Article pubs.acs.org/JPCC
Layer-by-Layer Assembled Thin Film of Albumin Nanoparticles for Delivery of Doxorubicin Vaishakhi Mohanta,† Giridhar Madras,‡ and Satish Patil*,† †
Solid State and Structural Chemistry Unit, and ‡Department of Chemical Engineering, Indian Institute of Science, Bangalore 560012, India S Supporting Information *
ABSTRACT: Protein nanoparticles (NPs) have found significant applications in drug delivery due to their inherent biocompatibility, which is attributed to their natural origin. In this study, bovine serum abumin (BSA) nanoparticles were introduced in multilayer thin film via layer-by-layer self-assembly for localized delivery of the anticancer drug Doxorubicin (Dox). BSA nanoparticles (∼100 nm) show a high negative zeta potential in aqueous medium (−55 mV) and form a stable dispersion in water without agglomeration for a long period. Hence, BSA NPs can be assembled on a substrate via layerby-layer approach using a positively charged polyelectrolyte (chitosan in acidic medium). The protein nature of these BSA nanoparticles ensures the biocompatibility of the film, whereas the availability of functional groups on this protein allows one to tune the property of the self-assembly to have a pH-dependent drug release profile. The growth of multilayer thin film was monitored by UV−visible spectroscopy, and the films were further characterized by atomic force microscopy (AFM) and field emission scanning electron microscopy (FESEM). The drug release kinetics of these BSA nanoparticles and their self-assembled thin film has been compared at a physiological pH of 7.4 and an acidic pH of 6.4.
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the multilayer assembly.12,13 A plethora of materials ranging from hydrogels,14 micelles,15−17 and even nanoparticles18,19 have been exploited for incorporation in multilayer assembly via LbL approach. Although various reports exist on incorporating inorganic nanoparticles into LbL assembly, very few polymeric nanoparticles have been deposited by this technique. Multilayer thin films of polymeric nanoparticles can be of interest as the nanoparticles have immense potential as drug delivery carriers due to high drug loading efficiency and their ability to show controlled and stimuli responsive drug release behavior.20,21 There are several polymers that can be brought into nanoparticles formulation, but many of them suffer from the issues related to their biodegradability and biocompatibility, limiting their applications in drug delivery. In this context, the polymers of natural origin such as polysaccharides22 and protein23,24 may prove to be a better choice. In addition, the deposition of nanoparticles into thin film via LbL approach requires the stability of the aqueous dispersion of nanoparticles along with the presence of functional groups on the surface that can interact with the underlying substrate. Soike et al.25 have successfully deposited PSS(polystyrene sulfonate)-functionalized poly-(DL-lactic-co-glycolic acid) (PLGA) NPs via LbL approach for the purpose of drug delivery and imaging. PSS being a strong polyelectrolyte
INTRODUCTION Drug delivery carriers have seen a breakthrough in recent years, and, although many polymeric nanovehicles have been evaluated and shown to be partially successful for cancer treatment in various in vivo models and clinical systems, there is a scope for tremendous improvement in their therapeutic effects.1−3 The need for fabricating new drug delivery carriers is driven by the fact that conventional modes of drug delivery systems are ineffective for selectively delivering the therapeutics in response to stimuli at the tumor ensuring administration of a smaller dose, thus alleviating the side effects associated with the drug.4 This problem may be addressed by localized delivery of therapeutics from polymer-coated surfaces or thin films.5 The polymeric thin films can be used as implants or scaffolds that can release their therapeutic content specifically at the site of lesions6 in a programmed manner, thus reducing the side effects of the drug. Localized therapeutic delivery is particularly useful in treatment of some kind of cancers like skin cancer7 or brain tumors8 where the intravenous injection of drug is ineffective due to the drug carriers failing to cross the barrier to reach the site of action, leading to low bioavailability of drug. Layer-by-layer (LbL) self-assembly technique based on the sequential adsorption of polyelectrolytes on a substrate has proven to be an extremely versatile approach for fabricating multilayer thin films for drug delivery.9−11 Caruso et al. extended the versatility of this approach in fabricating drug delivery vehicles via incorporation of dendrimers, acting as drug reservoirs, into © 2012 American Chemical Society
Received: October 1, 2011 Revised: January 16, 2012 Published: February 7, 2012 5333
