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Preparation and characterization of controlled release poly-ε-caprolactonemicroparticles of isoniazid for drug delivery through pulmonary route

Rajesh Parikh, Sonali Dalwadi

PII: S0032-5910(14)00407-0DOI: doi: 10.1016/j.powtec.2014.04.077Reference: PTEC 10238

To appear in: Powder Technology

Received date: 1 October 2013Revised date: 3 April 2014Accepted date: 20 April 2014

Please cite this article as: Rajesh Parikh, Sonali Dalwadi, Preparation and characteriza-tion of controlled release poly-ε-caprolactone microparticles of isoniazid for drug deliverythrough pulmonary route, Powder Technology (2014), doi: 10.1016/j.powtec.2014.04.077

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TITLE PAGE

Preparation and characterization of controlled release poly-ɛ -caprolactone

microparticles of isoniazid for drug delivery through pulmonary route

Rajesh Parikh1, Sonali Dalwadi*

1

1Ramanbhai Patel College of Pharmacy,

Charotar University of Science and Technology,

CHARUSAT Campus, Changa 388 421,

Ta. Petlad, Dist. Anand,

Gujarat, India

*Corresponding Author’s E-mail: sonali.dalwadi@yahoo.com

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ABSTRACT

In the present investigation inhaled isoniazid microparticles (IM) and isoniazid polymeric

microparticles (INH-PM) using poly-ɛ -caprolactone polymer were prepared through spray

drying techniques. The purpose of this investigation is to evaluate lung deposition of IM and

INH-PM through cascade impaction study. The drug release of IM and INH-PM was studies

using simulated lung fluids at pH 7.4 representing the interstitial site and at pH 4.5

representing phagocomal site after alveolar macrophage uptake. The kinetic models had also

been applied providing the drug release kinetics for IM and INH-PM in simulated lung fluids

at pH 7.4 and at pH 4.5. The results of the particles size and surface characteristics showed

the spherical shape and 1 - 5µ size of IM and INH-PM prepared using spray drying which can

be suitable for pulmonary drug delivery. The cascade impaction study with mass median

aerodynamic diameter ranging from 1.9 – 4 µ confirmed the inhaled characteristics of IM and

INH-PM with providing the deep lung deposition where tubercular bacilli reside. From in

vitro drug release studies done using simulated lung fluids and goodness of fit with kinetic

model applications, it can be concluded that the prepared poly-ɛ -caprolactone microparticles

of isoniazid provided the advantage of controlled release characteristics deep inside the lung

where tubercular bacilli reside and as suitable for pulmonary drug delivery it may help in

improving treatment of tuberculosis through direct administration to site of action.

Keywords: isoniazid; polymer; pulmonary; inhaled; tuberculosis; microparticles

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1. INTRODUCTION

Pulmonary route is widely employed to deliver drugs to the respiratory epithelium for the

treatment of local and systemic diseases. Drugs like antibiotics, antiviral agents, mucoactive

agents, corticosteroids, peptides, proteins and enzymes can be delivered through inhalation.

Pulmonary drug delivery may offers advantages like reduction in side effect as well as

enhanced therapeutic effect. Furthermore, the lungs can be targeted for delivery to specific

lung cells, such as alveolar macrophages1, for treatment of diseases such as tuberculosis

2.

However, the particles with aerodynamic diameter between 1 and 5 μ can only effectively

reach deep in the lungs to target alveolar macrophages. Devices used to deliver aerosolized

therapeutic agents are based on one of three platforms: pressurized metered-dose inhaler

(pMDI), nebulizer and dry powder systems3. Medication using dry powder inhalation can

result in high local levels of drug in epithelial lining fluid of the airways and lower

respiratory tract.

Tuberculosis (TB) is a chronic infectious disease. Lung is the primary site of infection. The

causative agent Mycobacterium tuberculosis (MTB) resides in Alveolar Macrophage (AM).

