Feasibility of haloperidol-anchored albumin nanoparticles loaded with doxorubicin as dry powder inhaler for pulmonary delivery.

http://informahealthcare.com/phd ISSN: 1083-7450 (print), 1097-9867 (electronic) Pharm Dev Technol, Early Online: 1–13 ! 2013 Informa Healthcare USA, Inc. DOI: 10.3109/10837450.2013.852576

RESEARCH ARTICLE

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Feasibility of haloperidol-anchored albumin nanoparticles loaded with doxorubicin as dry powder inhaler for pulmonary delivery Jaleh Varshosaz, Farshid Hassanzadeh, Amin Mardani, and Mahboubeh Rostami Department of Pharmaceutics, School of Pharmacy, Isfahan University of Medical Sciences, Isfahan, Islamic Republic of Iran

Abstract

Keywords

Haloperidol (Hal) is a ligand that can target sigma 2 receptors over-expressed in non-small cell lung cancer. Hal targeted nanoparticles of bovine serum albumin (BSA) were prepared for pulmonary delivery of doxorubicin (DOX). The conjugation was confirmed by Fourier transform infrared spectroscopy (FTIR) and 1H nuclear magnetic resonance (1H NMR) spectroscopic methods. Nanoparticles were prepared by desolvation method from BSA–Hal and were loaded with DOX. They were characterized for their morphology, particle size, zeta potential, drug loading and release efficiency. The optimized nanoparticles were spray-dried using trehalose, L-leucin and mannitol as dry powder inhaler (DPI) in different inlet temperatures between 80 and 120 C. The obtained nanocomposites were characterized for their aerodynamic diameter, specific surface area (cm2/g) and fine particle fraction (FPF) by a Cascade Impactor device. The optimized nanoparticles showed particle size of 218 nm, zeta potential of 25.4 mV, drug entrapment efficiency of 89% and release efficiency of 56% until 2 h. After spray drying of these nanoparticles, the best results were obtained from mannitol with an inlet temperature of 80 C which produced a mean aerodynamic diameter of 4.58 mm, FPF of 66% and specific surface area of 6302.99 cm2/g. The obtained results suggest that the designed DPI could be a suitable inhaler for targeted delivery of DOX in pulmonary delivery.

Albumin nanoparticles, doxorubicin, DPI, haloperidol, sigma 2 receptors

Introduction The lungs may be involved by primary or secondary tumors. The most common cause of deaths due to cancer is the lung cancer in men and the second cause in women. Worldwide 1.3 million deaths are reported due to the lung cancer and 300 000 new cases are diagnosed in Europe each year. There are two types of lung cancers: small cell and non-small cell lung cancer (NSCLC) that 80% of them are from the second type. In 70% of cases, the cancer is diagnosed too late that it has metastasized1. Surgery could be applied just in a few percent of the patients suffering from lung cancer. Thus the chemotherapeutic agents should be used in primary and secondary cancer1. When the drug is administered systemically the concentration of drug reached to the target organ may be so low that is not efficient for therapy2. It is proved that the concentration of drug in tumor is an important factor in drug efficacy3. There are lots of advantages in pulmonary drug delivery consisting: vast surface area, fairly low activity of enzymes, enhancing drug concentration at the site of action in pulmonary diseases, the extremely thin walls of the alveoli in the deep lungs provides rapidly systemic absorption of drugs which in turn enhances the bioavailability of drugs and prevention of the entrance of pathologic particles into the body4. Nebulization of chemotherapeutic agents has been recommended for the treatment of lung cancer. In this method, the Address for correspondence: Jaleh Varshosaz, Department of Pharmaceutics, School of Pharmacy, Isfahan University of Medical Sciences, Islamic Republic of Iran. E-mail: [email protected]

History Received 8 August 2013 Revised 29 September 2013 Accepted 30 September 2013 Published online 8 November 2013

anticancer drug is directly nebulized through an aerosol into the lungs. This method of drug administration not only reduces the unwanted side effects of the drug in non-target organs but also concentrates the drug at its site of action. Pulmonary delivery of 5-fluorouracil (5-FU) via inhalation is an example of the safe method of delivery of this antitumor drug with its accumulation in the trachea, bronchi and regional lymph nodes5. Sigma receptors are one of the marker receptors present in both normal and malignant cells like pituitary, liver, kidneys, lungs, gonads and ovaries. Different organs over-expressing these receptors may include nervous, immune and endocrine systems. In some malignancies they may be present as much as 105/cell, like in melanoma, NSCLC, breast and prostate cancer and neuroblastoma cell lines. Sigma ligands, i.e. haloperidol (Hal) and various other neuroleptics attached to drug carriers like liposomes can bind to these receptors with high affinity and inhibit malignant cells growth6. An anisamide derivatized ligand has been attached to liposomes for targeted delivery of drugs to prostate cancer cells that over-express sigma receptors7. Pulmonary drug delivery has benefits such as: fast effectiveness due to fast dissolution and diffusion of the drug, enhanced attachment of nanoparticles to pulmonary mucous tissue and decreased phagocytic clearance of nanoparticles compared to particles with micron size8. The best particle size for pulmonary drug delivery is 1–5 mm. It is expected that a large fraction of the inhaled dose will be exhaled and little particle deposition will take place in the lungs9. The objective of the present work was formulating a dry powder inhaler (DPI) formulation for targeted pulmonary delivery of a chemotherapeutic agent, i.e. doxorubicin (DOX) in the lungs for treatment of NSCLC to reduce its systemic

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side effects while concentrating it in the site of its action. Endocytic uptake by pulmonary alveolar macrophages constitutes the major clearance mechanism in the alveolar regions of the lung. For this purpose nanoparticles are advantageous and can escape endocytic clearance however, they will be exhaled during normal tidal breathing. Therefore, to deposit them in the lower respiratory tract providing a huge surface area due to the presence of alveoli and well perfusion with blood capillaries, bovine serum albumin (BSA) nanoparticles conjugated to Hal as a ligand for targeting sigma 2 receptors over-expressed in this disease were formulated. After optimization they were applied in production of nanocomposites as a DPI dosage form to minimize their exhaling while maximize their effect due to attaching to the malignant cells and preventing their endocytosis.