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Milli-Q water to remove the unabsorbed drug. The amount of drug loaded was calculated as:
renders high negative zeta potential to the nanoparticles, and further, by using a dilute solution of nanoparticles, interparticle interactions are minimized to favor random adsorption. In our study, we have chosen nanoparticles of a protein, BSA (molecular weight of 66.4 kDa), as they fulfill the biological criteria expected in a safe drug delivery system such as lack of toxicity, nonimmunogenicity, biocompatibility, biodegradability, stability, and controllable drug-release properties, etc.22,23 Because of their defined primary structure, and high content of charged amino acids, albumin-based nanoparticles allow the electrostatic adsorption of positively or negatively charged drug molecules, and the presence of hydrophobic pockets can facilitate incorporation of water-insoluble drugs as well.26 Moreover, BSA NPs can be easily prepared under mild conditions by simple desolvation, and their size distribution and surface potential can easily be engineered by controlling the process parameters.27 These BSA nanoparticles are stable in aqueous medium for a long period of time without agglomeration, as confirmed from the dynamic light scattering (DLS) study. The availability of functional groups on the nanoparticles makes them capable of showing pH responsive drug release behavior, the relevance of which is in anticancer-drug delivery as the extracellular environment of tumor cells has lower pH than that of healthy cells.28 Additionally, these multilayer thin film assemblies of polymeric nanoparticles also provide us with the opportunity of incorporating more than one drug into the film to overcome the multidrug resistance associated with tumor.25
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drug loaded(w/w) amount of drug added − amount of drug unloaded = weight of nanoparticles (1)
In vitro drug release was studied at the physiological pH of 7.4 as well as acidic pH of 6.4. Doxorubicin loaded nanoparticles were incubated with 1 mL of PBS (of pH 7.4 and 6.4) for a certain duration of time under vortexing. The suspension was then centrifuged, and the centrifugate was collected to calculate the amount of drug released by UV−vis spectroscopy, and it was then replaced with 1 mL of fresh PBS solution. The percentage of drug released was calculated as given below and plotted against time. %Dox release =
CRt × 100 CL
(2)
CL is the amount of drug loaded, and CRt is the cumulated amount of Dox released for time t. It should be noted that error is associated with this method as we may lose some particles during centrifugation. Layer-by-Layer Self-Assembly of BSA Nanoparticles. Multilayer thin film was prepared from BSA nanoparticles by layer-by-layer self-assembly of chitosan (CH) and BSA nanoparticles. Quartz plate and glass coverslips were used as substrates to fabricate the multilayer thin film assembly. At first, the substrate was cleaned by treating with piranha solution (3H2SO4:1.6H2O2) overnight. Caution: Piranha solution should be handled with care. It was then washed several times with Milli-Q water followed by drying under nitrogen flow. The treatment with piranha also serves the purpose of inducing more negative potential on the substrate. The first layer was deposited electrostatically by immersing the substrate overnight in acetic acid solution of chitosan (1 mg/mL). The substrate was then washed with Milli-Q water to remove the unadsorbed chitosan. It was then dried gently under nitrogen flow before immersing in BSA nanoparticles dispersion (0.7 mg/mL) for 6 h. It was then followed by a washing and drying step. In a similar fashion, multilayer assembly was formed by alternately depositing chitosan and BSA nanoparticles by immersing in the respective solution for 1 h. The layer-by-layer process is schematically shown in Scheme 1. A 10-bilayer assembly was fabricated for kinetic studies. To monitor the growth of the assembly by UV−visible spectroscopy, FITC conjugated-chitosan was used, which was prepared by a known method.35 Drug Loading and Release from Thin Film. For drug release study, thin film was fabricated on quartz plate as substrate. For drug loading, a 10-bilayer thin film was immersed in 30 mL of Doxorubicin solution (0.2 mg/mL). The amount of drug loaded as a function of time was monitored by UV−vis spectroscopy. The films were then dipped in Dox solution overnight to achieve saturation level. The loaded films were then immersed in PBS solution of pH 7.4 and 6.4 to study the release behavior in corresponding medium. The films were removed after regular intervals of time, washed with Milli-Q water, dried under gentle nitrogen flow,
EXPERIMENTAL SECTION
