Influence of Surface Modification and the pH on the Release

Feb 3, 2014 - Department of Pharmaceutical Sciences, School of Pharmacy, Thomas Jefferson University, 901 Walnut Street, Philadelphia,. Pennsylvania ...
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Influence of Surface Modification and the pH on the Release Mechanisms and Kinetics of Erlotinib from Antibody-Functionalized Chitosan Nanoparticles Asha R. Srinivasan and Sunday A. Shoyele* Department of Pharmaceutical Sciences, School of Pharmacy, Thomas Jefferson University, 901 Walnut Street, Philadelphia, Pennsylvania 19107. United States ABSTRACT: mAb-functionalized nanoparticles have shown great promise in targeting payload to specific cancer cells. However, very little information is available about the effect of such surface modification on the release of the loaded drug from these nanoparticles. The effects of surface modification by mAb on the release of erlotinib from chitosan nanoparticles (CNPs) were investigated using various mathematic models. Fickian diffusion was elucidated to be the main mechanism of release of erlotinib from nonfunctionalized CNPs irrespective of the pH. In contrast, a strong pH influence was observed in the release of erlotinib from the mAb-functionalized nanoparticles. The lack of a single model to definitely describe the release mechanism coupled to the slow release of erlotinib from antibody-functionalized CNPs in comparison to the nonfunctionalized CNPs strongly suggests that the diffusion pathway of erlotinib was being interfered with by the antibody coupled to the surface of CNPs.

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INTRODUCTION Drug-loaded chitosan nanoparticles (CNPs) are rapidly gaining prominence as an ideal technology for targeted drug delivery to cancer cells.1,2 This has been attributed to the fact that chitosan is a biodegradable, biocompatible, and nontoxic polymer of natural origin.2 Recently, active targeted drug delivery has allowed better targeting of payload to cancer cells. Such an active targeted drug-delivery system is normally achieved via covalent conjugation of a monoclonal antibody (mAb) to the nanoparticle surface, which can recognize and bind to a specific receptor expressed in cancer cells.1,3,4 While specific targeting has been achieved by these mAb-functionalized CNPs in comparison to nonfunctionalized CNPs in most of the studies conducted, very little information is available about the effect of such surface modification on the release of the loaded drug from the nanoparticles. Recent reports suggest that drugs are released from CNPs predominantly by a Fickian diffusion mechanism.5 Fickian diffusion has been established to be dependent on the porosity of the membrane barrier.6 To this end, we hypothesize that “surface modification of CNPs by mAb may inhibit the release of loaded drug from the nanoparticles probably by interfering with the diffusion pathway of the drug”. To test this hypothesis, trastuzumabfunctionalized erlotinib-loaded CNPs were prepared and the effect of surface modification on the release kinetics of erlotinib was investigated in comparison to the nonfunctionalized CNPs. Transtuzumab (herceptin) is a humanized mAb directed against the human epidermal growth factor receptor 2 (HER2) and is the only HER2-targeted therapy approved by the Food and Drug Administration for the treatment of breast cancer.3 HER2 is mostly overexpressed in most cancers, while it is weakly expressed in normal adult tissues.7,8 Erlotinib is an anticancer drug targeting the intracellular tyrosine kinase domain of the epidermal growth factor receptor (EGFR-TKI).9 It is therefore an exciting prospect for © 2014 American Chemical Society

intracellular delivery to tumor cells using trastuzumab-modified CNPs. The effect of the pH on the release kinetics of erlotinib from CNPs was also investigated because changes in the pH value may occur based on the physiological fluid of the target organ or the targeted area of a tumor. Tumor tissues normally have a pH value of 5−6 because of anaerobic glucose metabolism, which often leads to the production of lactic acid.5 Further, lower pH values of 3.0−5.5 have been confirmed in the endosomes and lysosomes of cancer cells.10 Hence, it is important to also investigate the effect of the pH on the release of erlotinib from CNPs. In the present study, the effects of surface modification by mAb and the pH on the release mechanisms and kinetics of erlotinib from CNPs were investigated using various mathematical models including zero-order, first-order, Higuchi, Hixson−Crowell, and Korsmeyer−Peppas models. A clear contrast in release kinetics was demonstrated between the nonfunctionalized and mAb-functionalized CNPs. Results also suggested that surface modification of CNPs with an antibody may influence the release of loaded drugs.