Pharm Dev Technol, Early Online: 1–13

Table 1. Composition of different formulations investigated in preparation of doxorubicin loaded in albumin–haloperidol (BSA–Hal) nanoparticles using a hybrid design. Formulation code E4G0.75B15 E2.6G0.75B11.5 E4G0.75B7.9 E3G0.3B18.5 E4G1.39B11.5 E5.4G0.75B11.5 E4G0.75B22.1 E3G1.2B18.5 E4G0.1B11.5 E5G0.3B18.5 E5G1.2B18.5

Et (ml)

Glu (ml)

BSA–Hal (mg)

4.0 2.6 4.0 3.0 4.0 5.4 4.0 3.0 4.0 5.0 5.0

0.75 0.75 0.75 0.3 1.4 0.75 0.75 1.2 0.1 0.3 1.2

15.0 11.5 7.9 18.5 11.5 11.5 22.1 18.5 11.5 18.5 18.5

Materials and methods Materials BSA was purchased from Merck Chemical Company (Darmstadt, Germany). Hal, toluene, dichloromethane, 4-dimethylaminopyridine (DMAP), glutaraldehyde, mannitol, trehalose, N-hydroxysuccinimide (NHS), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and succinic anhydride were from Sigma (St. Louis, MO). L-Leucin was purchased from Roth (Monroe, WI) and DOX from Hangzhou ICH Biopharm Co., Ltd. (Zhejiang, China). All other chemicals and solvents were from analytical grade. Conjugation of BSA to Hal (BSA–Hal) Synthesis of Hal hemisuccinate (Hal-Su) Two grams of Hal (0.0053 mol), 2.66 g of succinic anhydride (0.0266 mol) and 0.065 g of DMAP (5.32 104 mol) were dissolved in 20 ml of toluene and stirred for 24 h at 80 C, then the reaction mixture was cooled to room temperature, and evaporated to dryness under reduced pressure. The mixture was diluted in dichloromethane (30 ml) and washed with 0.5 N HCl (3 50 ml). The organic layer was dried over MgSO4 and filtered. The solvent was finally evaporated under reduced pressure to afford crude Hal-Su, then this residue was purified by crystallization from methanol:water (3:1). The yield of the product was 85% and its melting point (mp) was 290.5 C. The structure of the purified product was evaluated by Fourier transform infrared spectroscopy (FTIR) spectroscopy and 1H nuclear magnetic resonance (1H NMR) analysis. Conjugation of BSA to (Hal-Su) At first, 500 mg of BSA was dissolved in 5 ml of water. Then 100 mg of Hal-Su (0.2101 mmol) was dissolved in 5 ml of deionized water in the presence of 47 mg EDC (0.3 mmol) and 300 mg N-hydroxy succinimide (NHS) (2.6067 mmol) at 50 C and the solution was added to BSA solution. The mixture was stirred for 48 h. The resulting product was dialyzed (MembraCelÕ , Viskase, Darien, IL, Mw cutoff 12 kDa) against deionized water for 24 h. Then, the purified product was identified using FTIR spectroscopy after freeze drying. SDS-PAGE BSA samples were subjected to sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) analysis using precast gradient gels (12% Tris-HCl/glycine, Bio-Rad, Hercules, CA). Samples were diluted 3:1 in Laemmli sample buffer (62.5 mM Tris-HCl, pH 6.8, 25% glycerol, 2% SDS, 0.01% bromophenol blue), with 8% b-mercaptoethanol, and boiled for

5 min prior to loading. 5 ml of the sample was used. Gels were electrophoresed for 2 h at 100 V and then stained with Coomassie blue to visualize the protein bands. Preparation of nanoparticles of BSA–Hal The nanoparticles were prepared by desolvation method. For this purpose different amounts of BSA–Hal (according to Table 1) were dissolved in 3 ml of pure water and the solution was filtered by 0.22 m cellulose acetate filter. Then 4 mg of DOX was dissolved in this solution and mixed for 15 min. The pH of the solution was adjusted to 7.5–9. Then 3–5 ml of ethanol 96% (Et) (according to Table 1) was added with a rate of 1 ml/min and the mixture was stirred on a magnetic stirrer with the rate of 650 rpm. After desolvation of protein, the glutaraldehyde 8% solution was added for cross-linking of protein with different concentrations (according to Table 1) between 0.3 and 1.2 ml and stirred for 15 min in 25 C. Experimental design To evaluate the effect of processing variables on the particle size, zeta potential, encapsulation efficiency (EE%) and drug release efficiency (RE%) and screening the most effective ones a response surface statistical designing model based on the hybrid design was used. This is a design with minimum experiments for studying three variable factors each in five levels. This rotatable or nearly rotatable design is better than a small central composite design but is still highly sensitive to outliers or missing data. Three different variables including ethanol volume (as desolvating agent), glutaraldehyde volume (as the cross-linking agent of BSA) and the weight of BSA were studied each in five levels. The maximum and minimum concentration of each variable was determined in a preliminary study and then was studied in the design. An overview of the formulations proposed by Design Expert software (version 7.1, US) by a hybrid design is presented in Table 1. A run involved the corresponding combination of levels to which the factors in the experiment were set. All experiments were done in triplicate. The studied responses of nanoparticles included: particle size, zeta potential, drug loading efficiency and drug RE. The effects of the proposed experiments on the responses were then analyzed by the Design Expert software to obtain independently the main effects of these factors, followed by the analysis of variance (ANOVA) to determine which factors had statistically significant effect on the studied response. The optimum conditions were determined by the optimization method to yield a heightened performance.

