval into the mouth piece of cascade impactoroperated at a flow rate of 28.3 l min�1. The smallest particle sizecapable of depositing on a given stage, known as effective cutoffdiameter (ECD) and the cumulative percent less than size rangewere determined by comparing the deposited fractions of all stagesbelow a particular size limit to the total fraction collected on allstages. Aerodynamic properties, effective cutoff diameter (ECD)and the cumulative percent less than size range of LbL insulinformulation were plotted on a log-probability graph. Mass meanaerodynamic particle diameter (MMAD) and geometric standarddeviation (GSD) were quantified. The MMAD was calculated at the50th percentile acquired from the regression line and the GSD wasdetermined using the equation, GSD = (diameter at 84.13%/diame-ter at 15.87%)1/2 (Swift, 1993).

2.4. Zeta potential and in vitro drug release

To ensure the charge reversal after each polyion layering duringLbL process, the zeta potential of the suspended particles wasmeasured using Zeta-Plus photon correlation spectroscopy (Broo-khaven Instruments, NY, USA). The reported zeta potentialmeasurements represent the average of 10 measurements � S.E.Release experiments were performed by dipping 5 mg of insulinmicroparticles with or without polyelectrolyte multilayers in 1 mlof PBS (pH 7.4 at 37 �C) at 25 rpm, in a centrifuge tube. Afterimmersion for desired time, the microparticles were separated bycentrifugation and the protein concentration in the supernatantwas determined by the BCA protein assay kit.

2.5. Pulmonary absorption studies

Male Sprague-Dawley rats were obtained from Harlan Labora-tories (Houston, TX, USA). The rats used in this study were inbredin-house. Rats were housed in a 12 h light-dark cycle and aconstant temperature environment of 21 �C, provided withstandard diet ad libitum. On the day of the experiment, ratsweighing between 300 and 350 g were divided into three groupswith 3–4 rats in each group. Rats in the first and second group wereadministered 100 ml of PBS (pH 7.4) and 100 ml of insulin solution(10 U/kg), respectively, via intrapulmonary route. The third groupof rats received LbL insulin (10 U/kg) formulation particles layeredwith PDDA and PSS, via intrapulmonary route. The amount of theinsulin dose given to each rat was 0.35 mg/kg as determined by theBCA protein assay kit. The rats were anesthetized by anintramuscular injection of a mixture of ketamine (100 mg/ml)and xylazine (20 mg/ml). The dose of anesthetic mixture was1.0 ml/kg. Additional doses of ketamine and xylazine wereadministered as needed for the collection of blood samples. After40–45 min of initial anesthesia, intrapulmonary administrationwas done as reported previously (Hussain et al., 2006). The volumeof formulation administered was 90–110 ml (=10 U/kg of insulin)depending on animal body weight. Following pulmonary admin-istration, blood samples were collected from the tail vein at time 0(prior to LbL administration), 10, 20, 30 min, and 1, 2, 4, 6, 8, 12, and

24 h (post LbL administration). About 150 ml of blood was collectedat each time interval and centrifuged (2500 � g for 5 min) toseparate serum. Serum samples were stored at �20 �C for furtheranalysis. Serum insulin concentrations were determined using ahuman insulin-specific RIA kit and serum glucose levels weredetermined using a glucose assay kit.

According to Linco protocol, the properties of human insulinRIA were as follows: Inter-assay precision was 2.9–6.0% and intra-assay precision was 2.2–4.4%. Accuracy was 93–100%. Specificityfor rat insulin was less than 0.1%. All the serum samples werediluted with 50 ml of RIA buffer to fit the drug concentrationswithin the calibration curve. According to Pointe Scientific Inc., thelinearity of the glucose assay was between 0.6 and 600 mg/dl. Thelower limit of glucose detection was 0.6 mg/dl. For relativebioavailability studies, 100 ml of insulin solution (10 U/kg) wasgiven subcutaneously to fourth group of rats (n = 3–4). To study theeffect of number of layers on serum insulin profiles, 1, 2, 4, 6, 8, and16-LbL insulin (10 U/kg) microparticles layered with PDDA and PSSwere administered intrapulmonary. All animal experiments wereapproved by the Institutional Animal Care and Use Committee ofthe University of Louisiana at Monroe and all surgical andtreatment procedures were consistent with the IACUC policiesand procedures.