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MATERIAL AND METHODS Materials. Low-molecular-weight chitosan derived from crab shell was purchased from Sigma-Aldrich (St. Louis, MO). The degree of deacetylation was 75−85%. Sodium tripolyphosphate (TPP; purity 85%) was also purchased from SigmaAldrich (St. Louis, MO). Erlotinib hydrochloride (mol wt 429) was purchased from LGM Pharma (Boca Raton, FL), and trastuzumab (herceptin) was a kind gift from Genentech (San Francisco, CA). All other reagents were of analytical grade. Received: Revised: Accepted: Published: 2987

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Preparation of CNPs. Chitosan was dissolved in a 0.1 M acetic acid solution to make up chitosan concentrations at 1, 1.75, and 2 mg/mL. Chitosan solutions were then mixed with a 250 μg/mL erlotinib solution (erlotinib dissolved in 0.1% dimethyl sulfoxide and made up with methanol/water). Prepared erlotinib/chitosan solutions were flush-mixed with 1 mL of 1 mg/mL TPP. The CNP suspension was gently stirred for 10 min at room temperature. The drug-loaded CNPs were concentrated by centrifugal filters (30 kDa) at 4000g for 30 min at 10 °C. The unbound drug collected in the bottom of the filter was used to calculate the encapsulation efficiency (EE). The CNPs (with or without drug) were freeze-dried before further analysis. The blank CNPs were prepared under similar conditions as the drug-loaded nanoparticles. Thiolation of Trastuzumab. The trastuzumab thiolation reaction was adapted from a previously used method.3 Briefly, a 100-fold molar excess of 2-iminothiolane (Traut’s reagent) was used in the thiolation reaction. A total of 458 μL of Traut’s reagent was added to each 5 mL of a 2 mg/mL trastuzumab solution. The 2 mg/mL trastuzumab solution was prepared in a 50 mM phosphate buffer (pH 8) containing 0.15 M NaCl, and the reaction was stirred for 2 h at 25 °C. The mixture was then centrifuged three times to exclude unreacted Traut’s reagent using 30 kDa cutoff centrifugal ultrafilters (Millipore Corp.) at 4000g and 10 °C for 15 min. Ellman’s test was then used to quantify the number of thiol groups added to trastuzumab. Briefly, Ellman’s test was performed by incubating aliquots of a concentrated trastuzumab solution with 6.25 μL of Ellman’s reagent. Ellman’s reagent was prepared by dissolving 8 mg in 2 mL of phosphate for 15 min at 25 °C. When the absorbance was measured at 412 nm, the number of thiol groups introduced was calculated using Lcysteine as the standard solution. Preparation of Antibody-Functionalized Nanoparticles. mAb-funtionalized nanoparticles were prepared according to a previously reported method.3 Briefly, 5 mg/mL erlotinib-loaded nanoparticles dispersed in a 0.1 M phosphate buffer (pH 7.2) were functionalized using 2 mg of a heterobifunctional cross-linker, sulfosuccinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate. This reaction was performed for 3 h at room temperature, after which the nanoparticles were centrifuged at 4000g for 15 min at 10 °C with 100 kDa ultrafilters (Millipore Corp.). A total of 500 μL of 2 mg/mL thiolated trastuzumab was then added to 4 mL of a 5 mg/mL nanoparticle suspension and then incubated at 25 °C for 3 h. Trastuzumab-functionalized CNPs were centrifuged at 26000 rpm for 30 min at 10 °C in order to purify the nanoparticles. In order to quantify the unconjugated trastuzumab, trastuzumab in the supernatant was quantified with a Total Protein Kit. Fluorescence Spectroscopy. The presence of trastuzumab on the surface of CNPs was verified using a secondary antibody, fluorescein isothiocyanate (FITC)-labeled sheep antihuman immunoglobulin.4 FITC-labeled sheep antihuman IgG was incubated with the nanoparticles (both trastuzumabfunctionalized and nonfunctionalized CNPs) at 25 °C for 2 h at a ratio of 1:1000. Following the incubation, the mixture was centrifuged for 15 min at 13200 rpm. Unattached FITC-labeled sheep antihuman IgG was excluded by washing twice with phosphate-buffered saline (PBS). A suspension of the FITClabeled sheep antihuman IgG-labeled nanoparticles in PBS was used to obtain the fluorescent intensity. The fluorescent intensity of the nonfunctionalized nanoparticles treated with