The conventional treatment of TB involves systemic delivery of antitubercular drugs (antiTB)

through oral route. The major disadvantage associated with oral treatment is undesirable side

effects and toxic effects due to high doses. Delivery of antiTB drugs directly to the primary

infection site through pulmonary route may help in reduction of side effects as well as toxic

effects and provide an advantage of dose reduction 4-9

. Particulate systems are considered as

foreign bodies and are phagocytised by macrophages, this natural immunological response

can be utilized for targeting drugs to macrophages through their entrapment into particulate

systems10

. Microparticles for delivery of antitubercular drug through pulmonary route should

provide optimum deposition to deep lungs where the alveolar macrophages reside. The work

has been reported on antiTB drugs delivery system through pulmonary route are:

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microspheres employing PLG 7,11

and PLGA6,4

, nanoparticles employing alginate 12

, solid

lipid nanoparticles13

, liposomes formulated using Egg PC-and Chol- based liposomes14

,

Lung-specific stealth liposomes 15

, dry powder ‘porous nanoparticle-aggregate particle’

(PNAP) 16

. AntiTB drugs are always to be used in combination to avoid the chances of drug

resistance17

. In any combination of antiTB drugs, isoniazid is always recommended as first

line agent as per WHO guidelines. Isoniazid can be delivered through pulmonary route either

as polymeric or non-polymeric micro/nano particles18,19

or pro-liposome formulation20

.

Microparticles can be prepared using a variety of techniques like emulsion-solvent

evaporation21

, lyophilisation, supercritical fluid technique; spray drying 22

. Isoniazid and

poly-ɛ -caprolactone microspheres and nanospheres have been investigated by emulsion

solvent evaporation and freeze drying using tween80 as surfactant and ethyl acetate as

organic solvent21

. As compared to freeze drying, spray drying is one of the most preferred

techniques as it offers several advantages like easy scalability, wide applicability, reliability

under production conditions, the reproducibility, and the possible control of particles size

with potential to provide application for development of particles for nasal and pulmonary

delivery23

. The reports are not available for inhaled spray dried polymeric and non-polymeric

microparticles of isoniazid with inclusion of in vitro drug release studies using simulated lung

fluids. So, in the present investigation isoniazid microparticles (IM) and isoniazid polymeric

microparticles (INH-PM) using poly-ɛ -caprolactone polymer were prepared through spray

drying techniques. The inhaled characteristics of prepared formulations are important to

deposit the particles to deep lung where the alveolar macrophage resides. The mass median

aerodynamic diameter (MMAD) and the fine particle fraction (FPF) are important aerosol

properties for effective deposition of particles to target site22,24

. Thus the challenge to

formulation is to design a powder mass which meets required aerosol properties or qualities

for deposition of particles to appropriate site within lungs. The purpose of this investigation is

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to evaluate lung deposition of IM and INH-PM through cascade impaction study. The drug

release of IM and INH-PM was studied using simulated lung fluids at pH 7.4 representing the

interstitial site and at pH 4.5 representing phagosomal site after alveolar macrophage uptake.

The kinetic models have also been applied providing the drug release kinetics for IM and

INH-PM in simulated lung fluids at pH 7.4 and at pH 4.5.

2. MATERIALS AND METHODS

2.1 Materials

Isoniazid is gifted by IPCA, India. Poly-caprolactone was purchased from Sigma, USA.

Methanol, dichloromethane (DCM), poly vinyl alcohol AR grade (PVA), sodium

taurocholate, lecithin, pepsin, sodium chloride, hydrochloric acid, sodium hydroxide,

magnesium chloride, disodium hydrogen phosphate, sodium sulphate, calcium chloride

dehydrate, sodium citrate dehydrate, citric acid, glycine, sodium tartrate dehydrate, sodium

lactate, sodium pyruvate and phosphate buffer saline pH 7.4 (PBS) were purchased from

Merck Chemicals, India. All the chemicals and ingredients utilized for studies were of AR

grade. Double distilled water was utilized throughout the study.

2.2 Preparation of Isoniazid Microparticles (IM) through Spray Drying Process

Isoniazid solution (5%w/v) in double distilled water was prepared and subjected to drying

using Spray Dryer with high efficiency cyclone separator (LU-222 advanced, Labultima,

Mumbai, India) under following conditions: inlet temperature 45ºC, aspiration 45 Nm3/hr,

feed rate of 1mL/min. The powder in high efficiency cyclone separator was collected,

weighed (AUX220, Shimadzu, Japan) and stored in light resistant container till further

analysis.

