In Vitro Release of Lysozyme from Gelatin Microspheres - American

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In Vitro Release of Lysozyme from Gelatin Microspheres: Effect of Cross-linking Agents and Thermoreversible Gel as Suspending Medium Pradip Hiwale,† Sandrina Lampis,† Gabriele Conti,‡ Carla Caddeo,§ Sergio Murgia,*,† Anna M. Fadda,§ and Maura Monduzzi*,† †

Dipartimento di Scienze Chimiche, Universita di Cagliari, CNBS and CSGI, Cittadella Universitaria, s.s. 554 bivio Sestu, 09042 Monserrato (CA), Italy ‡ Dipartimento di Citomorfologia, Universita di Cagliari, Cittadella Universitaria, s.s. 554 bivio Sestu, 09042 Monserrato (CA), Italy § Dipartimento Farmaco Chimico Tecnologico, Universita di Cagliari and CNBS Via Ospedale 72, 09100 Cagliari, Italy ABSTRACT: This study was aimed to characterize the microstructure and the performance of gelatin microspheres (GMs) cross-linked by two different crosslinkers viz. D-glucose and glutaraldehyde. New formulations were obtained, suspending the GMs in a thermoreversible Pluronic F127 (PF127) liquidcrystalline gel. Lysozyme was used as a model biomacromolecular drug to evaluate release features. Both types of cross-linked GMs were prepared by thermal gelation method. The lysozyme-loaded microspheres were characterized by scanning electron microscopy (SEM) for size distribution, shape, and surface texture. SEM revealed that both types of lysozyme-loaded GMs were spherical in shape and that the surface of glutaraldehyde cross-linked GMs was smoother than that of the glucose cross-linked GMs. The degree of cross-linking of microspheres was investigated using ATR-FTIR technique. The prepared GMs were suspended in 20% w/v aqueous PF127 gel for which the usual sol
gel transition temperature of 22 °C did not change in the presence of GMs, as indicated by rheological measurements. SAXS study of the PF127 gel confirmed the occurrence of a discrete cubic liquid-crystalline phase of the Fm3m type whose lattice parameter slightly decreased as a result of GMs addition. The in vitro release of lysozyme from both types of cross-linked GMs was successfully controlled when they were suspended in PF127 gel, thus suggesting the potential use of this new combined formulation as a drug-depot system.

1. INTRODUCTION Nanostructured materials and nanotechnologies represent emerging tasks for nanomedicine applications. Thus, a major objective of formulation chemistry is to improve bioavailability, stability, and convenience to the patient. Indeed, a growing effort in the discovery of innovative therapies has led to an increasing demand for drug delivery vehicles whose capability should not be limited to a simple drug encapsulating and transporting affair. Protecting and selectively releasing the drug and, in particular, overcoming biological barriers that prevent the drug to reach the receptor represent novel, strict requirements. Actually, most drug delivery systems belong to the colloidal domain. Therefore, the awareness of the hydrophobic
hydrophilic intermolecular interactions along with the thorough knowledge of surfactant self-assembly is essential in engineering systems capable of achieving a well-defined biological target. In addition, a sustained drug release also at very long-term is of paramount importance in the development of modern drug delivery systems such as depot systems that can be very useful in the chronic therapeutical treatments. A variety of innovative drug delivery formulations have been proposed in the recent years.1 They embrace microsphere hydrogels based on polysaccharides, r 2011 American Chemical Society

emulsions and microemulsions, liposomes, micelles, lipid nanoparticles, as well as colloidal dispersions where liquid crystals may alternatively represent both the dispersed (cubosome and hexosomes)2 or the continuous phase.3
5 New materials, including nanoporous silica and polylactide nanoparticles, have been designed to accomplish the pressing demand for drug delivery systems with enhanced performance.6 Among the different types of drug delivery systems being proposed recently, microspheres composed of biodegradable polymers for controlled release applications are extensively studied.7 Gelatin is one of the common natural polymers used in the fabrication of particulate drug delivery systems such as microspheres.8
10 It is a well-known natural polymer derived from collagen that possesses good biodegradability and biocompatibility.8,11,12 Moreover, it has been extensively used in various contemporary pharmaceutical dosage forms, and it is included on the FDA list of inactive ingredients.13 Gelatin exhibits polyion complexation properties that can be utilized in Received: May 18, 2011 Revised: June 28, 2011 Published: August 02, 2011 3186