FITC-labeled sheep antihuman IgG, PBS, and FITC-labeled sheep. Antihuman IgG were also obtained using a fluorescent plate reader. Measurement of the Particle Size, Polydispersity, and ζ Potential. Prepared CNPs were analyzed for the particle-size distribution and ζ potential by photon correlation spectroscopy (PCS; Zetasizer Nano ZS, Malvern Instruments, Worcestershire, U.K.). Nanoparticles were dispersed in distilled deionized water (pH 7) for these measurements. The intensity autocorrelation was measured at a scattering angle (θ) of 173° at 25 °C. The Z average and polydispersity index (PDI) were recorded in triplicate. For ζ-potential measurement, samples were taken in a universal dip cell (Malvern Instruments) and the ζ potential was recorded in triplicate. Scanning Electron Microscopy (SEM). The morphology of CNPs was obtained using a Zeiss Supra 50VP system (Zeiss, Jena, Germany). A total of 1 drop of the liquid CNP preparation was layered on a SEM stub and allowed to airdry. The dried nanoparticles on the stubs were then coated with a thin layer of gold. The coated samples were then examined under a microscope operated at an acceleration voltage of 5 kV. Erlotinib EE and Loading Capacity (LC). LC and EE of erlotinib in the nanoparticles were measured using ultraperformance liquid chromatography−mass spectrometry (UPLC−MS) analysis of the filtrates obtained after centrifuging the nanoparticles in 30 kDa cutoff centrifugation tubes for 30 min. Drug EE and LC were calculated from the following equations: % EE = (A − B)/A × 100 and % LC = (A − B)/C × 100

where A = total amount of erlotinib, B = free drug, and C = weight of nanoparticles in grams. Erlotinib Release Studies. The release of erlotinib from CNPs was performed in three different pH buffers: pH 3 acetate buffer, pH 5 acetate buffer, and pH 7.4 phosphate buffer. The freeze-dried CNPs encapsulating erlotinib were suspended in 0.5 mL of a buffered solution and transferred to a tubular cellulose dialysis membrane secured tightly at both ends. The cellulose dialysis membrane was then incubated in 10 mL different pH-buffered reservoirs at 37 °C with gentle agitation. The amount of erlotinib released was determined at selected time intervals and analyzed for percentage cumulative drug release using UPLC−MS. UPLC−MS. Erlotinib was analyzed using a Waters Acquity UPLC BEH C18 column (1.7 μM × 2.1 mm) with a flow rate of 0.25 μL/min. A gradient system consisting of mobile phase A consisted of 0.1% formic acid in water (v/v), while mobile phase B consisted of 0.1% formic acid in methanol (v/v). A gradient started at 98% A reaching 2% A at 5 min followed by a linear gradient for 1 min and equilibrating to 98% A at 6.10 min with a total run time of 8.10 min. The sample injection volume was 5 μL. MS analysis was performed on a Thermo Fisher Scientific Exactive Orbitrap LC−MS. Analysis was performed in positiveion mode, and data were collected from m/z 200 to 600 at 100000 resolutions. The capillary and ion-spray voltage was kept at 4.5 V. For quantification, base peak chromatograms were extracted for the mass-to-charge of interest, applying a 5 ppm mass tolerance. Integrations and quantitation results were 2988