2.3 Preparation of Polymeric Microparticles of Isoniazid using Poly-ɛ -caprolactone

The polymeric microparticles of isoniazid were prepared by double emulsification method

and the microparticles were obtained using spray drying process. The weighed amount of

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polymer was dissolved completely in 5mL of DCM. Isoniazid (500mg) was dissolved in

double distilled water (3mL). The solution of INH was dispersed in polymer solution using

syringe with 23G needle to obtain w/o emulsion. This emulsion was again dispersed in

1.5%w/v PVA solution and homogenized for 1minute using high speed homogenizer at

10500rpm. The prepared w/o/w emulsion was spray dried to prepare INH-PM at following

conditions: Inlet temperature 120C, feed rate 2mL/min and aspiration rate 60Nm3/hr. The

microparticles were collected from high efficiency cyclone separator, weighed and stored at

room temperature and in air tight light resistant contained till further analysis.

2.4 22 Full Factorial Designs

22 full factorial designs was adopted to develop the optimized formulation. Using this design

one can determine the effect of independent variables - drug: polymer ratio (X1) and volume

of PVA (X2)on dependent variables - the particle size (µm) (Y1), drug entrapment (%w/w)

(Y2) and yield (%w/w) (Y3). As per the 22 factorial Design, total four batches of

microparticles were formulated having independent and dependent variables as shown in the

Table 1. Experimental trials of four batches were performed in triplicate using all possible

combinations as per the design layout shown in Table 2. The results obtained from the

experiment were statistically analyzed for response variables using Design Expert Version

8.0.7.1. Each experimental response can be represented by the following polynomial equation

(1) of the response surface.

Y = b0 + b1 X1 + b2 X2 + b12X1X2 [equation (1)]

Where, Y is the measured response; X1 & X2 are the experimental factors; b0 is the intercept;

b1, b2 & b12 are the coefficients of respective variables and their interaction terms.

2.5 Evaluation of Microparticles

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The pure INH, IM and optimized formulation of polymeric microparticles of isoniazid (INH-

PM) were evaluated for particle size, surface characteristics, entrapment efficiency, in vitro

aerosol performance and in vitro drug release.

2.5.1 Particle Size Analysis

The particle size analysis for 500 particles was performed using high resolution microscope

(Axio Lab.A1, Carl Zeiss MicroImaging, GmbH and Gottingen). The images were taken

using 3megapixel camera coupled with high resolution microscope. The image analysis was

carried out using the software [Biovis Image Plus (P+) V4.56 (Expert Vision Labs Pvt. Ltd.,

Mumbai, India)] and particle size as well as perimeter ratio or roundness of particles as

morphological character were determined.

2.5.2 Surface Characteristics through Scanning Electron Microscope

The surface characteristics were studied by scanning electron microscope (Model JSM-5610

LV, JEOL, Japan) from 100x to 2000x magnifications. The powder sample was sprinkled

onto the carbon tape affixed on aluminium stubs. The aluminium stubs were placed in the

vacuum chamber and observed for morphological characterization.

2.5.3 Entrapment Efficiency

The entrapment of drug in prepared INH-PM was determined. The particles equivalent to

2mg of INH was dispersed in double distilled water. The solution was sonicated for 5minutes

and then filtered through 0.5µ membrane filter. The filtrate was analyzed at 263nm for

estimation of INH.

2.5.4 Cascade Impaction Study

The in vitro aerosol performance of IM and INH-PM were evaluated using cascade impactor.

A turbuhaler type inhalation device was used for introduction of samples. A hydroxyl propyl

methylcellulose (HPMC) capsule (size no. 2) filled with equivalent to 5 mg of each samples

was used. The inhalation test was performed at an inhalation rate of 60 L/min for 4 s. Before

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inhalation study, the flow rate was calibrated several times with the inhaler containing an

empty HPMC capsule size no. 2. After aspiration, the particles that were deposited on the

capsule, device, throat, pre-separator, and each stage of cascade impactor were rinsed off

with double distilled water. The INH solution for each stage was diluted with double distilled

water to 10 mL. The amount of INH in the solutions was estimated at 263nm using uv-

vissible spectrophotometer (Model 1800 uv-vissible spectrophotometer, Shimadzu, Japan).