Materials. Bovine serum albumin (BSA, fraction V) and glutaraldehyde solution (25%) were obtained from S. D. Fine Chem Ltd. Ethanol was obtained from Brampton, Ontario, Canada. Chitosan (molecular weight 120 000; degree of deacetylation 85%) and FITC (Fluorescein isothiocyanate) were procured from Sigma Aldrich. Acetic acid was obtained from Qualigens, India. Preparation of BSA Nanoparticles. BSA nanoparticles were prepared by the desolvation technique as described previously27 with slight modification. Briefly, 100 mg of BSA was dissolved in 2 mL of Milli-Q water (pH 7). Eight milliliters of ethanol was added dropwise to this BSA solution under constant stirring at room temperature. The addition of ethanol resulted in the spontaneous formation of opalescent suspension. Thereafter, 100 μL of 8% glutaraldehyde was added to this colloidal suspension to induce cross-linking, and it was incubated for different time periods (5, 10, and 18 h) under constant stirring to stabilize the nanoparticles formed. The resulting nanoparticles were purified by 4-fold centrifugation (12 000 rpm, 15 min) and redispersed in Milli-Q water. The amount of BSA nanoparticles was calculated gravimetrically. Drug Release from Nanoparticles. For drug loading, 2.5 mg of BSA nanoparticles was incubated overnight in 1 mL of Doxorubicin solution (0.2 mg/mL). The loaded nanoparticles were then centrifuged, and the centrifugate was collected to calculate the amount of unloaded drug by UV− vis spectroscopy. The loaded nanoparticles were washed with 5334
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Scheme 1. Schematic Representation of Layer-by-Layer Self-Assembly of BSA Nanoparticles Employing Chitosan Polymer (in Acetic Acid) as the Alternate Component of the Self-Assembly
spectra were recorded on Perkin-Elmer FT-IR spectrophotometer, spectrum 1000.
and their UV−vis spectra were taken before reimmersing in PBS solution. Percentage of Doxorubicin is calculated as: % Dox release =
C 0 − Ct × 100 C0
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RESULTS AND DISCUSSION Fabrication of BSA Nanoparticles. BSA nanoparticles were prepared by the well-known desolvation method by inducing precipitation by a nonsolvent, ethanol. The method provides us with the freedom of varying a number of process parameters such as desolvating agent, rate of addition of desolvating agent, stirring speed, temperature, pH, and ionic strength of the media. This directly renders us with the opportunity of controlling the size of nanoparticles by having an effect on the rate of precipitation, which is the primary force that guides the formation of nanoparticles. In this study, we have used ethanol as the desolvating agent. BSA becomes insoluble above a certain concentration of aqueous ethanol (40%)23 due to aggregation of protein chains. This flocculation of proteins is prevented in solvating agent (water) due to the interaction of counterions of water molecules with the charged residues of the protein, leading to the formation of hydration sphere around the protein molecules. This generates repulsion between two protein molecules and masks any possible attraction (electrostatic, dipolar, or hydrophobic interactions) that may otherwise lead to aggregation. Thus, aggregationdriven creation of nucleation points leading to nanoparticle formation would require this repulsive force to be overcome that can be achieved on disruption of the hydration shell by the addition of water miscible organic solvents like ethanol. The organic solvent displaces the water molecules around the protein for the formation of its own hydration layer. With the depleted hydration layer, protein molecules can come together by attractive electrostatic and hydrophobic interaction, leading to aggregation. 29 This entire process is schematically represented in Scheme S1 (Supporting Information). It has been established that the pH of the solution also plays an important role in determining the size of the particles, as desolvation near the isoelectric point (where protein has minimum solubility in water) leads to faster precipitation and
(3)
C0 is the initial concentration of Dox in thin film, and Ct is the concentration of Dox in thin film after time “t” of incubation in PBS solution. Characterization. UV−Vis Spectroscopy. For following the layer-by-layer growth of the assembly as well as for the determination of Dox concentrations in kinetic studies, UV− visible absorbance spectra were recorded on Perkin-Elmer, Lambda 35 UV/vis spectrophotometer. Scanning Electron Microscopy. Size and morphology of BSA nanoparticles were examined from field emission scanning electron microscopy (FESEM), SIRION instrument (FEI Co., Eindhoven, Netherlands). Samples for SEM imaging were prepared by drop casting dilute sonicated aqueous dispersion of nanoparticles on silica substrate, which were then dried overnight at room temperature and kept under vacuum to ensure complete removal of moisture. Samples were gold sputtered prior to imaging. The images were taken at accelerating voltage of 5 kV. The sample chamber vacuum is maintained