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CNPs (erlotinib-free) were 65.8 ± 0.5 nm in size. Nonfunctionalized nanoparticles were predominantly positively charged, while antibody-functionalized nanoparticles were negatively charged. The charge on trastuzumab at this neutral pH (7) is negative (−14.8 mV). EE and LC. Figure 2 shows that increasing the chitosan concentration decreased the EE of erlotinib in the CNPs. The

obtained with Thermo Fisher Scientific Xcalibur software. A calibration curve for erlotinib was constructed with a range of concentrations (250−1 μg). Fourier Transform Infrared (FTIR). Spectra from trastuzumab-functionalized and nonfunctionalized CNPs were collected using a single-reflection attenuated total reflectance (ATR) with a diamond internal reflection crystal installed in a iS10 FTIR spectrometer (Thermo Fisher Scientific, Madison, WI). Powders were placed on the surface of the ATR crystal following the collection of background spectra. Spectra were collected after 64 scans at 4 cm−1. The system was continuously purged with nitrogen during spectra collection. Statistical Analysis. Results are expressed as mean ± standard deviation (SD), unless otherwise indicated. The difference between two groups was determined by two-tailed Student’s t test. A p value of 0.05 was taken as statistically significant.

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RESULTS Physicochemical Characterization of Nanoparticles. The structure of erlotinib-loaded CNPs (both mAb-functionalized and nonfunctionalized) was examined by SEM. The nanoparticles were spherical in shape with a smooth surface, as shown in Figure 1.

Figure 2. Influence of the chitosan concentration on erlotinib EE (erlotinib 250 μg/mL; n = 3).

EE of erlotinib was found to decrease from 80% to 58% as the concentration of chitosan increased from 1 to 1.75 mg/mL. This further decreased to 42% as the concentration of chitosan increased to 2 mg/mL. A similar trend was observed in the LC as the LCs of the nanoparticles of 41.3 ± 0.26, 22.80 ± 0.03, and 17.03 ± 0.01 when the concentrations of chitosan were 1, 1.75, and 2 mg/ mL, respectively. Trastuzumab Coupling to CNPs. 2-Iminothiolane (Traut’s reagent) was used to add the thiol groups required for the coupling of trastuzumab to CNPs. The number of reactive thiol groups linked to the lysine residues of trastuzumab was quantified as 38.6, according to the Ellman’s test performed. This is equivalent to 42% of the lysine residues available. Furthermore, 86% of the mAb molecules were attached to CNPs. A total of 43 mg of trastuzumab was calculated to be attached to each 1 g of trastuzumabfunctionalized CNPs. Verification of Antibody Coupling to Nanoparticles. The covalent linkage of the antibody to CNPs was verified using fluorescence spectroscopy and FTIR. The fluorescent intensity obtained from the coupling of FITC-labeled sheep antihuman IgG to trastuzumab on the surface of the mAbfunctionalized nanoparticles was compared to that of the nonfunctionalized nanoparticles. Table 2 demonstrates the increase in the fluorescent intensity of the trastuzumab-functionalized nanoparticles in comparison to the nonfunctionalized nanoparticles. This indicates that trastuzumab is attached to the nanoparticles. FTIR was used to confirm the covalent linkage of trastuzumab to the nanoparticles. Figure 3 shows the FTIR spectra generated for both nonfunctionalized CNPs and trastuzumab-functionalized nanoparticles. The distinctive differences between these set of spectra further indicated that trastuzumab was successfully linked to the CNPs. In Vitro Release of Erlotinib from Antibody-Functionalized and Nonfunctionalized CNPs. The release of

Figure 1. Representative SEM micrograph of nonfunctionalized erlotinib-loaded CNPs. The scale bar represents 200 nm.

The particle size and ζ-potential analysis by PCS in Table 1 demonstrated that the nonfunctionalized erlotinib-loaded nanoparticles were 237.5 ± 1.8 nm in size, while the mAbfunctionalized nanoparticles were 345.6 ± 2.6 nm in size. Blank Table 1. Particle Size and ζ-Potential Characteristics of Antibody-Functionalized and Nonfunctionalized CNPs (Mean ± SD, n = 3)

sample blank CNPs erlotinib-loaded CNPs trastuzumabfunctionalized nanoparticles

mean diameter (nm)

PDI

ζ potential (mV)