The fine particle fraction, which is the total percentage deposition at stages 3–6 of the

cascade impactor, was used to evaluate the aerosol performance. A higher fine particle

fraction deposition is thought to indicate a higher in vitro aerosol performance.

2.5.5 In vitro Drug Release

A USP Type II tablet dissolution test apparatus (TDT-08L, Electrolab, Mumbai, INDIA) was

used for in vitro drug release. The stirring speed was kept 150rpm25

. A dialysis membrane (

Himedia, molecular weight cut-off > 900 kDa) was cut into equal pieces of about 6

cm×2.5cm and pre-treated as suggested by the manufacturer. Equivalent to 5mg each of INH,

IM and INH-PM were dispersed in 1ml of PBS pH 7.4 and filled in the pre-treated dialysis

membrane and sealed with clips. The pouch thus formed was attached to the paddles of the

apparatus using rubber bands wound over the clips. Nine-hundred millilitres of Gamble’s

solution pH 7.4 and Alveolar Lung Fluid pH 4.5 (ALF) were used for the study. The

composition of Gamble’s solution and ALF are shown in Table 3. Gamble’s solution

represents the interstitial fluid deep within the lung while ALF is analogous to the fluid with

which inhaled particles would come in contact after phagocytosis by alveolar macrophages26

.

Samples of 5ml were drawn and 5ml fresh medium was replaced at each time interval 5, 10,

15, 30, 45, 60, 90, 120 min, 12 hr and 24hr. The drug was estimated at 263 nm using uv-

vissible spectrophotometer (Model 1800 uv-vissible spectrophotometer, Shimadzu, Japan).

All the samples were tested in triplicates. The drug release profiles were evaluated for drug

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release kinetics and compared by regression value of the kinetic model using functions of

Microsoft Office Excel 2007 27

.

3. RESULT AND DISCUSSION

3.1 Effect of Formulation Variables on Response Variable

A polynomial model was individually fitted to all the response variables. In order to make

prediction, the responses were measured and polynomial equations were derived by ANOVA

and regression analysis (Table 4). The polynomial equations obtained are as follow:

Y1 (Particle Size)= 8.455 + 2.728X1 - 3.505X2 - 1.745X12 (R2

= 0.9958; p < 0.0001)

[equation (2)]

Y2 (Drug Entrapment)= 76.083 + 7.25X1 + 0.5833X2 + 5.083X12 (R2 = 0.9366; p <

0.0001) [equation (3)]

Y3 (Yield)= 22.33 -7.4 X1 + 11.68X2 – 8.6 X12 (R2 = 0.982; p <0.0001) [equation (4)]

Where, Y1, Y2 and Y3 are dependent variable. The main effect X1 (drug:polymer ratio) and

X2 (volume of PVA) represent the average results of changing one factor at a time from its

low to high value. The interaction (X12) shows how the response changes when two factors

are changed simultaneously. Co-efficient with one factor represents the effect of that

particular factor on responses. Positive sign in front of the terms indicates synergistic effect

while negative sign indicates antagonistic effect upon the responses.

For response Y1 (Particle Size (µ)), the effect of X1 and X2 was found significant as p was

less than 0.05. From the polynomial equation (2), it can be qualitatively concluded that X1

had synergistic effect on the response of Y1, which indicated that X1 (drug : polymer ratio)

was a more important parameter for particle size. While the antagonistic effect was found of

X2 (volume of PVA). It can be inferred that to decrease the particle size the drug : polymer

ration should be kept minimum and the volume of PVA should be kept at maximum level. As

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drug: polymer ratio increases, the particle size increases and as volume of PVA increases, the

particle size decreases28

.

For response Y2 (% Drug Entrapment), the main effect X1 was found significant as p was less

than 0.05. The X2 was found insignificant as p was more than 0.05. Further, from the

coefficient values, all formulations variable had synergistic effect over the response Y2

[equation (2) and Table 4]. As drug: polymer ratio decreases, the drug entrapment decreases

because it increases the viscosity of polymer phase which prevents the drug diffusion into the

droplet28

.

For response Y3 (yield), the main effect X1 and X2 was found significant as p was less than

0.05. The X1 had antagonistic effect while X2 had synergistic effect over yield [equation (3)

and Table 4]. It can be inferred that as drug: polymer ratio decreases and as volume of PVA

increases, the yield increases.