at 10−5 mbar, and the IGP vacuum is 10−10 mbar. Atomic Force Microscopy. AFM measurements were carried out on an Agilent 5500, Agilent Technologies, Inc., in tapping mode. Samples were prepared on glass coverslips, and film morphology was determined for different layers of the multilayer assembly. Dynamic Light Scattering and Zeta Potential Measurement. Particle size and electrophoretic mobilities were determined from the ZetaPALS Potential Analyzer, Brookhaven Instruments Corp., Holtsville, NY. The electrophoretic mobilities were converted to zeta potential (ζ) using Hückel’s model. All experiments were performed at 25 °C. FTIR Analysis. The dried samples were mixed with KBr, ground, and then made into pellets under high pressure. FTIR 5335
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Figure 1. SEM micrograph of BSA nanoparticles. Inset shows the magnified image of BSA nanoparticles and size distribution of BSA nanoparticles. Maximum distribution of BSA nanoparticles lies in the size range of 75−100 nm.
Figure 2. Variation of zeta potential (a) and drug loading capacity (b) of BSA NPs as a function of amount of cross-linking varied by incubation time of NPs in glutaraldehyde.
spherical morphology of the particles is clearly depicted in the magnified FESEM image of the particles as shown in the inset of Figure 1a. The size distribution of the particles is also calculated from dynamic light scattering (DLS) measurement. The observed size of the particles from DLS is in the range 164.8 ± 8.5 nm with a polydispersity index (PDI) of 0.0895 ± 0.0397. The amount of cross-linking is known to have a marked effect on the surface potential of the nanoparticles and their drug loading and release behavior.31 In our experiment, the amount of cross-linking was varied by incubating the nanoparticles for different time duration (5, 10, and 18 h) with the same concentration of glutaraldehyde (1 mL of 8% glutaraldehyde/mg of BSA), and its effects on surface potential and drug loading capacity were studied. BSA nanoparticles show a negative zeta potential at neutral pH in water, implying the excess of carboxylate anions on the surface over amine groups. Further, we observed an increase in the negative zeta potential value with increasing time of incubation in
formation of larger aggregates. We have performed the synthesis at the neutral pH of 7 (which is well above the isoelectric point of BSA (pH 4.4)) with a high stirring speed, using a BSA concentration of 10 mg/mL and controlling the rate of ethanol addition at 1 mL/min. By this technique, we were able to obtain small size particles with the maximum size distribution less than 100 nm as determined from FESEM (Figure 1). The nanoparticles formed by desolvation approach are not stable and redissolve in water and therefore need to be hardened by thermal or chemical cross-linking. Glutaraldehyde is a commonly used cross-linker for proteins. The cross-linking mechanism is supposedly the reaction between aldehydic groups with amine groups present on the protein chain and also on lysine residues leading to the formation of Schiff’s base as one of the expected products.30 The FESEM image of the cross-linked nanoparticles shown in Figure 1 shows nearly uniform size distribution. The maximum distribution of the nanoparticles was found to be in size range of 75−100 nm (inset of Figure 1). The 5336
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glutaraldehyde as shown in Figure 2a, which is a manifestation of the progress of cross-linking reaction in which amine groups are involved in Schiff base formation leading to an excess of carboxylate anions over amine groups. This high zeta potential value results in very stable dispersions of BSA nanoparticles in water and does not show any agglomeration in water for a long period of time. We studied the drug loading behavior of these nanoparticles as a function of amount of cross-linking. Figure 2b shows the variation of amount of drug loading with amount of crosslinking. We observed maximum Dox loading in case of nanoparticles incubated in glutaraldehyde for 18 h. This may denote that a significant amount of drug is held on the surface of the nanoparticles via electrostatic interaction of the positively charged Dox with the negative surface of the nanoparticles, and this interaction is enhanced with increase in the negative zeta potential value of the nanoparticles. There is a possibility of increasing hydrophobicity of the nanoparticles on incorporation of hydrophobic glutaraldehyde moieties, which may lead to greater inclusion of Dox through the hydrophobic interaction with the matrix.32 Layer-by-Layer Self-Assembly of BSA Nanoparticles. We next fabricated the multilayer thin film via LbL approach employing nanoparticles with 18 h of incubation in glutaraldehyde as the highest zeta potential of these would assist the stable film formation through electrostatic