65.8 ± 0.5 237.5 ± 1.8 345.6 ± 2.6

0.21 ± 0.01 0.11 ± 0.01 0.07 ± 0.02

29.2 ± 0.5 27.26 ± 1.88 −17.90 ± 0.73

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Table 2. Fluorescent Intensity of Nonfunctionalized Nanoparticles, Antibody-Functionalized Nanoparticles, PBS as the Solution, and Sheep Antihuman IgG Conjugated to FITC sample nonfunctionalized nanoparticle antibody-functionalized nanoparticles PBS sheep antihuman IgG-FITC

fluorescent Intensity 587.5 2339.75 496.25 1095.25

± ± ± ±

1.29 2.65 6.34 5.56

erlotinib from CNPs (both mAb-funtionalized and nonfunctionalized) was investigated using pH 3 and 5 acetate buffer and pH 7.4 phosphate buffer. Released erlotinib was quantified by UPLC-MS. As demonstrated in Figure 4, a burst release was observed for erlotinib from the nonfunctionalized nanoparticles at pH 3, leading to the release of all encapsulated erlotinib within the first 200 min. In contrast, the mAbfunctionalized nanoparticles produced an initial burst release within the first 200 min. However, a slower controlled release was demonstrated thereafter. In pH 5 (Figure 5), both mAbfunctionalized and nonfunctionalized nanoparticles demonstrated an initial burst release of erlotinib within the first 200 min. Nevertheless, while the release from mAb-functionalized nanoparticles was much slower and sustained thereafter, reaching an optimum after 12000 min, the release from nonfunctionalized nanoparticles was relatively faster thereafter, reaching an optimum after 4000 min. In pH 7.4 (Figure 6), nonfunctionalized nanoparticles demonstrated a release profile similar to that of pH 5. However, mAb-functionalized nanoparticles demonstrated a much slower release profile, showing no release of erlotinib until after 12000 min.

Figure 4. In vitro erlotinib release profile from drug-encapsulated CNP and antibody-functionalized drug-loaded CNP (pH 3).

Release Kinetics and Mechanisms of Drug Release. The release kinetics and mechanisms of erlotinib release from CNPs were evaluated by several mathematical models including zero-order, first-order, Higuchi, Hixson−Crowell, and Korsmeyer−Peppas models. Correlation values (R2) and release parameters determined from the results of model fitting of the release profiles are presented in Tables 3 and 4.

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DISCUSSION In the present study, we aimed to investigate the effects of surface modification by mAb and the pH on the release mechanisms and kinetics of erlotinib from drug-loaded CNPs using various mathematic models including zero-order, first-

Figure 3. FTIR spectra of (a) nonfunctionalized and (b) trastuzumab-functionalized CNPs. 2990