The validation of polynomial equation model was performed through check point analysis

using Design Expert Version 8.0.7.1. The check point batch (C1) was prepared and observed

responses were compared to that of predicted responses. The results showed that there was no

significant difference (p > 0.05) between predicted and experimentally performed responses -

particle size, drug entrapment and yield during check point analysis (Table 5). It can be

inferred that the polynomial equation model obtained were found to be valid for responses.

The optimization of formulation was carried out using desirability function on the Design

Expert software. The result showed that the desirability factor for optimized formulation

obtained was 0.849. The optimized formulation (INH-PM) was prepared and observed

responses were compared to that of predicted responses. The results (Table 5) showed that

there was no significant difference (p>0.05) found between the observed responses and

predicted responses for optimized formulation. Therefore, the optimized formulation (Table

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5) was utilized for further evaluations considering as optimum formulation of polymeric

particles of isoniazid (INH-PM).

3.2 Particle Size Analysis

Particle size for INH, IM and INH-PM were found to be 20.6 µ, 4.3 µ and 3.8 µ, respectively.

The Figure 1 shows the photo micrographs obtained using high resolution microscope for

INH, IM and INH-PM respectively. The morphological evaluation suggested that the IM and

INH-PM prepared using spray dryer were of spherical in shape with shape factor 0.82 and

0.91, respectively. This indicated that being spherical in shape and with size less than 5 µ,

they may phagocytose by AM 29-31

. The size less than 5 µ also depicted that the particles were

also suitable for delivery through pulmonary route.

3.3 Entrapment Efficiency

The percentage drug entrapment of INH-PM was found to be 64.83%w/w. The yield ~drug

content obtained for IM was 60%w/w.

3.4 Surface Characteristics through SEM

Figure 2 shows the SEM photomicrographs of INH, IM and INH-PM respectively. INH was

found of rod and columnar shape crystalline particles. The particles of INH-PM and IM were

found with smooth and spherical in shape. From SEM photomicrographs it can be depicted

that the polymeric microparticles of smooth surface characteristics spheres were produced

using spray drying. SEM photomicrographs of IM depicted the agglomeration of particles

which were found to be spherical in shape. It can be inferred that the spherical particles may

likely to be favourably taken up by AM by phagocytosis30

.

3.5 Cascade Impaction Study

Figure 3(A) and 3(B) show the results of in vitro aerosol performance of IM and INH-PM.

Figure 3(C) and 3(D) show the plot of cumulative percentage deposition of emitted dose vs

cut-off diameter of stages of cascade impactor for IM and INH-PM respectively. The mass

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median aerodynamic diameter (MMAD) calculated from the plot were 1.9 and 3 for IM and

INH-PM respectively. The fine particle fraction (%FPF<4µm) and emitted dose (%ED) of the

IM was 54.85% and 57.17% while %FPF<4µm and %ED of INH-PM was 51.83% and 26.9%,

respecively. The lower MMAD and high FPF may be obtained due to spray drying process

used for preparation of particles with lower density32,33

. The results showed that IM and INH-

PM were found to be compatible with dry powder for inhalation, as evident from MMAD.

The %FPF<4 depicted that the larger dose was available to the lungs after inhalation. Though

the deposition was found over the throat (Figure 3(A) and (B)) which may be due to

agglomeration of particles (Figure 2), the favourable MMAD and FPF depicted the retention

of drug over stages 4-5-6 suggested the deep lung deposition can be possible. These may help

to target the alveolar macrophage cells where the tubercular bacilli reside34

.

3.6 In vitro drug release study using simulated lung fluids

Figure 4(A), 4(B) and 4(C) show the drug release profile of INH-PM, IM and INH in

Gamble’s solution pH 7.4. For IM and INH-PM the 50% drug release was found at about 300

minutes and 10 minutes respectively in Gamble’s solution pH 7.4. The release of INH and IM

was significantly higher (p < 0.05) as compared to that of INH-PM. Figure 4(D), 4(E) and

4(F) show the drug release profile of INH, IM and INH-PM in Alveolar Lung Fluid pH4.5.