2.6. Pulmonary toxicity studies

2.6.1. Bronchoalveolar lavage studiesMale Sprague-Dawley rats were obtained from Harlan Labora-

tories (Houston, TX, USA). The rats used in this study were inbredin-house. Rats were housed in a 12 h light-dark cycle and aconstant temperature environment of 21 �C, provided withstandard diet ad libitum. For this set of experiments, the ratsweighing between 300 and 350 g were divided into four groups(n = 3–4). Bronchoalveolar lavage (BAL) studies were conducted toassess the biochemical and histological changes in the lungs afterpulmonary administration of LbL formulation for 30 days (chronictreatment). The first group of rats received 100 ml of PBS (pH 7.4),intrapulmonary. The second group received positive control,lactose (5 mg/kg), and the third group received 100 ml of 6-LbLinsulin/PDDA/PSS (5 U/kg) intrapulmonary, once-a-day for 30 days.On the 31st day, lungs were surgically removed and wet lungweight was recorded. The lungs were lavaged and BAL fluid wascollected according to the method described previously (Rawatet al., 2008). Activities of lactate dehydrogenase (LDH), N-Acetylglucosaminidase (NAG), and alkaline phosphatase (ALP) inthe BAL fluid were determined using commercially available kits.Total protein content in the BAL fluid was determined usingbicinchoninic acid (BCA) protein assay kit. According to PointeScientific, alkaline Phosphatase assay kit has linearity of 1000 U/l.Thermo scientific reports that the linearity of BCA protein assay kitis in the range of 20–2000 mg/ml with sensitivity of 5 mg/ml.Similar experiments were conducted in untreated (control) groupof rats to determine the activities of these marker enzymes.

2.6.2. Extravasation of evans blueMale Sprague-Dawley rats were obtained from Harlan Labora-

tories (Houston, TX, USA). The rats used in this study were inbredin-house. Rats were housed in a 12 h light-dark cycle and aconstant temperature environment of 21 �C, provided withstandard diet ad libitum. For this set of experiments, the ratsweighing between 300 and 350 g were divided into four groups(n = 3–4). In vivo lung permeability assays were performed using EBextravasation as an indicator of pulmonary vasculature leakagebecause of possible inflammation caused by the administration ofLbL formulations over a period of three days (short-termtreatment). The procedure used to perform these experiments

Fig. 1. Scanning electron microscopic image of uncoated insulin crystals.

Fig. 2. Confocal image of insulin microparticles encapsulated with 3 bilayers ofPDDA/PSS. PDDA – Polydimethyldiallyl ammonium chloride; PSS – Polystyrenesulfonate.

K.P. Amancha et al. / International Journal of Pharmaceutics 466 (2014) 96–108 99

was described previously (Christou et al., 1998; Patterson et al.,1992). Rats in the first group received 100 ml of sodiummonododecyl sulfate (SDS, 1% w/v, positive control), and thesecond group received 100 ml of 6-LbL formulation (5 U/kg)intrapulmonary, once-a-day for three consecutive days. The thirdgroup of rats was untreated (control). On day 3, one hour afterpulmonary administration, rats were injected with 0.5 ml of EB (1%v/v) via external jugular vein and kept under anesthesia foradditional 30 min. The lungs were removed surgically and BAL wasperformed as mentioned previously. The BAL fluid (750 ml) wasmixed with an equal portion of acetone and centrifuged (3900 � gfor 10 min). The concentration of EB in the supernatant wasmeasured spectrophotometrically at 620 nm. Similarly, EB con-centration in the lung homogenate was determined.

2.7. Pharmacokinetic analysis

Standard non-compartmental pharmacokinetic analysis wasperformed for the serum insulin concentration–time profiles usingKinetica1. Area under the curve (AUC0–t) for serum insulin–timeprofiles was determined by trapezoidal method. The area underthe first moment curve (AUMC0–t) for serum insulin–time profilewas estimated from a plot of the product of serum insulinconcentration and time (c � t) vs. time. The mean residence time(MRT) was calculated by dividing AUMC0–t with AUC0–t. Percentminimum blood glucose concentration (%MBGC) and time atwhich percent minimum blood glucose concentration occurred (T%MBGC) were obtained from serum glucose concentration–timeprofile. The relative bioavailability (Frel) was assessed by comparingthe AUC0–t for serum insulin–time curve obtained after pulmonaryadministration to that obtained after subcutaneous administra-tion. For all of the assays, pharmacokinetic parameters werecalculated after subtraction of the initial serum insulin concentra-tion analyzed prior to formulation administration (at T = 0) fromserum protein concentrations analyzed following formulationadministration (T = 10, 20, 30 min, and 1, 2, 4, 6, 8,12, and 24 h). Thisallowed us to minimize the effects of unknown interferingcomponents with antibodies used in the immunoassays.

2.8. Statistical analysis

The serum insulin, serum glucose and various pharmacokineticparameters obtained after different treatments were analyzed byusing one-way ANOVA. When the differences in means weresignificant, a post-hoc pair wise comparison was conducted usingNewman–Keuls multiple comparison (GraphPad Prism, version5.0, GraphPad Software, San Diego, CA). Results were consideredstatistically significant if p-value was <0.05.