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Biomacromolecules the formulation of the sustained release of numerous charged pharmacologically active molecules including proteins.11,12 Gelatin microspheres (GMs) are normally prepared by the simple method of thermal gelation, also called emulsification
solvent
extraction method. To control the immediate quick dissolution of gelatin and hence a burst release of the drug encapsulated within the microspheres, several cross-linking methods have been proposed. Chemical cross-linking of gelatin is frequently adopted by many researchers to decrease the solubility of GMs and consequently to decrease the rate of release of drugs from the microspheres. Di- or polyaldehydes used as cross-linking agents are known to give excellent microspheres for controlling release properties of encapsulated drug. However, these cross-linking agents lead to several undesirable toxic side effects if they remain after cross-linking, if they form toxic products after reaction with the encapsulated drug, or both. To overcome this problem, several “natural” cross-linking agents, for example, sugars or sugar-derived compounds, have been attempted for the preparation of microspheres with modified release profiles of the encapsulated drugs. Different cross-linking methods have been reported for GMs to alter the toxicity associated with cross-linking agents.14 The two representative compounds of chemical cross-linkers (i.e., di- or polyaldehydes and sugars) for GMs selected for this study are glutaraldehyde and D-glucose. In this context, it should be remarked that GMs, because of their biodegradability also in the presence of cross-linkers, are not expected to fulfill all requirements of an effective controlled release in terms of pharmacokinetic protocols. Burst release cannot be avoided unless a protection that slows the degradation process is adopted. To obtain sustained release drug delivery systems, combinations of microspheres and gel systems have been attempted previously by several authors. Examples include 5-fluorouracil in chitosan microspheres and Pluronic F127 (PF127) gel,15 insulin in calcium
alginate microspheres and PF127 gel,16 Baclofen in poly(lactide-co-glycolide) microspheres dispersed in chitosan and PF127 gels,17 timolol maleate in poly(adipic anhydride) microspheres and gelrite gel,18 and oxybenzone in GMs and aloe vera gel.19 Among the different polymers showing gelling properties and sol
gel transition temperatures in convenient ranges, PF127 has widely been investigated. PF127 is a poloxamer-type block copolymer having the composition-block sequence EO100PO70EO100, where EO denotes poly(ethylene oxide) and PO denotes poly(propylene oxide). Above the Krafft temperature, which was found to be ∼12 °C, 20,21 PF127 molecules (concentration ∼20% w/v) form oil-in-water micelles. The micelle structure consists of almost spherical arrangement of copolymer with a dehydrated polypropylene oxide core and an outer shell of hydrated swollen polyethylene oxide chains. In the presence of different oils, PF127 can self-assemble into lyotropic liquid crystalline structures, having one- (lamellar), two- (cylindrical), or three-dimensional (cubic) order, “normal” (oil-in-water, o/w) or “reverse” (water-in-oil, w/o) morphology, and discrete (e.g., spheres) or continuous (e.g., interconnected bilayer) topology, depending on the ternary copolymer
water
oil composition.22
24 The present work focuses on GMs dispersed in the thermosensitive PF127 gel matrix. Indeed, the concentrated aqueous solutions (20
40% w/v) of PF127 show thermoreversible properties due to interactions between different segments of the copolymer along with hydration strength. With increasing