interaction. Further, due to the highest amount of cross-linking, we expect these nanoparticles to be stable in aqueous medium for a long time. Because the nanoparticles carry negative zeta potential, naturally occurring and biocompatible polymer chitosan having positive charge in acidic media (due to the protonation of amine groups present on the polymeric chain) was exploited as the complementary polyelectrolyte for the LbL assembly. The assembly formation is favored mainly by electrostatic interaction, but a possibility of hydrogen bonding between the various function groups (amine, carboxylic, etc.) present on the surface of BSA nanoparticles and the amine groups of chitosan polymer exists. The lack of prominent signature in UV−vis region for both BSA NPs and chitosan was deterrent in monitoring the growth of the film by UV−vis spectroscopy, so chitosan was conjugated with a fluorescent dye, FITC. The absorption spectrum of FITC-chitosan is shown in Figure S1 (Supporting Information). The absorbance maximum of FITC-chitosan in the film at 490 nm is taken as the reference for monitoring the film growth. The amount of FITC conjugated was kept very small (2.9% w/w) so as to avoid any significant effect on the growth of the assembly, and we may expect the results to be close to those in case of unconjugated chitosan. Figure 3 shows the layer-by-layer growth of the film (as monitored via UV−vis spectroscopy). We have further investigated the effect on surface coverage of nanoparticles with increasing number of depositions from atomic force microscopy (AFM) (shown in Figure 4) and scanning electron microscopy (SEM) (shown in Figure 5). On increasing the number of layers of albumin nanoparticles from 1 to 6, we observe a significant deposition of BSA nanoparticles and an increase in the surface coverage of the film. The broadness of the peak in the line profiles of AFM images can give an indication of the size of the nanoparticles within the multilayer self-assembly. This is approximately around 100 nm and is very similar to the size of the nanoparticles used for the multilayer construction. Spherical morphology of the nanoparticles in-
Figure 3. Layer-by-layer growth of multilayer thin film assembly of BSA nanoparticles and chitosan as monitored by UV−visible spectroscopy. The peak intensity at 490 nm corresponding to FITCchitosan is taken as reference for monitoring the growth. The inset shows the linear relationship between the peak intensity and number of layers.
corporated in thin film is further confirmed from the FESEM image of the surface of the thin film as shown in Figure 5. FESEM image for sixth layer of BSA nanoaparticles shows a uniform surface coverage. Complete coverage of nanoparticles on the film cannot be ensured because of the drying effect, which may lead to shrinkage of nanoparticles. The incomplete coverage may provide room for the NPs to swell in different medium, which may be advantageous in drug release. Figure 6 shows the cross-sectional FESEM image of the 10bilayer film. The magnified image shown in the inset clearly shows the incorporation of nanoparticles in the multilayer assembly. It is quite evident from the FESEM image that spherical morphology of the nanoparticles is retained. The thickness of the multilayer film was observed to be 260 nm for 10-bilayer film. FTIR Analysis of Thin Film. Further, the self-assembled multilayer assembly of BSA NPs and chitosan (CH) has been characterized by FTIR spectral analysis. The FTIR spectra of BSA NPs, chitosan, and the multilayer thin film are compared in Figure 7. We observe a large overlap between the FTIR spectra of BSA NPs and chitosan. The characteristic amide I (due to CO stretching) and amide II bands (out of phase combination of NH in-plane bending and CN stretching) of BSA NPs are observed as a broad peak around 1682 and 1535 cm−1, respectively.33 In case of chitosan (dry powder), the peaks at 1657 and 1597 cm−1 are assigned as amide I and amide II bands.33 The presence of these two amide bands of both BSA NPs and chitosan is expected in the FTIR spectra of thin film, which we observe at 1657 and 1535 cm−1. The band at 1657 cm−1 for thin film shows overlap with amide I bands of both BSA and chitosan. However, the amide II band of pure chitosan (powder) at 1597 cm−1, which is due to NH bending vibration of primary amine, is not observed in the FTIR spectra of thin film. We propose that it is due to the presence of the protonated form of chitosan in the film34 or the formation of amide bond, which decreases the frequency of vibration to 1535 cm−1. Amide III34 band (in-phase combination of NH in-plane bending and CN stretching) at around 1260 cm−1 is enhanced 5337
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Figure 4. AFM images showing the surface morphology of thin film (left) as well as the line profiles (right) for the (a) first layer and (b) sixth layer of BSA nanoparticles.