3. Results and discussion

3.1. Particle size and morphology

Scanning electron photomicrograph of micronized insulinpowder is shown in Fig. 1. This image indicates that the powdershows aggregation of small drug particles due to the interparti-culate bonding, hence, the diameters were in the range 5–20 mm.During formulation, uncoated drug particles were subjected tosonication, which resulted in particle size reduction. In the absenceof polyions, the cohesive insulin particles adhere to each other toform large aggregates and when sonicated in a solution containingpolyions, the particles are coated with layers of oppositely chargedpolyions and the coated particles tend to repel with each other.Fig. 2 represents confocal image of LbL particles. From this image, itis evident that insulin particles were intact and LbL assemblingprocess did not cause changes in particle morphology. The shell

formed around the microcrystals preserved the integrity and theshape of the formulated drug particles. On the whole, these imagesindicate that the particles were covered with LbL assembly ofpolyions.

Particle size distribution of 6-LbL insulin formulation wasquantified using eight-stage non-viable cascade impactor and thedata is presented in Fig. 3. The mass median aerodynamic diameter(MMAD) of formulation particles was found to be 2.7 mm. Both theparticle shape and particle density are taken into account for themeasurement of mass median aerodynamic diameter. MMADrepresents the average particle size of the formulation where fiftypercent of the particles by weight are larger than the MMAD andfifty percent of the particles by weight are smaller than the MMAD(Swift, 1993). The GSD calculated from the linear regression of theparticle size distribution was 1.9 mm. A GSD value greater than 1.22indicates a heterogeneous mixture of particle sizes.

From Fig. 3, it is evident that the particles were having the sizesin the range of 0.4–9.0 mm. A linear correlation between ECD andcumulative percent less than size range represents an evenlydistributed range of particle sizes. Previous reports demonstratedthat good distribution throughout the lungs requires particles withan aerodynamic diameter between 1 and 5 mm, particles greaterthan 5 mm are deposited in upper respiratory tract while particlesless than 1 mm are exhaled (Lee et al., 1995).

From these results, it is evident that the MMAD and GSD of 6-LbL formulation were within the range of an ideal particle sizedistribution accessible to the lower airways. MMAD and GSDvalues of 1, 2, 4, 8, and 16-LbL formulations did not significantlydiffer from that of 6-LbL formulation (data not shown). This is due

Fig. 3. Particle size distribution of 6-LbL formulation in the cascade impactor. LbL –

layer-by-layer.

Fig. 5. Release profiles of unencapsulated insulin microcrystals and encapsulatedinsulin microcrystals. LbL – layer-by-layer.

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to the fact that LbL assembly of polyelectrolytes introducesnanometer thick shell around drug particles which does notcontribute significant changes in particle size distribution. In fact,Pargaonkar et al. (2005) used quartz crystal microbalance (QCM) tomeasure the layer thickness for different polyelectrolytes ondexamethasone particles. They found that each PDDA monolayerhad a thickness of about 2–3 nm and each PDDA/PSS bilayer had athickness of about 4–6 nm.

3.2. Zeta potential and in vitro drug release

A change in zeta potential of LbL insulin microparticles aftereach layering with polyions is shown in Fig. 4. The uncoated insulincrystals were negatively charged, with a zeta potential of �21.1� 2.4 mV. After the adsorption of first PDDA layer, the zetapotential obtained was 38.2 � 1.3 mV, indicating a charge reversal.At this stage, cationic PDDA was preferentially adsorbed on to thenegatively charged insulin particles resulting in PDDA monolayeraround the drug. The first PSS layering changed zeta potential frompositive to negative (�22.2 � 1.5 mV). This trend of charge reversalwas observed after each sequential layering. The alternatingsurface charge of drug particles after each layering demonstratesthat the LbL assembly process was successful.

The in vitro release profiles of uncoated and LbL insulinmicroparticles in PBS (pH 7.4 at 37 �C) are presented in Fig. 5. Thehalf release times (T1/2) were 0.52, 0.65, 0.76, and 0.94 h for 1, 2, 4,and 6-LbL formulations, respectively. The T1/2 of uncoated drugcrystals was �0.05 h. The release T1/2 of 1, 2, 4, and 6-LbLformulations were 10, 13, 15 and 19 times longer compared to

Fig. 4. Zetapotential as a function of adsorbed polyelectrolyte layer, PDDA and PSS,for 6-LbL insulin microparticles. PDDA – Polydimethyldiallyl ammonium chloride;PSS – Polystyrene sulfonate.

uncoated drug crystals. This suggests that the release rate of insulincan be controlled by increasing the number of assembled layers onthe drug particles. Insulin coated with 8 and 16 layers did notdemonstrate a significant increase in the release T1/2 compared to6-LbL formulation.