For INH-PM and IM the 50% drug release was found at 90 minutes and 15 minutes

respectively in ALF. The release profile for INH was found irregular in Alveolar Lung Fluid

pH 4.5. From the in vitro drug release study, it can be inferred that INH-PM showed higher

drug release in Alveolar Lung Fluid pH 4.5 representing the phagosomal site as compared to

drug release in Gamble’s solution pH 7.4 representing the cytosol. IM showed significantly

higher drug release in Gamble’s solution pH 7.4 as compared to that in Alveolar Lung Fluid

pH 4.5. Therefore, it can be depicted that INH-PM provided the drug release inside AM at

phagosomal pH where the tubercular bacilli resides. Further for polymeric particles the drug

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release is dependent on polymer degradation. The drug release kinetic models has been

applied to the drug release profile27

. The results of kinetic model and goodness of fit showed

that IM followed zero order drug release kinetic while that of INH-PM followed HIGUCHI

model of diffusion in both Gamble’s solution and in ALF (Table 6). It can be concluded that

INH-PM releases drug inside AM at late phagosomal phase of phagocytosis in controlled

manner by erosion mechanism which may help in reduction in dosing frequency of drug35

.

3. CONCLUSION

From the results of experimental design it can be concluded that optimized formulation can

be obtained from low number of experimental trial with having significant effect over

dependant / response parameters. From the particles size and surface characteristics, it can be

concluded that the spherical particles of IM and INH-PM were of 1 - 5µ can be prepared

using spray drying which can be suitable for pulmonary drug delivery. The cascade impaction

study confirmed the inhaled characteristics of IM and INH-PM with providing the deep lung

deposition where tubercular bacilli reside. From in vitro drug release studies using simulated

lung fluids, it can be concluded that the prepared poly-ɛ -caprolactone microparticles of

isoniazid provided the advantage of controlled release characteristics deep inside the lung

where tubercular bacilli reside and with suitable for pulmonary drug delivery it may help in

improving treatment of tuberculosis. In future prospect, the in vivo experimentation should be

required to study the target potential of prepared microparticles of isoniazid.

DECLARATION OF INTEREST

There is no declaration of interest

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Figure 1: Photomicrograph of (A) INH, (B) IM and (C) INH-PM at 40x using high resolution

microscope and 3MP camera

Figure 2: SEM photomicrograph of (A) INH, (B) IM and (C) INH-PM

Figure 3: (A) Percent mass deposited of drug over the cascade stages from total emission of

INH-PM, (B) Percent mass deposited of drug over the cascade stages from total emission of

IM, (C) The plot of cummulative percent drug deposition against the cut-off diameter for

INH-PM and (D) The plot of cummulative percent drug deposition against the cut-off

diameter for IM

Figure 4: (A)Drug Release Profile of INH-PM in Gamble’s Solution, (B) Drug Release

Profile of IM in Gamble’s Solution, (C) Drug Release Profile of INH in Gamble’s Solution

(D)Drug Release Profile of INH-PM in ALF, (E)Drug Release Profile of IM in ALF and (F)

Drug Release Profile of INH in ALF

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Table 1: Independent and Dependent variables of 22 Full Factorial Design

Independent Variables Levels

Low (-1) High (+1)

Drug: Polymer ratio X1 1:1 1:5

Volume of PVA(mL)X2 25 75

Dependent Variables Particle size(µ) (Y1), Drug Entrapment(%w/w) ( Y2) and Yield(%w/w) (Y3)

Table 2: 22 Full Factorial Design Matrix with Response Variables

X1:Drug to

Polymer ratio

X2:Volume of

PVA(mL)

Y1:Particle Size

(µ)

Y2:Drug Entrapment

(%w/w)

Y3:Yield

(%w/w)

-1 -1 7.16 70 9.3

-1 -1 7.5 75 10

-1 -1 7.8 75 9

1 -1 16 80 12

1 -1 16.5 75 11

1 -1 16.8 78 12.5

-1 1 4.1 60 45

-1 1 4 65 50

-1 1 3.8 68 55

1 1 5.5 89 16

1 1 5.8 90 20

1 1 6.5 88 18

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Table 3: Composition of Simulated Lung Fluids -Gamble’ Solution and Alveolar Lung Fluid

for In Vitro Drug Release Study

Composition of Simulated Lung Fluid (g/L)