Haloperidol-targeted albumin nanoparticles

DOI: 10.3109/10837450.2013.852576

Table 2. The DPI formulations prepared by spray drying technique containing BSA–Hal nanoparticles loaded with doxorubicin.

Particle size and zeta potential measurements The mean particle size and zeta potential of BSA–Hal nanoparticles were measured by photon correlation spectroscopy (PCS) at a fixed angle of 90 (Zetasizer, ZEN 3600, Malvern Instrumental, Worcestershire, UK). Nano-dispersion was suitably diluted to measure mean particle size and poly-dispersity index.


Entrapment efficiency A 600 ml of BSA–Hal nano-dispersion was centrifuged (Microcentrifuge Sigma 30 k, UK) at 10 000 rpm for 5 min in Amikon microcentrifugation tubes (cutoff 10 000 Da, Ireland). The supernatant, containing the free drug was diluted 1:7 with deionized water and the UV absorbance of resulting solution was evaluated spectrophotometrically (UV-mini 1240, Shimadzu, Kyoto, Japan) at max ¼ 247 nm. The solution containing all components but the drug was used as the blank. The difference between the total and the free drug showed the amount of entrapped drug. The entrapment efficiency (EE) of DOX in BSA– Hal nanoparticles was calculated using Equation (1): EEð%Þ ¼

entrapped drug in nanoparticles 100 total amount of drug added

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Formulation code L80 L120 M80 M120 T80 T120

Carrier type

Inlet air flow temperature ( C)

L-Leucin L-Leucin

80 120 80 120 80 120

Mannitol Mannitol Trehalose Trehalose

Determination of particle size of nanocomposites The particle size (d) of nanocomposites was determined by a HELOS Particle Size Analyzer (Sympatec GmbH, H2047, Clausthal-Zellerfeld, Germany). Other parameters measured by this device contained: surface mean diameter (SMD, mm), surface to volume ratio (SV, m2/cm3), volume mean diameter (VMD, mm), specific surface area (Sm, cm2/g), density (, g/cm3) and shape factor (SF). Aerodynamic diameter of particles of spray-dried nanocomposites was measured by Equation (3): daer ¼ d1=2

ð1Þ

ð3Þ

Drug loading determination in nanocomposites Drug release studies One milliliter of each formulation of nanoparticle dispersions was transferred to a dialysis bag (Mw cutoff 12 000, Membra-Cel) and the bag was placed in phosphate buffer solution (pH 7.4) containing 2% Tween 20 while stirred at 37 1 C. Seven hundred microliters of samples were taken and DOX absorbance of each sample was measured at max ¼ 499.4 nm at specific time intervals until 70% of entrapped drug was released into the media. The parameter of RE within 120 min (RE120%) was used to compare the release profiles: Rt y dt 100 ð2Þ RE120 % ¼ 0 y100 t Optimization of the BSA–Hal nanoparticle formulations Data processing was done using Design expert software and the effects of each independent variable on the studied responses were determined. All responses were fitted in a linear model. The constraints of particle size was 100 Y1 633 nm with particle size targeted on minimum, for zeta potential it was 35 Y2 4.22 mV while it was desired to be in minimum of the obtained results, for loading efficiency the constraints were 84 Y3 91.75% with the goal set at the maximum and RE120% had constraints of 36.41 Y4 68.69% with desired target set at the maximum. Preparation of nanocomposites of DPI The optimized formulation of nanoparticles was used to prepare the nanocomposites of DPI by spray drying technique. For this purpose 880 mg of mannitol or trehalose or L-leucin was dissolved in 100 ml of deionized water and then 120 mg of the BSA–Hal nanoparticles dispersion was added to this solution and spry-dried (Lab Plant SD-05, UK). The inlet and outlet air flow temperature changed between 80–120 C and 51–93 C, respectively. The air flow rate was set at 70% (30 m3) and the pump flow rate was 5% (2.5 ml/min). Accordingly by changing two variables including carrier type of the powder and the inlet air flow temperature, six different formulations were designed according to Table 2.

Fifty milligrams of nanocomposites were dispersed in phosphate buffered solution (pH 7.4) and stirred for 5 h then 600 ml of nanocomposite dispersion was centrifuged (Microcentrifuge Sigma 30 k, UK) at 9838 g for 15 min in microcentrifugation tubes (cutoff 10 000 Da). The supernatant, containing the free drug was diluted 1:7 with deionized water and the UV absorbance of the resulting solution was evaluated spectrophotometrically (UV-mini 1240, Shimadzu, Kyoto, Japan) at max ¼ 499.4 nm. Scanning electron microscopy (SEM) Morphology of the nanoparticles and also the nanocomposites was characterized by SEM. The nanoparticles or nanocomposites were mounted on aluminum stubs, sputter-coated with a thin layer of Au/Pd and examined using an SEM (Hitachi F41100, Tokyo, Japan). Fine particle fraction (FPF) and emitted fraction (ED) FPF and emitted fraction (EF) of the powder were assessed using an Andersen Cascade Impactor (Copley, UK) through a DPI device (Aerolizer, Novartis). Fifteen milligrams of M80 nanocomposite DPI powder were packed in a capsule of size 2 and placed into the inhaler device. The capsule was punctured and the powder was delivered at a flow rate of 60 l/min for 5 s to simulate an inspiration. This process was repeated for four times. Particles were separated between the stages based on their aerodynamic diameter. The cutoff diameters were comprised between 1.1 and 4.7 mm as FPF, 51.1 mm as extra fine particle fraction (EPF) and 44.7 mm as coarse particle fraction (CPF). The amount of powder deposited on cascade stages, as well as the amount of retained by the filter were dissolved in deionized water and assayed spectrophotometrically at max ¼ 247 nm. The EF was defined as the percent of total loaded powder mass exiting the capsule.