It is important to note that the insulin release from the LbL-coated microparticles exhibited a biphasic pattern: an initial burstrelease of about 60–80% within the first 2 h and a subsequentsteady release. Basically, insulin LbL particles are micrometer sized(1–9 mm) drug particles coated with nanometer thick (1–3 nm)polymer layers, forming a porous membrane around the drug core.The membrane around the LbL particles basically controls themovement of the liquid medium across it. The drug release is dueto the dissolution of the core and diffusion across the membrane(Whelan, 2001). As reported earlier, the release mechanism of thedrug from the microparticles involves two processes: a) bulksolution diffuses into the capsules to dissolve the drug crystals andb) the dissolved drug diffuses out of the shell. It is expected that atthe initial stage, drug concentration inside the shell is high (close tosaturation). The high concentration of drug inside the shell causedby the dissolution of core can result in a large concentrationgradient across the shell leading to a high osmotic pressure insidethe capsule. Therefore, the diffusion of drug through the capsuleshell is accelerated. During later stages of release, the concentra-tion gradient across the shell decreases causing reduction in thediffusion of drug from the microparticles (Adi and Moiwia, 2007;Siegel and Rathbone, 2012). Hence, irrespective of the numberlayers fabricated onto the drug particles, we have found thatapproximately fifty percent of the drug was released within onehour in to release medium we used in this study. In contrast to LbLlayers, polymers used in the traditional matrix systems usuallyswell to form a gel layer. Depending on the polymer concentrationused, these gel layers sometimes have a thickness more than thethickness of the central drug core. Release mainly occurs either bydrug dissolution and diffusion through the gel layer or bycombination of polymer degradation, dissolution and diffusion(Gothoskar et al., 2006).

3.3. Pulmonary absorption studies

In general, the preferred method of aerosol administration inanimals is direct intrapulmonary administration of formulation.The other modes of pulmonary administration include nose-onlyexposure and whole body exposure chambers (Stephenson et al.,1988). Compared to nose-only and whole body exposure to drugaerosol, the intrapulmonary administration could reduce thevariations in the amount of the drug delivered to lungs. In case ofnose-only and whole body exposure studies, the amount of the

K.P. Amancha et al. / International Journal of Pharmaceutics 466 (2014) 96–108 101

dose reaching the lungs depends on the breathing pattern of theanimals. Hence, the amount of the dose reaching the lungs couldvary between individuals (Bivas et al., 2005; Wright et al., 2008).

To the best of our knowledge, we have evaluated the in vivoperformance of various LbL formulations following pulmonarydelivery for the first time. Pulmonary absorption studies wereconducted to estimate the pharmacokinetic and pharmacodynam-ic parameters of various insulin formulations by measuring thechanges in serum insulin as well as serum glucose concentrations.Rats were administered PBS, insulin solution, and various LbLinsulin formulations intrapulmonary. For relative bioavailabilitystudies insulin solution was administered through subcutaneousroute.

Pulmonary absorption experiments were conducted in normalfed rats. The glucose levels in normal rats are 80 � 16 mg/dl(Kowluru et al., 2008). In our case, the average serum glucoseconcentrations following anesthesia and prior to formulationadministration were 360 � 93 mg/dl. It was reported that acutehyperglycemic effect of ketamine/xylazine in rats was associated

Fig. 6. Changes in serum insulin concentrations after pulmonary administration of 1, 2, 4mU – microunits. *Results were significantly different from the initial insulin concentr

with decreased plasma levels of insulin, adrenocorticotropichormone (ACTH), and corticosterone and increased levels ofglucagon and growth hormone (Saha et al., 2005). In this study, theauthors concluded that the hyperglycemic effect of ketamine/xylazine anesthesia may be mediated by modulation of theglucoregulatory hormones through stimulation of a-2 adrenergicreceptors.

In a separate study, rats anesthetized with ketamine/xylazinedisplayed hyperglycemia as a result of marked decrease in thepancreatic blood flow (Hindlycke and Jansson, 1992). Theseauthors found that intraperitonial injection of ketamine andxylazine mixture decreased both the whole pancreatic and isletblood flow. They also found that the pancreatic blood flow and isletblood flow in rats injected with ketamine/xylazine were0.33 � 0.04 ml/min/g and 21 � 3 ml/min/g pancreas, respectively.However, the reported pancreatic blood flow and islet blood flow inunanesthetized rats were 2.2 � 0.2 ml/min/g, and 121 � 4 ml/min/gpancreas, respectively, (Iwase et al., 2001) indicating that therewas a significant decrease in the blood flow. Moreover, the effect of

, 6, 8, 16-LbL insulin (PDDA/PSS) formulations (10 U/kg) (n = 4). LbL – layer-by-layer;ation (at zero time point) (p < 0.001).

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different anesthesia on glucose tolerances was compared. Inanimals injected with ketamine/xylazine mixture, hyperglycemicconditions were observed for a period of five hours. In our case,following anesthesia in PBS treatment group, the hyperglycemiawas observed until 6 h. Hence, the animal model used in thepresent study mimics the temporary diabetic model.