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demonstrates a significant increase (P < 0.05) in the size of erlotinib-loaded nanoparticles following the coupling of the antibody to the surface of the nanoparticles. The increase in size may be attributed to the conjugation of mAb to the surface of the nanoparticles. Further, the involvement of several centrifugation and lyophilization steps may also contribute to the increase in the particle size of the mAb-functionalized nanoparticles.12 A drastic increase in the size of the erlotininbloaded nanoparticle in comparison to the blank nanoparticles could be attributed to the bulk effect of the encapsulated erlotinib. This is because the formation of CNPs is primarily due to the ionic interaction between the positive charges of chitosan and the negative charges of TPP at room temperature. The inclusion of erlotinib during the nanoparticle formation may affect this electrostatic interaction, hence leading to a larger particle size. To achieve the conjugation of trastuzumab to prepared CNPs, the lysine residue in trastuzumab was thiolated using 2iminothiolane, as previously reported by Yousefpour et al.3 The binding of trastuzumab to CNPs was probed by using the fluorescent intensity of mAb-functionalized nanoparticles in comparison to the nonfunctionalized nanoparticles. mAbfunctionalized nanoparticles demonstrated a significantly higher fluorescent intensity because of the presence of trastuzumab on the surface of the nanoparticles, which was able to bind to sheep antihuman IgG-FITC. In order to exclude the possibility of trastuzumab merely adsorbing on the surface of CNPs and to confirm that a covalent conjugation actually occurred, FTIR analysis was performed on both trastuzumab-functionalized CNPs and nonfunctionalized nanoparticles. Figure 3 shows that the spectrum of the nonfunctionalized CNPs has an absorption peak at 3416 cm−1. This peak can be attributed to the group stretching vibration of both NH2 and OH in the chitosan.13,14 However, following the conjugation of trastuzumab to CNPs, the peak became broader, moving to a lower wavenumber at 3219 cm−1. This indicated a strong interaction between NH2 in chitosan and trastuzumab. The peak at wavenumber 2925 cm−1 observed in the spectrum for the nonfunctionalized CNPs can be attributed to the asymmetric stretching of CH3 and CH2 of chitosan.14 This peak moved slightly to a lesser wavenumber of 2482 cm−1 and became broader in the trastuzumab-functionalized CNPs, indicating the existence of a covalent interaction with another group in trastuzumab. The peak at 1561 cm−1 attributed to the amide II carbonyl stretch in the nonfunctionlized CNPs shifted to a lower wavenumber of 1077 cm−1 in the trastuzumab-functionalized CNPs, indicating the presence of −NH2 and C−O stretching from trastuzumab attached to the CNPs.14 The conspicuous differences in the FTIR spectra of the nonfunctionalized CNPs in comparison to the trastuzumab-functionalized CNPs suggest that the conjugation of trastuzumab to the surface of the CNPs was successful. In this study, we hypothesize that “surface modification of CNPs by mAb may inhibit the release of loaded drug from the nanoparticles probably by blocking the diffusion pathway of the drug”. To test this hypothesis, trastuzumab-functionalized erlotinib-loaded CNPs were prepared and the effect of this surface modification on the release kinetics of erlotinib was investigated in comparison to the nonfunctionalized CNPs. Different pH values were applied in this study to simulate the pH of different body fluids including that of a cancer microenvironment.

Figure 5. In vitro erlotinib release profile from drug-encapsulated CNP and antibody-targeted drug-encapsulated CNP (pH 5).

Figure 6. In vitro erlotinib release profile from drug-encapsulated CNP and antibody-targeted drug-encapsulated CNP (pH 7.4).

Table 3. Mathematical Models and Parameters Based on Release Data from Nonfunctionalized Nanoparticles correlation (R2) pH

zeroorder

firstorder

3 5 7.4

0.7380 0.8504 0.8843

0.5690 0.5156 0.6739

Korsmeyer−Peppas

Higuchi

Hixson− Crowell

correlation (R2)

n

0.9112 0.9577 0.9475

0.6511 0.7181 0.8164

0.8114 0.9079 0.9321

0.53 0.66 0.70

Table 4. Mathematical Models and Parameters Based on Release Data from Antibody-Functionalized Nanoparticles correlation (R2)

Korsmeyer−Peppas

pH

zeroorder

firstorder

Higuchi

Hixson− Crowell

correlation (R2)

n

3 5 7.4

0.8846 0.9531 0.5454

0.9185 0.7768 0.5582

0.814 0.8997 0.3836

0.9096 0.8876 0.5587

0.7185 0.8732 0.297

0.26 0.40 0.26

order, Higuchi, Hixson−Crowell, and Korsmeyer−Peppas models. CNPs were spontaneously formed following the addition of TPP because of the ionic cross-linking between the TPP and chitosan solutions.5 TPP was used in the preparation of CNPs in this study as a gelling agent because of its ability to interact electrostatically with ammonium ions in chitosan.11 Table 1 2991