Results Conjugation of BSA–Hal Synthesis of Hal hemi-succinate (Hal-Su) The conjugation of Hal with succinic anhydride was confirmed by analysis of FTIR (Figure 1) and 1H NMR (Figure 2) spectrums.

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Figure 1. FTIR spectra of (a) haloperidol, (b) haloperidol hemi-succinate (Hal-Su), (c) BSA and (d) BSA–Hal-Su.



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Figure 2. 1H NMR spectrums of (a) haloperidol and (b) haloperidol hemi-succinate (Hal-Su).

FTIR absorption spectrum feature (Figure 1) reveals the chemical bonding between Hal and succinic anhydride. Figure 1(a) and (b) shows the FTIR spectra of Hal and Hal-Su. In spectrum of Hal (Figure 1a), the aliphatic and aromatic C–H stretching band of Hal are seen around 2820–3130 cm1, the stretch vibration band at 1681.6 cm1 was assigned to the C¼O bands of Hal, furthermore, the absorption bands around 1221 cm1 can be assigned to stretching vibration mode of C– N and C–O. The formation of esteric band between Hal and succinic anhydride is evident in Figure 1(b), in this spectrum a broad stretching band at 3452.9 cm1 was assigned to the O–H group of Hal-Su carboxylic group and the absorption band at 1737.55 cm1 was attributed to formed esteric band. Also the presence of new absorption bands at 1686 cm1

(C¼O of carboxylic group), 1361.5 cm1 and 1409 cm1 (CH2–CH2 of succinate group) along with other reference bands of Hal in Figure 1(b) convinced us that the conjugation had occurred. Figure 2 shows the 1H NMR spectra of Hal and of Hal-Su. Presence of the reference picks of both Hal (7.97–8.00 ppm, m, 2, a), (7.25–7.29 ppm, m, 6, b, k, l), (4.74 ppm, s, 1, m), (2.89 ppm, t, j ¼ 7.60, 2, c), (2.41–2.49 ppm, m, 2, i), (2.19–2.27 ppm, m, 4, e, h), (1.73–1.76 ppm, m, 2, d), (1.53–1.60 ppm, m, 2, g), (1.34– 1.38 ppm, m, 2, f) and succinate subunits (2.41–2.51 ppm, m, 4, CH2CH2) in the spectrum of the Hal-Su, beside the disappearance of O–H of Hal in the product spectrum around 4.74 ppm, convinced us that the chemical bonding has occurred between two ingredients.

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increased (less negative values) with increasing of ethanol. Increasing the glutaraldehyde content of nanoparticles also increased the zeta potential of nanoparticles, i.e. less negative values were seen (Figure 5). This figure also indicates that increasing of BSA–Hal content in nanoparticles increased the negative zeta potential.


Drug loading efficiency The drug loading efficiency values of nanoparticles ranged between 84% and 91.75% (Table 3). Increasing of ethanol decreased the drug loading efficiency (Figure 6). Glutaraldehyde enhanced the loading efficiency of drug in the nanoparticles (Figure 6). As this figure indicates increasing in the BSA–Hal content increased the loading efficiency of drug in nanoparticles. Figure 3. SDS-PAGE (12% gradient gel) of (1) standard proteins (approximate molecular weights are indicated on the left), (2) BSA and (3). BSA–Hal. Protein bands were visualized by Coomassie blue staining. Table 3. Physicochemical properties of BSA–Hal nanoparticles loaded with doxorubicin.

Formulation code

Particle size (nm) SD

Zeta potential (mV)

Drug loading efficiency (%) SD

RE120 (%) SD

E4G0.75B15 E2.6G0.75B11.5 E4G0.75B7.9 E3G0.3B18.5 E4G1.39B11.5 E5.4G0.75B11.5 E4G0.75B22.1 E3G1.2B18.5 E4G0.1B11.5 E5G0.3B18.5 E5G1.2B18.5

344.0 21.5 248.3 11.5 633.2 11.5 164.5 4.2 144.2 6.8 100.1 14.7 341.6 19.4 246.3 9.4 143.4 6.2 222.5 8.8 387.3 9.4

6.2 28.6 14.0 17.5 21.0 8.7 35.0 4.2 21.0 23.0 5.7

90.2 0.2 91.2 0.4 85.7 1.9 89.2 1.4 90.0 0.2 87.5 0.4 91.5 0.5 90.2 0.0 91.7 0.4 84.0 0.7 85.7 0.7

45.6 4.3 38.5 1.3 42.1 1.2 47.8 2.2 36.4 0.7 65.4 2.1 42.5 1.3 50.2 0.8 64.8 0.1 68.7 2.4 64.9 1.1

Conjugation of BSA to (Hal-Su) In the FTIR spectrum of both BSA (Figure 1c) and BSA–Hal-Su (Figure 1d), all spectrum features of albumin can be seen in the spectrum of final product, beside a newly formed band around 1718 cm1 that can essentially result in a good conjugation between BSA and Hal-Su. SDS-PAGE SDS-PAGE test was used to evaluate the fragmentation of BSA and BSA–Hal, which showed the formation of several peptide species with different molecular weights between 35 and 45 kDa (Figure 3). The small spot at the 66.2 kDa indicates the presence of some intact BSA monomer. Particle size The size of nanoparticles changed between 100 and 663 nm in different studied formulations (Table 3). Figure 4 shows the effect of the studied variables on the particle size of BSA–Hal nanoparticles. As this figure indicates addition of ethanol had no significant effect on the size of nanoparticles (p40.05), while enhanced amounts of glutaraldehyde increased the particle size (p50.05). With increasing of BSA–Hal content of nanoparticles their particle size decreased (Figure 5) (p50.05).