Figs. 6 and 7 represent changes in serum insulin and serumglucose concentrations after intrapulmonary administration of 1, 2,4, 6, 8 and 16-LbL formulations. Pharmacokinetic and pharmacody-namic parameters obtained in this study are listed in Table 1. Insulinparticles coated with one layer produced a substantial increase inserum insulin concentration (Cmax = 2385 � 358 mU/ml). This couldbe due to the rapid release and subsequent absorption of the drugfrom 1-LbL formulation. When the number of layers in the LbLassembly was increased to two, the serum drug concentrations(Cmax = 1912 � 286 mU/ml) were reduced by more than 20%compared to one layered formulation. There was significantdecrease in Cmax after the administration of 4-LbL formulationand further increasing number of layers did not contribute to the

Fig. 7. Changes in serum glucose after pulmonary administration of 1, 2, 4, 6, 8, 16-LbL insignificantly different from the initial glucose concentration (at zero time point) (p < 0

reductions in Cmax. On the other hand, Tmax of 6-LbL formulation(0.94 � 0.1 h) was significantly higher compared to all other LbLformulations (p < 0.0002). As the number of layers in the LbLassembly increased from 1 to 6, following trend in pharmacoki-netic parameters was observed; increase in the T1/2, MRT, T %MBGC, and decrease in %MBGC. The T1/2, MRT, %MBGC and T %MBGC for 6-LbL formulation were 14.2 � 2.1 h, 13.4 � 3.8 h, 2 � 1.4,and 8 h., respectively, indicating that 6 layers in the shellcontributed to better in vivo performance of the formulation.The changes in serum glucose concentrations after pulmonaryadministration of various LbL formulations were consistent withthe changes in serum insulin concentrations. At T = 0 glucose levelsfor 1-, 2-, 4-, 6-, 8-, and 16-LbL formulations were 492 � 14 mg/dl,397 � 93 mg/dl, 249 � 200 mg/dl, 407 � 46 mg/dl, 389 � 61 mg/dl,and 225 � 117 mg/dl, respectively. However, at T = 8 h postformulation administration, the glucose levels for 1-, 2-, 4-, 6-,8-, and 16-LbL formulations were 80 � 15 mg/dl, 21 � 20 mg/dl,33 � 3 mg/dl, 8 � 6 mg/dl, 88 � 24 mg/dl, and 35 � 32 mg/dl, re-spectively. The %MBGC was lower and T %MBGC was longer for

sulin (PDDA/PSS) formulations (10 U/kg) (n = 4). LbL – layer-by-layer. *Results were.001).

Table 1PK and PD parameters of insulin after I.P. administration of 1, 2, 4, 6, 8, and 16-LbL-insulin/PDDA/PSS formulations. Data represent Mean � SD, n = 3–4.

1-LbL 2-LbL 4-LbL 6-LbL 8-LbL 16-LbL

Cmax (mU/ml) 2385 � 358 1912 � 286 403 � 74.4 297 � 43.2 348 � 115 305 � 94.5Tmax (h) 0.16 � 0.05 0.16 � 0.07 0.2 � 0.07 0.94 � 0.1a 0.2 � 0.15 0.2 � 0.1AUC0–24 h (mU/ml h) 2266 � 426 2476 � 516 1384 � 527 1791 � 176 860 � 349 1084 � 358T1/2 (h) 6.22 � 1.3 5.24 � 3.57 7.6 � 3.33 14.2 � 2.1b 6.8 � 2.56 11.3 � 0.65MRT (h) 1.75 � 2.12 3.52 � 2.59 5.10 � 1.97 13.4 � 3.8b 8.43 � 2.89 10.5 � 2.12Frel (%) 12.9 14.1 7.9 10.2 4.9 6.2%MBGC 9.6 � 2.2 5.3 � 3.8 8.0 � 0.6 2 � 1.4 19.8 � 3.6 14.4 � 5.27T %MBGC (h) 4 6 8 8 6 6

a Pharmacokinetic and pharmacodynamic parameters obtained after intrapulmonary administration of 6-LbL formulation were significantly different from those obtainedafter intrapulmonary administration of 1, 2, 4, 8, and 16-LbL formulations, (p < 0.0002).

b Pharmacokinetic and pharmacodynamic parameters obtained after intrapulmonary administration of 6-LbL formulation are significantly different from those obtainedafter intrapulmonary administration of 1, 2, and 4-LbL formulations, (p < 0.002). Cmax – Maximum concentration of the drug in the serum; Tmax – Time at which Cmax wasobserved; AUC – Area under the curve; MRT – Mean residence time; Frel – Relative bioavailability.

K.P. Amancha et al. / International Journal of Pharmaceutics 466 (2014) 96–108 103

6-LbL formulation, indicating prolonged hyperglycemic control.All these findings suggest that 6 layers of polyions around the drugparticle resulted in optimum formulation performance. However,there was no significant improvement in the in vivo performanceof 8 and 16-LbL formulations. In fact, it has been stated that releaseof the drug from these formulations could be controlled with acertain number of layers in the LbL assembly above which there isno improvement in controlling the release of drug from theformulation (Ai et al., 2003). It is worthwhile to mention that thereleased insulin from the microparticles within the lungs may besubjected to enzymatic inactivation by proteases (Shen et al., 1999;

Fig. 8. Changes in serum insulin concentrations after pulmonary administration of PBS (pof insulin solution (10 U/kg) (n = 3). PBS – Phosphate buffer saline; mU – Microunits, I.P.different from the initial Insulin concentration (at zero time point) (p < 0.001).