Figure 5. SEM images of self-assembled multilayer thin film with first (a) and sixth (b) layer of BSA nanoparticles.
in the multilayer thin film, which is an indication of formation of amide bonds between carboxylate groups of BSA and amine moieties of CH contributing to the C−N stretching. Hence, apart from the electrostatic interaction, covalent bond contributes to the stability of the assembly. The peak at around 1450 cm−1 corresponding to CH2 bending33 in BSA is preserved in the FTIR spectra of thin film. The band at around 1098 cm−1 is from the saccharide structure of chitosan, which is observed at around 1073 cm−1 in case of chitosan. The comparison of the FTIR spectra of thin film with those of BSA
NPs and chitosan gives an elementary idea of the presence of both of the constituents in the self-assembled multilayer thin film. Loading and Release Kinetics. We have studied the drug loading and release behavior of multilayer thin film assembled out of these BSA nanoparticles. Loading of Doxorubicin was confirmed by the development of reddish coloration of the film and also from UV−vis absorbance spectra (taken after blank correction with unloaded film) showing a characteristic peak of Doxorubicin in the region 490−500 nm as shown in the inset 5338
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Figure 6. Cross-sectional SEM micrograph of layer-by-layer selfassembled multilayer thin film of BSA nanoparticles (10 bilayers). The magnified image in the inset clearly depicts the incorporation of BSA nanoparticles in the multilayer self-assembly. Figure 8. Loading of Doxorubicin in multilayer thin film assembly as followed by UV−vis spectroscopy. The broad peak around 500 nm is characteristic of Doxorubicin.
behavior similar to that of BSA NPs, burst release followed by sustained release. We have further observed pH-dependent drug release behavior for the BSA nanoparticles. At an acidic pH of 6.4, we observed a greater release of the drug than that at the physiological pH of 7.4. We assumed that this can be due to weakening of the interaction of the Dox adsorbed on the particle surface due to the protonation of free amine groups of BSA NPs in acidic medium. To account for this, we measured the zeta potential of the BSA nanoparticles in PBS solution of 7.4 as well as 6.4. Unlike the zeta potential value of −55 mV when the medium is Milli-Q water, the negative zeta potential of the nanoparticles has drastically reduced to −17.6 mV when the medium is PBS solution with pH 7.4. This can be due to adsorption of ions and creation of ionic sphere in medium of high ionic strength. We observed a decrease in the negative zeta potential to −14.8 mV when the pH is decreased to 6.4, which is an indication of protonation of amine groups present on the surface of BSA nanoparticles. This pH responsive drug release behavior is observed to be retained on incorporating the NPs in thin film assembly. The pH-dependent drug release behavior in case of multilayer thin film can be attributed to either swelling of the NPs and/or the swelling of the matrix due to protonation of amine groups on chitosan backbone as well those on BSA NPs, leading to repulsion between the NPs and chitosan backbone and release of drug entrapped between the layers. The greater solubility of Dox in water at acidic pH may also contribute to the release. Hence, multilayer thin film assembly of BSA NPs is capable of releasing its payload in a controlled manner, and the drug release behavior is pH dependent as well. The difference in the release with respect to pH is clearly represented when the desorption kinetics of release of Doxorubicin is fitted with a second-order model (Figure 9c,d):
Figure 7. FTIR spectra for BSA NPs (black), chitosan (CH) (red), and LbL self-assembled multilayer thin film of BSA NPs and chitosan (green).