In vitro drug release studies Figure 7 shows DOX release profiles versus time for each studied formulation of nanoparticles. The values of RE of DOX after 2 h (RE120%) of release test are summarized in Table 3. RE120% has direct relation with the rate of drug release. As this table shows the RE120% in different formulations changed between 36.4% and 68.7%. Figure 7 shows that nanoparticles of E4G0.1B11.5 with the lowest glutaraldehyde as cross-linking agent showed the fastest drug release pattern. However, formulation of E4G1.39B11.5 with maximum amount of glutaraldehyde could release just 80% of the loaded drug within about 6 h. Figure 8 also shows a reverse relationship between the RE120% and the glutaraldehyde content of nanoparticles. This figure also shows a direct relation between ethanol content and RE120%. Optimization of the BSA–Hal nanoparticle formulations Considering the results of the experiments done on the formulations of nanoparticles (Table 3), optimization was done using Design Expert software and E3.55G0.3B20 was suggested as the optimum formulation which showed a good particle size of 218 nm, zeta potential was 25.4 mV, an acceptable entrapment efficiency of 89% and relatively high RE of 56% until 2 h. Determination of particle size of nanocomposites According to Table 4, the nanocomposites of mannitol with 80 C inlet temperature (M80) had minimum diameter of 4.12 mm and consequently the highest specific surface area while L-leucin nanocomposites prepared at the same temperature (L80) had maximum diameter of 5.52 mm. Increase of temperature from 80 to 120 C caused almost no increase in diameter of trehalose nanocomposites from 4.18 to 4.23 mm and mannitol from 4.12 to 4.28 mm but decreased the diameter of L-leucin ones from 5.52 to 5.28 mm. The density of all obtained nanocomposite powders were 3.22 g/cm2 and their SF was 1.00 showing a symmetric shape (Table 4). SEM studies Figure 9 shows the morphology of different nanocomposites (a–f) and nanoparticles of Hal-BSA loaded with DOX (g). As this figure indicates the nanocomposites of mannitol are approximately round and gashed. FPF and EF

Zeta potential Zeta potential of nanoparticles changed between 4.22 and 35 mV (Table 3). Figure 5 shows that zeta potential values

Influence of the carrier on dried powders FPF, EF dose percent (EF%) and the aerodynamic diameter (daer) are summarized in Table 5.


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Figure 4. Effect of different studied variables on the particle size of BSA–Hal nanoparticles loaded with doxorubicin.

Figure 5. Effect of different studied variables on the zeta potential of BSA–Hal nanoparticles loaded with doxorubicin.

The results of Table 5 show that EF% of M80 was higher than other nanocomposites while these powders showed the lowest stickiness. EF% of T80 was the lowest among the studied powders. The CPF% of trehalose nanocomposites was more than mannitol.

Figure 10 shows distributions of DOX in each fragment obtained from Cascade Impactor. The throat fraction traps DOX more than other stages. Stages 3, 4 and 5 were considered as FPF% while stages 6, 7 and filter as EPF%. Table 5 and Figure 10

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Figure 6. Effect of different studied variables on the loading efficiency of doxorubicinin BSA–Hal nanoparticles loaded with doxorubicin.

120

Doxorubicin released %


8

E4 G0.75 B15 E2.6 G0.75 B11.5 E4 G0.75 B7.9 E3 G0.3 B18.5 E4 G1.39 B11.5 E5.4 G0.75 B11.5 E4 G0.75 B22.1 E3 G1.2 B18.5 E4 G0.1 B11.5 E5 G0.3 B18.5 E5 G1.2 B18.5

100 80 60 40 20 0

0

50

100

150

200

250

300

Time (min) Figure 7. Release profiles of doxorubicin HCl from different studied formulations of Hal–BSA nanoparticles.

show that there is significant difference between FPF of M80 with T80 and T120 (p50.05). However, M120 showed no significant difference with M80 (p40.05).

Discussion

the FTIR and 1H NMR results (Figures 1 and 2). Then Hal-Su was covalently linked to BSA (Figures 1 and 2) and the BSA–Hal was used as a nano carrier for DOX. Various other neuroleptics6 and anisamide7 have been also attached to liposomes with high affinity to these receptors.

Conjugation of BSA–Hal

SDS-PAGE

Successful conjugation of Hal with succinic anhydride as a ligand of sigma 2 receptors over-expressed in NSCLC was confirmed by

SDS-PAGE test was used to evaluate the fragmentation of BSA and BSA–Hal. The small spot at the 66.2 kDa indicates the



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Figure 8. Effect of different studied variables on the release efficiency of doxorubicin from BSA–Hal nanoparticles loaded with doxorubicin after 2 h of release test. Table 4. Characteristics of spray-dried nanocomposite DPI of doxorubicin. Formulation code L80 L120 M80 M120 T80 T120

d (mm)

SMD (mm)

SV (cm2/cm3)

VMD (mm)

Sm (cm2/g)

(g/cm3)

Shape factor

Drug content (%)