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temperature, PF127 micelles undergo a sol
gel transition forming a gel characterized by an ordered discrete cubic arrangement.25 PF127 gels can be useful to enhance the stability and the protection of proteins. They do not show any inherent myotoxicity with single or multiple injections. 26 Interestingly, this polymer has widely been used as an efficient o/w emulsifier2,27 and, in particular, for the delivery of various protein/peptide drugs such as insulin,28 urease,29 interleukin-2,30 epidermal growth factor,31 bone morphogenic protein,32 and basic fibroblast growth factor.33 Most release profiles show sustained release kinetics over several hours. The aim of the present work was to investigate the effect on morphology and release performance of two different crosslinking methods in the preparation of GMs loaded with lysozyme chosen as a model protein. To the best of our knowledge, the combination of GMs and PF127 gel has never been reported. Lysozyme is a small antibacterial protein with molecular weight of 14.4 kD useful for the treatment of prosthetic valve endocarditis.34 Another important aim of this work was to investigate the release performance modifications produced by suspending the lysozyme-loaded GMs in PF127 gel and to ascertain that lysozyme does not lose its activity because of the preparation conditions of the formulation or during the release. Scanning electron microscopy (SEM), ATR-Fourier transform infrared (FT-IR) spectroscopy, and small-angle X-ray scattering (SAXS) techniques along with rheological measurements were used to characterize morphology and physicochemical properties of the formulations. The quantification of lysozyme loading and release was performed through lysozyme activity assay using Micrococcus lysodeikficus bacterial suspension.35

2. MATERIALS AND METHODS 2.1. Materials. Hen egg white lysozyme (isoelectric point ∼11), gelatin B (isoelectric point 4.7
5.2), D-glucose, glutaraldehyde, Span 80, acetone, Pluronic F127, potassium dihydrogen orthophosphate, and dipotassium hydrogen phosphate were purchased from Sigma Aldrich. Millipore-filtered and distilled water was used wherever required. 2.2. Preparation of the GMs. Glucose cross-linked gelatin microspheres (GluGMs) and glutaraldehyde cross-linked gelatin microspheres (GAGMs) were prepared in the absence and in the presence of lysozyme. Both types of GMs were prepared by thermal gelation method, as reported in the literature.8,36,37 For GluGM, 400 mg of D -glucose was dispersed in 20 mL of an aqueous gelatin B solution (20%, w/v, preheated at 40 °C), and the dispersion was added dropwise to a mixture of 200 mL of soya oil and 2 mL of Span 80 (1% v/v) while the mixture was stirred at 1000 rpm at 80 °C for 10 min. This gave w/o emulsion. Stirring was continued for further 10 min while the temperature was decreased to 15 °C. Then, 150 mL of acetone was added to dehydrate and flocculate the microspheres. After 10 min of stirring, microspheres were filtered through sintered glass filter and washed with 250 mL of acetone to remove residual oil. GluGMs were dried under vacuum to remove acetone. For lysozyme-loaded GluGM (LGluGM), 100 mg of lysozyme was dissolved in gelatin and D-glucose solution. Here w/w ratio between lysozyme and gelatin was about 1:0.025. In the case of GAGM, first GMs without cross-linking were obtained through the same procedure as that of GluGM without the addition of sugar. The GMs (500 mg) were then mixed with 50 mL of 0.22 M glutaraldehyde of an aqueous ethanol solution (90% v/v) with stirring for 45 min and then with 99.8% ethanol for 4 h to remove excess of glutaraldehyde. GAGMs were vacuum-dried to evaporate residual ethanol. For lysozyme-loaded GAGMs (LGAGMs), 100 mg GAGMs were suspended in 10 mL of lysozyme solution (10 mg/mL) prepared in phosphate buffer pH 7.4 for 3187

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24 h. The resulting LGAGMs were separated from the lysozyme solution and freeze-dried. In this case, w/w ratio between lysozyme and gelatin was about 1:1. All microspheres were stored in a closely packed glass vial. 2.3. Lysozyme Activity. Micrococcus lysodeikficus (Sigma) bacterial cell suspension was used to determine quantitatively the bioactivity of the enzyme.35 Lysozyme is known to digest the cell wall of bacteria. The bacterial cells were dispersed in potassium phosphate buffer (66 mM, pH 6.24) in the concentration of 0.015% w/v. It was further diluted with the same buffer so that initial absorbance was ∼1.50 at 450 nm. An appropriately diluted lysozyme solution (0.1 mL) was added to 2.5 mL of bacterial suspension, and the absorbance at 450 nm was recorded every 15 s during a total incubation period of 5 min at 25 °C. The bioactive lysozyme concentration was determined by the following equation units=mL lysozyme ¼

ðΔE450nm =min test
ΔE450nm =min blankÞðdf Þ ð0:001Þð0:1Þ

ð1Þ where ΔE450nm/min is the reduction in the absorbance at 450 nm per minute, df is the dilution factor, 0.001 is the change in absorbance at 450 nm as per the unit definition of lysozyme activity, and 0.1 is the volume (in milliliters) of lysozyme used for the assay.