Okumura et al., 1992). It is very unlikely that proteases degradethe insulin molecules which are yet to be released from themicroparticles. However, it is not clear that the relationshipbetween the in vivo release rate of insulin from these micro-particles and degradation rate by proteases.

Figs. 8 and 9 represent changes in serum insulin and serumglucose after pulmonary administration of PBS, insulin solution, 6-LbL insulin and subcutaneous administration of insulin solution.The pharmacokinetic and pharmacodynamic parameters obtainedin this study are listed in Table 2. Rats administered with PBSshowed no increase in serum insulin concentration (Fig. 8). The

H 7.4), insulin solution, 6-LbL insulin (PDDA/PSS) and subcutaneous administration – Intrapulmonary; S.C. – Subcutaneous; Sol – Solution. *Results were significantly

Fig. 9. Changes in serum glucose concentrations after pulmonary administration of PBS (pH 7.4), insulin solution, 6-LbL insulin (PDDA/PSS) and subcutaneous administrationof insulin solution (10 U/kg) (n = 3). PBS – Phosphate buffer saline; I.P. – Intrapulmonary; S.C. – Subcutaneous; Sol – Solution. *Results were significantly different from theinitial glucose concentration (at zero time point) (p < 0.002).

104 K.P. Amancha et al. / International Journal of Pharmaceutics 466 (2014) 96–108

presence of human insulin in rat serum following PBS administra-tion may be due to cross-reactivity of rat insulin with the humaninsulin specific antibody. As per the Linco’s radioimmunoassayprotocol, a cross-reactivity of 0.1% can be expected.

Pulmonary administration of insulin solution resulted in a rapidincrease in serum insulin levels (Cmax = 1345 � 74.8 mU/ml) com-pared to pulmonary administered 6-LbL formulation (Cmax = 297� 43.2 mU/ml). The rapid increase in serum insulin levels afterinsulin solution administration was transient and followed a steepdecrease. The observed higher Cmax of insulin solution is due tofaster rate of insulin absorption from solution form. This can be

Table 2PK and PD parameters of insulin after pulmonary administration of PBS, insulin solutirepresent Mean � SD, n = 3–4.

PBS Ins. So

Cmax (mU/ml) 48 � 15.4 1345 �Tmax (h) 9.5 � 10.3 0.44 �AUC0–24 h (mU/ml h) 564 � 46 2619 �T1/2 (h) – 5.54 �MRT (h) – 1.69 �Frel (%) 100 –

%MBGC 71 � 7.5 18.2 �T %MBGC (h) – 2

a Pharmacokinetic and pharmacodynamic parameters were significantly different fromI.P. – Intrapulmonary; Cmax – Maximum concentration of the drug in the serum; Tmax

residence time; Frel – Relative bioavailability.

further substantiated by the fact that LbL formulation resulted intwo-fold increase in Tmax (0.94 � 0.10 h, p < 0.015) compared toinsulin solution (Tmax = 0.44 � 0.08 h). On the other hand, subcuta-neous administration of insulin solution resulted in a higher Cmax

(3673 � 327 mU/ml), compared to pulmonary administered insulinsolution (Cmax = 1345 � 74.8 mU/ml) and 6-LbL formulation (Cmax =297 � 43.2 mU/ml). AUC0–24 h value from serum insulin–time curveafter subcutaneous administration was significantly higher (p< 0.001) compared to pulmonary administration of insulinsolution and 6-LbL. In general, subcutaneous administration ofdrug formulations acts as “depot” and results in drug absorption

on, 6-LbL-insulin/PDDA/PSS and after S.C. administration of insulin solution. Data

l. I.P. 6-LbL-Ins. I.P. Ins. Sol. S.C.

74.8 297 � 43.2a 3673 � 327a

0.08 0.94 � 0.10a 0.8 � 0.2a

457 1791 � 176 17503 � 1254a

1.9 14.2 � 2.1a 17.6 � 4.2a

2.29 13.4 � 3.8a 11.5 � 3.54a

13.7 10.2 4.6 2 � 1.4a 17 � 6.7

8a 8a

intrapulmonary administration of Insulin solution, (p < 0.015). S.C – Subcutaneous;– Time at which Cmax was observed; AUC – Area under the curve; MRT – Mean

K.P. Amancha et al. / International Journal of Pharmaceutics 466 (2014) 96–108 105

for extended periods. It is worthwhile to mention that thesubcutaneous administration of insulin loaded glucose sensitiveLbL multilayer films provided glycemic control varying between 2weeks and about 42 weeks (Chen et al., 2011; Chen et al., 2012; Luoet al., 2012). The prolonged insulin release from LbL systemcombined with slow absorption from the subcutaneous site mighthave contributed to glycemic control over several weeks. However,in our case, pulmonary administration of LbL insulin microparticlesresulted in glycemic control for hours as opposed to weeks. Thesedifferences can be due to the variations in the LbL fabrication, andthe physiological and morphological variations between these tworoutes of administration.