of Figure 8. The amount of Dox loading is studied as a function of time as shown in Figure 8. Availability of large number of functional groups arising from both BSA nanoparticles as well as chitosan makes this multilayer thin film efficient to load a substantially high amount of Dox molecules via electrostatic interaction, hydrogen bonding, and hydrophobic interaction, which is evident from the peak intensity of Dox in UV−vis absorbance spectra of loaded film as shown in Figure 8. In this multilayer thin-film assembly, the drug has two sites for being entrapped, either in the nanoparticles or in the matrix of the film. Release kinetics of Dox from BSA nanoparticles and thin film at physiological pH of 7.4 and acidic pH of 6.4 is shown in Figure 9a,b. From the plots, it can be concluded that the release of Doxorubicin from the nanoparticles shows two distinct behaviors, burst release followed by a sustained release. We hypothesize that the burst release within the initial time period of 1 h can be accounted for the release of Doxorubicin molecules that are adsorbed on the surface of the nanoparticles and those that are interacting weakly with the matrix. The subsequent slow release can be due to the release of Doxorubicin, which is entrapped deeper into the matrix and, therefore, has to travel a longer distance in the diffusion controlled release. The drug release behavior of the multilayer thin film showed
dqt dt
= k s(qe − qt )2
where qt is the percentage of drug released at time t, and qe is the percentage of drug released when equilibrium is reached. ks is the second-order rate constant. Integrating the above 5339
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Figure 9. Drug release profile of BSA NPs (a) and multilayer thin film (b) at a physiological pH of 7.4 and an acidic pH of 6.4. Parts (c) and (d) show the second-order fitting of the release profiles of nanoparticles and thin film, respectively.
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CONCLUSION We have shown that nanoparticles of protein, BSA, can be successfully deposited as a thin film by the LbL technique. The high zeta potential and stability of aqueous dispersion of these BSA NPs facilitate their LbL assembly via electrostatic interaction with complementary polymer, chitosan. The surface coverage of NPs on the surface is improved on increasing number of depositions as shown from AFM and FESEM. In addition, BSA NPs show pH-dependent drug release behavior, and this pH-dependent release behavior is retained in the NP assembly. Hence, localized pH-responsive drug delivery can be accomplished by LbL assembled thin film of BSA nanoparticles.
differential equation with initial conditions, qt = 0 at t = 0, yields:
t 1 1 = + t 2 qt qe k sqe Thus, a plot of t/qt with desorption time t would be linear with intercept, 1/ksqe2, and slope, 1/qe. Clearly, there is a marked difference between the slopes at the two different pH values. The values of qe and ksqe2 are listed in Table 1. The value of Table 1. Values of qe and ksqe2 for Release Kinetics of Doxorubicin from the Nanoparticles and Multilayer Thin Film at pH 7.4 and 6.4 pH 7.4
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S Supporting Information *
pH 6.4
system
qe
ksqe2
qe
ksqe2
BSA nanoparticles multilayer thin film
0.36 0.15
1.23 0.32
0.63 0.22
4.35 1.16
ASSOCIATED CONTENT
Schematic representation of preparation of BSA nanoparticles. Absorbance spectra of FITC-chitosan. This material is available free of charge via the Internet at http://pubs.acs.org.
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qe indicates that equilibrium release is attained, and this is found to be greater at acidic pH of 6.4 in case of nanoparticles as well as thin film.
AUTHOR INFORMATION
Corresponding Author
*Tel.: +91-80-22932651. Fax: +91-80-23601310. E-mail: satish@ sscu.iisc.ernet.in. 5340
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Notes
(33) Freddi, G.; Anghileri, A.; Sampaio, S.; Buchert, J.; Monti, P.; Taddei, P. J. Biotechnol. 2006, 125, 281−294. (34) de Vasconcelos, C. L.; Bezerril, P. M.; Dantas, T. N. C.; Pereira, M. R.; Fonseca, J. L. C. Langmuir 2007, 23, 7687−7694. (35) Qaqish, R.; Amiji, M. Carbohydr. Polym. 1999, 38, 99−107.
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We would like to acknowledge Nano Centre and CENSE, IISc. for FESEM and AFM. We would like to thank Siram Raja Bhaskar Kanth, SSCU, IISc. for carrying out the FTIR measurements. We thank Defence Research and Development Organisation (DRDO), India, for funding through project number ERIP/ER/0901110/M/1184.
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