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log Q t = log Q 0 + K1t

Figure 4 shows a comparison between the release of erlotinib from nonfunctionalized CNPs and antibody-functionalized CNPs at pH 3. While the release of erlotinib from nonfunctionalized nanoparticles was relatively very fast, an initial burst release was initially demonstrated in the mAbfunctionalized nanoparticles before a more sustained release. The initial burst release demonstrated by both nonfunctionalized and mAb-functionalized nanoparticles may be attributed to the fact that some amounts of erlotinib were adsorbed on the surface of the nanoparticles, which were released easily by diffusion.5 The seemingly very fast release of erlotinib from the nonfunctionalized nanoparticles was somewhat expected because this could be attributed to the fact that erlotinib has its greatest aqueous solubility at pH of less than 5 because of protonation of the secondary amine group in erlotinib.15 Further, it has been previously suggested that drug release from CNPs can be suddenly accelerated at lower pH (less than 5) because of the pH-responsive nature of CNPs.5 In contrast, the slower sustained and controlled release demonstrated by the mAb-functionalized nanoparticles following an initial burst release may be attributed to the presence of the antibody on the surface of the nanoparticles, inadvertently interfering with the diffusion pathway of the drug from the inner core of the nanoparticles. In both pH 5 and 7.4, a similar trend showing faster release of erlotinib from the nonfunctionalized nanoparticles and a much slower release from the mAb-functionalized nanoparticles was conspicuously demonstrated in Figures 5 and 6. A release lag time was observed for mAb-functionalized nanoparticles at pH 7.4 up to approximately 11000 min. This lag time may be attributed to two major factors: a combination of the fact that the drug erlotinib has a very pure solubility at neutral pH and the interference with the diffusion pathway due to the presence of antibody on the surface of the nanoparticles. In order to have a clearer understanding of the effect of antibody functionalization on the mechanisms and kinetics of the release of erlotinib from CNPs, the release was evaluated by mathematical models such as zero-order, first-order, Higuchi, Hixson−Crowell, and Korsmeyer−Peppas models. Zero-order kinetics (eq 1) describes systems in which the release of the drug is not dependent on the drug concentration16 Q t = Q 0 + K 0t

where K1 is the first-order release constant. The Hixson−Crowell cube root law (eq 5) describes the release from systems by dissolution where there is a change in the surface area and diameter of the particles:20 3√ Q t − 3√ Q 0 = KHCt

(1)

(2)

where KH is the Higuchi constant. The Korsmeyer−Peppas model (eq 3) describes drug release from a polymeric system:18 Q t = KKPt n

(5)

where KHC is the Hixson−Crowell constant. Table 3 shows that the correlation values of release data from nonfunctionalized CNPs fitted to the Higuchi model for all pH values. This suggests that erlotinib is released from the nanoparticles by diffusion.17 Further, the Korsmeyer−Peppas release model exponent, n, describing a pure diffusion mechanism is above 0.5 for all pH values.5 This confirms that the Fickian diffusion is the controlling mechanism in erlotinib release.18 In contrast, the correlation values obtained from release of erlotinib from mAb-functionalized nanoparticles strongly indicate that the pH of the environment may have an effect on the mechanisms of release. In Table 4, the correlation values indicate that the release of erlotinib from antibodyfunctionalized CNPs at pH 3 well fitted to a combination of first-order kinetics (R2 = 0.9185) and the Hixson−Crowell cube root law (R2 = 0.9096). In first-order kinetics, the release rate of a drug from nanoparticles is concentration-dependent,19 while in the Hixson−Crowell cube root law, the release of the drug from the nanoparticle is dependent on dissolution/decomposition of the polymer, which ultimately leads to a change in the surface area.20 This suggests the involvement of multiple release mechanisms (none of the models tested was adequate) in the release of erlotinib from mAb-functionalized CNPs unlike the corresponding nonfunctionalized nanoparticles. However, a conspicuous difference in the release rate exists at both pH 3 and 5. Previous reports suggest that, at pH5, CNPs tend to swell.5 This is because of protonation of the primary amino group on the chitosan, resulting in an increased repulsion force between ammonium cations.5 However, at lower pH (≤3), a screening effect of negatively charged counterions shields the charges of the ammonium cations and prevents an efficient repulsion.21 The swelling of CNPs could possibly explain the reason for the slower release demonstrated by the mAbfunctionalized CNPs at pH 5 in comparison. A definite mechanism of release of erlotinib from mAb-functionalized CNPs at pH 7 could not be elucidated because the release failed to fit to any of the mathematical models used in the present study. Nevertheless, the difference in the release rate of erlotinib from mAb-functionalized chitosan based on the pH value is quite obvious. In contrast, Fickian diffusion was the main mechanism of erlotinib release from the nonfunctionalized CNPs irrespective of the pH value. The possible involvement of multiple release mechanisms coupled to the obvious late/ slower release of erlotinib from mAb-functionalized CNPs in comparison to the nonfunctionalized CNPs strongly suggests that the diffusion pathway of erlotinib was being interfered with by the antibody coupled to the surface of CNPs.