5.52 5.28 4.12 4.28 4.18 4.23

3.90 3.76 2.96 3.10 2.97 3.01

1.54 1.59 2.03 1.94 2.02 1.99

6.31 5.96 4.58 4.87 5.24 5.11

4780.32 4955.01 6302.99 6021.72 6276.56 6189.69

3.22 3.22 3.22 3.22 3.22 3.22

1.00 1.00 1.00 1.00 1.00 1.00

75 81 98 95 93 90

presence of some intact BSA monomer, which suggests the deficient hydrolysis of the peptide. BSA–Hal molecular weights ranged from approximately 85 to 100 kDa, possibly due to formation of Hal-Su with different rate of substitution that is attached to BSA (Figure 3). Particle size The size of nanoparticles changed between 100 and 663 nm in different studied formulations (Table 3). Figure 4 shows the effect of the studied variables on the particle size of BSA–Hal nanoparticles. As this figure indicates addition of ethanol had no significant effect on the size of nanoparticles (p40.05), while enhanced amounts of glutaraldehyde increased the particle size (p50.05). With increasing of BSA–Hal content of nanoparticles their particle size decreased (Figure 5) (p50.05). It seems that with increasing of BSA–Hal the effect of glutaraldehyde in crosslinking decreased as glutaraldehyde was not adequate to cross-link all the BSA–Hal and consequently the particle size of nanoparticles decreased. Rahimnejad et al.10 reported that increasing the BSA concentration, decreased the particle size of nanoparticles. They showed that beyond a certain BSA concentration, i.e. 30 mg/ ml, the particle size was unaffected with the concentration of BSA. A similar result was reported on fabrication of human serum albumin (HSA) nanoparticles using. Langer et al.11 showed that glutaraldehyde concentrations had no influence on particle size. The reason for the opposite effect of the glutaraldehyde on the particle size in our study with Rahimnejad et al.’s10 study maybe

due to the use of ethanolamine which was added to block the nonreacted aldehyde functional group and consequently the size of nanoparticles remained unchanged by addition of the glutaraldehyde but in our study ethanol was used which could not block these groups and therefore cross-linking of BSA–Hal produced a three dimensional network which could increase the particle size of nanoparticles as seen in Figure 4. Sebak et al.12 showed the influence of the cross-linking process on the size of albumin nanoparticles. Their results revealed that increasing the concentration of glutaraldehyde caused particle size growth of nanoparticles but had little or no effect on their polydispersity. Similar to our study which showed ethanol had no significant effect on the particle size of BSA–Hal nanoparticles, Rahimnejad et al.’s10 study also confirmed that the particle size of BSA nanoparticles was not affected by the ethanol concentration. Zeta potential Zeta potential of nanoparticles changed between 4.22 and 35 mV (Table 3). Figure 5 shows that zeta potential values increased (less negative values) with increasing of ethanol which may be due to covering of negative end group of BSA–Hal by ethanol and consequently decrease of negative surface charge of nanoparticles has increased the zeta potential values. Sebak et al.12 studied the effect of different pH on the electrical behavior of the HAS nanoparticles. They showed the higher the pH of the HSA solution the higher the surface charge of HSA nanoparticles. At higher pH than 7, the zeta potential of nanoparticles was


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Figure 9. Scanning electron micrographs of nanocomposites of (a) L80, (b) L120, (c) M80, (d) M120, (e) T80, (f) T120 and (g) the nanoparticles of HalBSA loaded with doxorubicin before spray drying.

Table 5. Aerodynamic characteristics of spray-dried nanocomposite DPI of doxorubicin by cascade impactor. Nanocomposite T80 T120 M80 M120

ED

CPF

FPF

EPF

daer

89.03 87.59 92.78 91.86

40.60 44.15 35.04 38.56

30 29.11 35.53 35.08

17.92 14.16 20.04 17.21

7.48 7.58 7.37 7.66

EF ¼ emitted fraction, CPF ¼ coarse particle fraction, FPF ¼ fine particle fraction, EPF ¼ extra fine particle fraction.

reduced so that at pH 8 it reduced of 47 mV. Higher pH led to steric effect among the HSA molecules and their repulsion. In our study also the nanoparticles of BSA–Hal were prepared at pH of 7.5–9 which produced the higher zeta potential and better stability of the particles. Wang et al.13 worked on the BSA nanoparticles for encapsulation of bone morphogenetic protein 2 (BMP-2). They reported the zeta potential of 26 mV for uncoated nanoparticles but after coating them with cationic polymers of poly(L-lysine) and poly(ethyleneimine) the zeta potential significantly increased to 16 mV which was due to neutralization of some carboxylic end groups of BSA by amine residues. The same


DOI: 10.3109/10837450.2013.852576


Mass deposited %

16

11

T80

14

T120

12

M80

10

M120

8 6 4 2 0

Cascade impactor collecon-plate stage Figure 10. Particle deposition patterns of spray-dried nanocomposites of trehalose and mannitol dried at 80 and 120 C containing Hal–BSA nanoparticles in each fragment of Cascade Impactor.

effect like ethanol on zeta potential was seen by increasing the glutaraldehyde concentration as seen in Figure 5. Increasing the glutaraldehyde content of nanoparticles increased the zeta potential of nanoparticles, i.e. less negative values were seen. Glutaraldehyde cross-links the surface of nanoparticles of BSA– Hal by reacting with amine end groups of BSA and therefore the absolute value of zeta potential of nanoparticles are decreased or less negative charge is observed. The reason may be interpreted so that; increasing of glutaraldehyde content increased the particle size of nanoparticles (Figure 4) which in turn caused to distribution of less charge on a bigger surface and therefore reduction of the charge density on the nanoparticles. Although in the study of Adami and Rice14 the changes of zeta potential of glutaraldehyde cross-linked peptide DNA condensates was very mild and changed between 34 and 41 mV, but in our study it increased from 23 to about 6 mV. Figure 5 indicates that increase of BSA–Hal content in nanoparticles increased the negative zeta potential due to its carboxylic group which induced a negative surface charge to nanoparticles in pH 812. Drug loading efficiency The drug loading efficiency values of nanoparticles ranged between 84% and 91.75% (Table 3). Increasing of ethanol decreased the drug loading efficiency (Figure 6). BSA in natural configuration has capacity for loading the drugs; but additional ethanol can deform the BSA and therefore decreases the capacity of albumin for drug loading. Through increase in the volume of ethanol, nanoparticles acquire more stability–rigidity hence mislay less drug during separation steps15. Glutaraldehyde enhanced the loading efficiency of drug in the nanoparticles (Figure 6) as it can cross-link the BSA–Hal which results in trapping the drug within the nanoparticles and the rising trend in loading efficiency is describable. Arnedo et al.16 used albumin nanoparticles as carriers for a phosphodiester oligonucleotide. They showed that increase in glutaraldehyde content up to 3 mg/mg of BSA, enhanced the incorporated and/or adsorbed oligonucleotide in nanoparticles. As Figure 6 indicates increase in