Materials Gamble’s Solution pH 7.4 Alveolar Lung Fluid pH 4.5

Magnesium Chloride 0.095 0.050

Sodium Chloride 6.019 3.21

Potassium Chloride 0.298 -

Disodium Hydrogen Phosphate 0.126 0.071

Sodium Sulphate 0.063 0.039

Calcium Chloride Dihydrate 0.368 0.128

Sodium Acetate 0.574 -

Sodium Hydrogen Carbonate 2.604 -

Sodium Citrate Dihydrate 0.097 0.077

Sodium Hydroxide - 6.000

Citric Acid - 20.8

Glycin - 0.059

Sodium Tartrate Dihydrate - 0.090

Sodium Lactate - 0.085

Sodium Pyruvate - 0.086

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Table 4: Results of ANOVA and Regression Statistics for 22 Full Factorial Design

ANOVA of 22 Full Factorial Design

Y1(Particle Size) Y2 (Drug Entrapment) Y3(Yield)

R Square 0.995973 0.936566 0.981589

Adjusted R Square 0.994462 0.912778 0.974685

Standard Error 0.371663 2.828427 2.731605

Observation 12 12 12

Regression Statistics for responses of 22 Full Factorial Design

Responses

Co-efficient

Intercept b1 b2 b12

Y1 8.455 2.738 -3.705 -1.745

P <0.0001(Model significance) <0.0001 <0.0001 <0.0001

Y2 76.08333 7.25 0.58333 5.08333

p <0.0001(Model significance) <0.0001 0.4953 0.0003

Y3 22.31667 -7.4 11.6833 -8.6

p <0.0001(Model significance) <0.0001 <0.0001 <0.0001

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Table 5: Results of Responses of Check Point Batch and Predicted Optimized Formulation

Check point batch

(C1)

Optimized batch

(INH-PM)

Drug: Polymer Ratio X1 0.5 1:1

Volume of PVA (mL) X2 0.75 75

Predicted Response of Particle Size (µ) Y1 6.53 4.05

Observed Response of Particle Size (µ) Y1’

6.25* 3.8

*

Predicted Response of Drug Entrapment (%w/w) Y2 82.05 64.5

Observed Response of Drug Entrapment (%w/w)Y2’

82± 64.83

±

Predicted Response of yield (%w/w)Y3 24.15 48.86

Observed Response of yield (%w/w)Y3’

25# 50.3

#

*indicates no significant difference (p > 0.05 ) between observed particle size (µ) and predicted particle size (µ) of C1 and INH-PM.

± indicates no significant difference (p > 0.05 ) between observed drug entrapment (%w/w) and predicted drug entrapment (%w/w) of C1

and INH-PM.

# indicates no significant difference (p > 0.05 ) between observed yield (%w/w) and predicted yield (%w/w) of C1 and INH-PM.

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Table 6: Regression for Drug Release Kinetic Model Application to In vitro Drug Release

Profile of INH, IM and INH-PM in Gamble’s Solution and in Alveolar Lung Fluid

(A) Regression (r2) for Drug Release Kinetic Model Application for Gamble’s solution pH 7.4

Zero order First order Hixson Crowel Higuchi Krosmeyer-Peppas

INH 0.956 0.9673 0.933 0.916 0.9135

IM 0.9774 0.3292 0.837 0.833 0.8055

INH-PM 0.8705 0.5898 0.8592 0.9815 0.8793

(B) Regression (r2) for Drug Release Kinetic Model Application for Alveolar Lung Fluid pH 4.5

Zero order First order Hixson Crowel Higuchi Krosmeyer-Peppas

INH 0.9459 0.8548 0.9333 0.9545 0.9653

IM 0.9574 0.8408 0.9519 0.8355 0.901

INH-PM 0.8424 0.7825 0.8575 0.9688 0.8912

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Fig 1

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Fig 2

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Fig 3

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Fig 4

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Graphical abstract

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HIGHLIGHTS FOR REVIEW

Spray dried poly-ε-caprolactone microparticles of isoniazid provided the advantage of

controlled release characteristics deep inside the lung where tubercular bacilli reside and due

to inhaled characteristics suitable for pulmonary drug delivery it may help in improving

treatment of tuberculosis through direct administration to site of action.