Fig. 10. A. Changes in wet lung weights for different treatment groups. *Wet lung weigh(n = 3). PBS – Phosphate buffer saline. LbL-Ins – LbL-insulin. B. Changes in the lactate dehwere significantly different from those obtained with control group, (p < 0.004) (n = 4)phosphatase (ALP) levels in the BAL fluid of different treatment groups. *ALP levels were s– Phosphate buffer saline; LbL-Ins – LbL-insulin. D. Changes in N-Acetylglucosaminidasignificantly different from those obtained with control group, (p < 0.001) (n = 4). PBS –

levels in the BAL fluid of different treatment groups. *Total protein content levels were sigPhosphate buffer saline; LbL-Ins – LbL-insulin.

Serum glucose profiles (Fig. 9) mimicked the serum insulinprofiles following formulation administration. For example, atT = 12 h post formulation administration the serum insulinconcentrations for PBS, 6-LbL, insulin solution I.P., and insulinsolution S.C. were 23 � 10 mU/ml, 70 � 8 mU/ml, 23 � 9 mU/ml and258 � 27 mU/ml and corresponding glucose levels were289 � 7 mg/dl, 38 � 11 mg/dl, 197 � 54 mg/dl, and 67 � 21 mg/dl.From these results it was evident that 6-LbL formulation resultedin better hyperglycemic control than PBS and insulin solution I.P. Itis also worthwhile to mention that the effect of the ketamine/xylazine anesthesia on the endogenous levels of insulin, glucagonand ACTH which in turn increase the endogenous glucose levels. It

ts were significantly different from those obtained with control group, (p < 0.001)ydrogenase (LDH) levels in the BAL fluid of different treatment groups. *LDH levels. PBS – Phosphate buffer saline; LbL-Ins – LbL-insulin. C. Changes in the alkalineignificantly different from those obtained with control group, (p < 0.006) (n = 4). PBSse (NAG) levels in the BAL fluid of different treatment groups. *NAG levels were

Phosphate buffer saline; LbL-Ins – LbL-insulin. E. Changes in total protein contentnificantly different from those obtained with control group, (p < 0.001) (n = 4). PBS –

Fig. 11. A. Changes in wet lung weights for different treatment groups. *Wet lungweights were significantly different from those obtained with control group,(p < 0.001) (n = 3). SDS – Sodium monododecyl sulfate.B. Changes in the Concentration of EB in Bronchoalveolar lavage fluid and lunghomogenate. *Evans blue concentrations were significantly different from thoseobtained with control group, (p < 0.001) (n = 3). BAL –

Bronchoalveolar lavage fluid; SDS – Sodium monododecyl sulfate; LbL-Ins – LbL-insulin.

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should be noted that the positive effect of ketamine and xylazineon endogenous glucose levels could not be negated for prolongedperiod unless if there were sustained release of insulin frommicroparticles. The controlled release property of LbL micro-particles could be attributed to the presence of nanometer thickshell around the drug reservoir (Qiu et al., 2001). After LbL particledeposited in alveoli, lung surface lining fluids including lungsurfactant may migrate through the shell and cause drugdissolution followed by diffusion of the dissolved drug throughthe shell. Moreover, in diabetic patients, short-acting pulmonaryinsulin formulations focus on postprandial glycemic control, whilelong-acting formulations focus on overnight glycemic control. Thetarget duration of glycemic control for a long-acting formulation isbetween 8 and 10 h (Ralf and Glen, 1999). If we consider the 6-LbLformulation used in the present study, then achieving this targetappears promising.

3.4. Pulmonary toxicity studies

Failures of marketed inhaled insulin and other inhaled insulinproducts in clinical trials were great concerns (Black et al., 2007;Heinemann, 2008; Siekmeier and Scheuch, 2008). Exubera, thefirst commercial inhaled insulin, was pulled from the market in2008. This product was manufactured by Pfizer in collaborationwith Nektar therapeutics. According to the manufacturers, Exuberafailed because of poor sales and high cost compared to injectablealternatives. There were cases of lung cancer identified in inhaledinsulin patients compared to the control group. However, all thepatients who were diagnosed with lung cancer had a prior historyof cigarette smoking. Pfizer was required to conduct extensivepost-marketing safety clinical trials, which was part of FDA’sapproval of the product. Failure of Exubera led Lilly (in collabora-tion with Alkermes) and Novo Nordisk (in collaboration withAradigm) to end their advanced clinical trials. More recently,Mannkind Corporation submitted a new drug application for itsinhalable drug, Afrezza, which is under review by the FDA. On thewhole, inhaled insulin was as effective as injectable alternatives;however, underlined safety concerns needs to be addressed.

Since treatment of diabetes requires chronic use of insulinformulations, the safety/toxicity of LbL formulation on the lungswas investigated. Pulmonary formulations were administeredonce-a-day for 30 consecutive days in male Sprague-Dawley rats.