the BSA–Hal content increased the loading efficiency of drug in nanoparticles. BSA has potential to loading drug thus increase in BSA concentration increased the loading efficiency due to the increase in capacity to accommodate higher quantity of adsorbed drug on BSA–Hal molecules. Yang et al.17 also showed when drug ratio to the BSA content of nanoparticles increased the loading efficiency of 10-hydroxycamptothecin in nanoparticles decreased significantly. This finding is quite in accordance with our results which show at constant level of the applied drug, the higher the BSA–Hal content, the higher the drug loading efficiency. But when the drug content increased compared to BSA level, there was not enough space for the drug to be laid in the nanoparticles as reported by Yang et al.17 In vitro drug release studies Figure 7 shows DOX release profiles versus time for each studied formulation of nanoparticles. Drug release from four formulations was approximately 100% in less than 2 h with a considerable burst release however; in other formulations it was slower and more gradual due to changes in different studied variables such as ethanol, glutaraldehyde and BSA–Hal contents. In these cases, a possible sustained behavior was seen in the DOX release profile and after 6 h of release test less than 80% of the drug was released. Cross-linking of nanoparticles with a cross-linking agent can limit their degradation rate and hydration potential, thereby attaining slow-release kinetics18,19. The values of RE of DOX after 2 h (RE120%) of release test are summarized in Table 3. RE120% has direct relation with the rate of drug release. As this table shows the RE120% in different formulations changed between 36.4% and 68.7%. Figure 7 shows that nanoparticles of E4G0.1B11.5 with the lowest glutaraldehyde as cross-linking agent showed the fastest drug release pattern. However, formulation of E4G1.39B11.5 with maximum amount of glutaraldehyde could release just 80% of the loaded drug within about 6 h. Figure 8 also shows a reverse relationship between the RE120% and the glutaraldehyde content of nanoparticles. Li et al.20 who prepared sodium ferulate loaded BSA nanoparticles for liver targeting also showed the in vitro drug


12

J. Varshosaz et al.

release behaved with an initial burst effect and then sustainedrelease stage. To some extent, the drug release rate could be adjusted by cross-linking with increasing amounts of glutaraldehyde. Comparing the drug release profile of E3G0.3B18.5 nanoparticles with E5G0.3B18.5 it is apparent that with increasing of ethanol as denaturing agent the drug release got faster (Figure 7). Figure 8 also shows a direct relation between ethanol content and RE120%. This may be because ethanol caused denaturation of BSA–Hal and consequently reduced the binding site of drug to BSA which in turn expelled it and caused faster drug release. According to Maghsoudi et al.15 addition of ethanol made nanoparticles more rigid and reduced their elasticity to keep the drug thus drug was released faster. As shown in Figure 7, formulation E4G0.75B7.9 compared to E4G0.75B15 with increasing the BSA–Hal content of nanoparticles drug release was faster probably because in constant amounts of glutaraldehyde increasing the BSA–Hal was not enough to crosslink it and the three-dimensional network was not produced to prevent drug release20. The same effect is seen in Figure 8 for the direct relationship between BSA–Hal content and RE120%. Optimization of the BSA–Hal nanoparticle formulations In many formulations, not just pharmaceutical in nature, it is necessary to balance several different measures of quality (i.e. properties) in order to find the best overall product. Changes to the formulation to improve one property may have a deleterious impact on another property. The process of finding the best compromise has been more rigorous by the process of desirability optimization, to produce numerical value of a desirability function. Computer optimization of the results from a response surface statistical designing model based on the hybrid design used, will allow the estimation of a specific combination of the variables that will optimize the individual responses and will yield a product with desirable qualities. The criteria for the optimization of all studied factors are shown in Table 1. Eleven different formulations were designed with Design Expert software by hybrid design. Then considering the results of the experiments done on these formulations (Table 3), optimization was done using Design Expert software and E3.55G0.3B20 was suggested as the optimum formulation which showed a good particle size of 218 nm, zeta potential was 25.4 mV, an acceptable entrapment efficiency of 89% and relatively high RE of 56% until 2 h. Comparing the results predicted by this software for the optimum formulation of nanoparticles with the actual values showed that the error percent was 3.7% for particle size, 9.4% for zeta potential, 1.1% in entrapment efficiency and 3.6% in RE. This indicates a good correlation between the actual and predicted values and success of the designed model in formulating the nanoparticles. Determination of particle size of nanocomposites To overcome the disadvantages of both microparticles and nanoparticles for inhalation, BSA–Hal nanocomposites were designed as drug carriers for targeting the lungs. The prepared nanocomposites having the particle size of about 2.5 mm were composed of sugar or amino acids and drug-loaded BSA–Hal nanoparticles with the aim to reach deep in the lungs, and to decompose into drug-loaded BSA–Hal nanoparticles in the alveoli. After spray drying of the optimized nanoparticles of Hal-BSA loaded with DOX (E3.55G0.3B20) by using mannitol, L-leucin or trehalose in different temperatures, the obtained nanocomposites were tested for their particle size. According to Table 4, the nanocomposites of mannitol with 80 C inlet