where Q0 = initial amount of drug, Qt = cumulative amountof drug release at time “t”, and K0 = zero-order release constant. The Higuchi model (eq 2) describes the release of drugs from an insoluble system as a square root of the timedependent process based on the Fickian diffusion17 Q t = Q 0 + KHt 1/2

(4)

(3)

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where KKP is the Korsmeyer−Peppas constant and n is the release exponent describing the drug-release mechanism. For this model, pure diffusion is the controlling release mechanism when n ≥ 0.5. The first-order release (eq 4) describes a system where the release rate is concentration-dependent:19

CONCLUSIONS In this study, the effect of functionalization of the surface of CNPs with trastuzumab (antibody) and the pH on the release mechanism and kinetics of erlotinib was investigated using different mathematical models. Fickian diffusion was elucidated 2992

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(12) Ishida, O.; Maruyama, K.; Sasaki, K.; Iwatsuru, M. Sizedependent extravasation and interstitial localization of polyethylene liposomes in solid tumor-bearing mice. Int. J. Pharm. 1999, 190, 49− 56. (13) Guo, M.; Diao, P.; Cai, S. Hydrothermal growth of well aligned ZnO nanorod arrays: dependence of morphology and alignment ordering upon preparing conditions. J. Solid State Chem. 2005, 178, 1864−1873. (14) AbdElhady, M. Preparation and characterization of chitosan/ zinc oxide nanoparticles for imparting antimicrobial and UV protection to cotton fabric. Int. J. Carbohydr. Chem., 2012, Article ID 840591. (15) Tarceva (erlotinib hydrochloride) Tablet Drug Insert; OSI Pharmaceuticals Inc.: New York, 2006. (16) Hadjiioannou, T. P.; Christian, G. D.; Koupparis, M. A.; Macheras, P. E. Quantitative calculations in Pharmaceutical Practice and Research; VCH Publishers: New York, 1993. (17) Higuchi, T. Mechanism of sustained-action modification. Theoretical analysis of rate of release of solid drugs dispersed in solid matrices. J. Pharm. Sci. 1963, 52, 1145−1149. (18) Gao, Z. Mathematical modeling of variables involved in dissolution testing. J. Pharm. Sci. 2011, 100, 4934−4942. (19) Bourne, D. W. A. Pharmacokinetics. In Modern Pharmaceutics; Banker, G. S., Rhodes, C. T., Eds.; Marcel Dekker: New York, 2002; pp 67−93. (20) Hixon, A. W.; Crosswell, J. H. Dependence of reaction velocity upon surface and agitation. Ind. Eng. Chem. 1931, 23, 923−931. (21) Bajpai, J.; Maan, G. K.; Bajpai, A. K. Preparation, characterization and water uptake behavior of polysaccharide based nanoparticles: swelling behavior of nanoparticles. Prog. Nanotech. Nanomater. 2012, 1, 9−17.

to be the main mechanism of release of erlotinib from nonfunctionalized CNPs irrespective of the pH value of the environment. However, for the mAb-functionalized nanoparticles, none of the models could adequately describe the release mechanism of erlotinib. This could be because of the involvement of multiple release mechanisms, which could not be described by a single model. Finally, the possible involvement of multiple release mechanisms coupled to the obvious late/slower release of erlotinib from antibody-functionalized CNPs in comparison to the nonfunctionalized CNPs strongly suggests that the diffusion pathway of erlotinib was likely being interfered with by the antibody coupled to the surface of CNPs.

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AUTHOR INFORMATION

Corresponding Author

*Tel: 215-503-3407. Fax: 215-503-9052. E-mail:sunday. shoyele@jefferson.edu. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors are grateful to Thomas Jefferson University for providing start-up grant for Dr. Shoyele to carry out this project.

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REFERENCES

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dx.doi.org/10.1021/ie402807y | Ind. Eng. Chem. Res. 2014, 53, 2987−2993