3.4.1. Bronchoalveolar lavage studiesAmong all the pulmonary formulations 6-LbL formulation

showed optimum in vivo performance hence we investigated itsshort-term and chronic toxicity to the lungs. Bronchoalveolarlavage studies and EB extravasation studies were performed toevaluate pulmonary toxicity following chronic and short-termadministration of the formulation, respectively. Biochemicalchanges in the lungs may occur after treatment with these LbLformulations. The BAL fluid analysis can be used to assess suchbiochemical changes in the lungs. Although there is data on thetoxicity of pulmonary formulations containing absorptionenhancers and protease inhibitors, there are no reports availableon the toxicity of these LbL formulations following chronic and/orshort-term pulmonary administration. Lactose and PBS were usedas positive and negative controls, respectively. The wet lungweights of the rats showed significant differences (p < 0.001)between untreated and lactose treated group (Fig. 10 A–E). Theincrease in the wet lung weight of lactose treated group is mainlydue to the accumulation of physiological fluids because ofinflammation. The levels of LDH, ALP, NAG and protein contentin the BAL fluids of rats treated with lactose were comparativelyhigher than those of untreated, PBS and LbL treated groups. Forinstance, lactose produced approximately 2-fold increase in LDH

levels,1.3-fold increase in ALP, 6-fold increase in NAG levels, and 3-fold increase in total protein levels compared to both PBS and LbLtreated groups (Fig. 10 A–E). However, it is not clear that why therewas a decrease in the levels of lung injury markers of PBS treatmentgroup compared to the control (untreated) group.

The biochemical changes in the lungs after intrapulmonaryadministration of formulations could be predicted by measuringthe levels of injury markers in the BAL fluid (Henderson et al.,1985). The increased concentration of LDH in the BAL fluid aftertreatment has been considered as an indicator of lung injury(Henderson et al., 1978). Elevated levels of NAG, a lysosomalenzyme, could be attributed to increased phagocytic activity orlung toxicity (Hickey and Garcia-Contreras, 2001). ALP levels inBAL fluid increase in response to type 1 cell damage in pulmonaryepithelium (DeNicola et al., 1981). Elevated total protein content inthe BAL fluid is an indicator of capillary-alveolar barrier leakagebecause of lung inflammation (Beck et al., 1982). On the whole, themarker enzyme levels and protein content in the BAL fluid of PBSand LbL treated groups were not significantly different demon-strating that LbL insulin particles did not cause lung damage.

3.4.2. Extravasation of evans blueThe levels of protein content in the BAL fluid mainly depend on

the integrity of capillary-alveolar membrane barrier. As a result,there has been considerable interest in qualitative measurement ofthe barrier function of capillary-alveolar membrane after treat-ment with pulmonary formulations. EB dye has been used as asensitive but qualitative marker of protein leakage from vascular

K.P. Amancha et al. / International Journal of Pharmaceutics 466 (2014) 96–108 107

space of a variety of inflammatory tissues (Chuang et al., 1990;Finck et al., 1989; Lin et al., 1988). It has been reported that theextravasation of EB as an indicator of protein movement throughcapillary-alveolar membrane in inflamed rabbit lungs (Mason andEffros, 1983).

Changes in the wet lung weights for different treatment groupsare illustrated in Fig. 11A. Variations in the concentrations of EB inBAL fluid and lung homogenate for different treatment groups arepresented in Fig. 11B. Wet lung weight of LbL treated group was notsignificantly different compared to that of untreated (control)group. However, there was more than 25% increase in the wet lungweights of SDS treated rats compared to control group, indicatinginflammation. Data on changes in wet lung weights after shortterm treatment is consistent with that of chronic treatment.

In SDS treated rats, there was more than 3-fold increase in EBconcentration in BAL fluid compared to the untreated group. SDS isan anionic surfactant, could cause inflammation to biologicalmembranes. The increased concentration of EB in the BAL fluid ofSDS treated group could be attributed to increased extravasation ofthe dye into BAL fluid because of the barrier membraneinflammation. However, there were no significant differences inEB concentrations in both BAL fluid and lung homogenate of LbLtreated group compared to the untreated (control) group. Theseresults indicate that short term treatment with LbL formulation didnot cause any damage to the lung capillary-alveolar membrane.

4. Conclusion

The present study examined the pulmonary delivery of LbLinsulin microparticles. Insulin microparticles were prepared usingthe layer-by-layer approach. Zeta potential measurements con-firmed successful application of LbL approach to formulate insulin.MMAD and GSD measurements strongly supported the suitabilityof LbL microparticles for pulmonary delivery. Intrapulmonaryadministration of 6-LbL formulation produced sustained seruminsulin profiles, and also resulted in sustained lowering of serumglucose. Analysis of BAL and EB extravasation revealed that therewas no lung damage after short-term and chronic treatment. Thedistribution of microparticles in the lungs as well as the rate andmechanism of clearance from the lungs is a subject of futurestudies.

Acknowledgement

The work described in this manuscript was supported by aninternal fund from the University of Louisiana, Monroe.

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