temperature (M80) had minimum diameter of 4.12 mm and consequently the highest specific surface area while L-leucin nanocomposites prepared at the same temperature (L80) had maximum diameter of 5.52 mm. Increase of temperature from 80 to 120 C caused almost no increase in diameter of trehalose nanocomposites from 4.18 to 4.23 mm and mannitol from 4.12 to 4.28 mm but decreased the diameter of L-leucin ones from 5.52 to 5.28 mm. Tomoda et al.21 used trehalose and lactose for preparation of spray-dried nanocomposites. They reported when the inlet temperatures were 80 and 90 C, nanocomposite particles with average diameters of about 2.5 mm were obtained but, those prepared above 100 C were not decomposed into nanoparticles in water, while the average diameter was almost 2.5 mm. On the other hand, nanocomposite particles prepared at lower inlet temperatures had larger sizes. The density of all obtained nanocomposite powders were 3.22 g/cm2 and their SF was 1.00 showing a symmetric shape (Table 4). SEM studies The morphological characteristics of the nanoparticles and also the nanocomposites were studied by SEM. Figure 9 shows the morphology of different nanocomposites (a–f) and nanoparticles of Hal-BSA loaded with DOX (g). As this figure indicates the nanocomposites of mannitol are approximately round and gashed. Increasing the inlet temperature of spray dryer from 80 to 120 C had almost no effect on the shape of these and L-leucin nanocomposites. As seen in Figure 9(a) and (b), L-leucin in both temperatures produced hollow microparticles with imperfect coating. Lei Wang et al.22 also reported such a round shape for leucin spray-dried particles. Mannitol nanocomposites (Figure 9c and d) were nearly round and smooth23. Trehalose nanocomposite particles (Figure 9e and f) were almost in cubic form and it seems at high temperature they are crystallized in almost rod or trapezoid and rectangle forms24. Nanoparticles of Hal–BSA (Figure 9g) were also in spherical shape. FPF and EF The pulmonary deposition of the spray-dried powders was investigated in vitro using an Andersen Cascade Impactor. Influence of the carrier on dried powders FPF, EF% and the aerodynamic diameter (daer) are summarized in Table 5. L-leucin nanocomposites were omitted from this study due to their low drug content, high average particle size (Table 4) and the defected imperfect microcapsules (Figure 9a and b). The results of Table 5 show that EF% of M80 was higher than other nanocomposites while these powders showed the lowest stickiness. EF% of T80 was the lowest among the studied powders. The CPF% of trehalose nanocomposites was more than mannitol which explains the reason of higher mean particle size of trehalose powder than mannitol. The FPF% of mannitol nanocomposites was between 35.08% and 35.53% and that of trehalose were between 29.11% and 30% that shows mannitol is more suitable for pulmonary drug delivery. The EPF% of mannitol is also more than trehalose that shows its nanocomposites are finer than trehalose. As Table 5 indicates mannitol with its smoother surface (Figure 9b and c) compared to trehalose had higher EF (ED). In other words ED and FPF of the coated powders decreased as the surface roughness increased which is hypothesized as mechanical interlocking between the surface asperities25. By the measurements of aerodynamic diameters of the nanocomposite particles prepared with trehalose at 70, 80 and



DOI: 10.3109/10837450.2013.852576

90 C, it was shown that the particles prepared at 80 C had the highest FPF value and were suitable for pulmonary delivery of bioactive materials deep in the lungs. Tomoda et al.21 reported that meanwhile in the case of lactose, the particles prepared at 90 C had almost the best FPF value but they had many particles larger than 11 mm. Littringer et al.26 showed that increase in the inlet temperature of the spray drier caused more irregularity in the shape of mannitol particles and hence the FPF was decreased with surface roughness. However, in our study no change in the shape of mannitol particles was seen by increased temperature and thus their FPF was almost the same (Table 5). Table 5 also indicates that mannitol with the lowest daer showed the highest FPF which is also reported by Schu¨le et al.27 who used mannitol in preparation of spray-dried powder of IgG1 for inhalation. Figure 10 shows distributions of DOX in each fragment obtained from Cascade Impactor. The throat fraction traps DOX more than other stages. Stages 3, 4 and 5 were considered as FPF% while stages 6, 7 and filter as EPF%. Table 5 and Figure 10 show that there is significant difference between FPF of M80 with T80 and T120 (p50.05). However, M120 showed no significant difference with M80 (p40.05). Kumon et al.28 produced a co-spray-dried solution containing an inhaled corticosteroid, a long-acting-2-agonist, and mannitol as a crystalline excipient. The FPF of our spray-dried mannitol powder was lower than Kumon’s study due to the higher particle diameter of our mannitol powder. The ED in our nanocomposite of M80 was also higher due to its finer and less adhesive characteristics.

Conclusions Processing variables for formation of BSA–Hal nanoparticles of DOX can be optimized by using a response surface statistical model based on a hybrid design. The size of nanoparticles was more affected by concentration of BSA–Hal and glutaraldehyde while zeta potential and drug loading efficiency were more affected by the concentration of BSA– Hal, glutaraldehyde and ethanol. Ethanol and glutaraldehyde were the most effective variables on the DOX RE from the nanoparticles. Increase in inlet temperature during spray drying of nanocomposites had less effect on the particle size and FPF%. Nanocomposites of M80 were more suitable for drug delivery by inhalation route. Overall, these preliminary results suggest that it is possible to obtain the accumulation of NPs in the microenvironment of the tumor with subsequent intracellular targeting and drug delivery into the cytosol due to the ligand of Hal attached to the nanoparticles. Pulmonary delivery of nanoparticles corresponds to a novel concept that allows a selective target delivery to the deep lung region and represents a potential system for regional delivery to the lung region, able to overcome current clinical strategy problems. It provides for a better retention of active ingredients in the lungs and minimizes their penetration into the systemic circulation. However, further in vivo studies in lung cancer patients are needed to prove the safety and capability of the developed formulation to eradicate the malignant cells.

Acknowledgements The authors wish to thank the Vice Chancellery of Isfahan University of Medical Sciences that supported this work. The technical assistance of Mr Firooz Mardani, Dr Farzin Firoozian and Dr Nabi Maybody is appreciated.

13

Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.

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