2.4. Drug Loading and Encapsulation Ratio of the GMs. Both types of lysozyme-loaded GMs (5 mg) were dispersed in 10 mL of distilled water with the help of a magnetic stirrer. The concentration of bioactive lysozyme was monitored until it becomes constant by the method described in Section 2.3. The drug loading (LD) and encapsulation ratio (ED) were determined using the following equations LD ¼

actual loaded amount of lysozyme obtained by extraction  100 amount of microspheres

ð2Þ ED ¼

actual loaded amount of lysozyme obtained by extraction100 amount of lysozyme added to the preparation

ð3Þ

2.5. Preparation of PF127 Gel and Gelatin Microsphere/ PF127 Gel Suspension. PF127 gel was prepared by dissolving

20% w/v of PF127 powder in distilled water for 12 to 24 h at 4 °C in a refrigerator according to the “cold method”.38 The lysozyme was dissolved in distilled water for preparing lysozyme-loaded PF127 gel. Microspheres in gel formulations were obtained by adding 0.5% w/v lysozyme-loaded GMs to PF127 solution (below sol
gel transition temperature) just before the release experiment. 2.6. Microscopy. The prepared GMs were observed under SEM to study shape and surface properties. The GMs were sprinkled onto a double adhesive tape fixed on aluminum stage. Fixed microspheres were spattered with platinum film. Examination of microspheres was performed with a SEM FE Hitachi S4000 operating at 15
20 kV. 2.7. ATR-FTIR. ATR-FTIR studies were conducted with a Bruker Tensor 27 spectrophotometer equipped with a diamond ATR accessory and DTGS (deuteriotriglycine sulfate) detector. Each spectrum was an average of 128 scans at resolution of 4 cm
1 from wavenumber 4000 to 400 cm
1. The Opus spectroscopic software was used for data handling. 2.8. Rheology. Rheological measurements were performed with rheometer (Malvern Kinexus, Worcestershire, U.K.) using cone
plate geometry CP4/40 and a 200 μm gap operating in the oscillation mode. The frequency and shear stress was set as 0.1 Hz and 0.5 Pa. The temperature ramp was performed from 5 to 40 °C at 1 °C/min rate. The sampling interval was set to 5 s. The oscillatory measurements gave the information about the rheological parameters such as elastic or

storage modulus (G0 ) and the loss or viscous modulus (G00 ). Sol
gel transition temperature was determined for all samples. 2.9. SAXS Experiments. Small X-ray diffraction was recorded with a S3-MICRO SWAXS camera system (HECUS X-ray Systems, Graz, Austria). Cu KR radiation of wavelength 1.542 Å was provided by a GeniX X-ray generator, operating at 50 kV and 1 mA. A 1D-PSD-50 M system (HECUS X-ray Systems, Graz, Austria) containing 1024 channels of width 54.0 μm was used for detection of scattered X-rays in the small-angle region. The working q range (Å
1) was 0.003 e q e 0.6, where q = 4π sin(θ)λ
1 is the modulus of the scattering wave vector. Solutions were usually taken from storage under refrigerator, heated to the required temperature, and allowed 15 min to equilibrate. A few milligrams of the sample was enclosed in a stainless-steel sample holder using a polymeric sheet (Bratfolie, Kalle) window. The diffraction patterns were recorded at 37 °C for 3 h. To minimize scattering from air, we kept the camera volume under vacuum during the measurements. Silver behenate (CH3
(CH2)20
COOAg) with a d-spacing value of 58.38 Å was used as a standard to calibrate the angular scale of the measured intensity. The lattice parameters (a) were determined for the cubic phases from the linear fits of the reciprocal spacing (1/dhkl) of the various reflections versus the sum of the Miller indexes (h2 + k2 + l2)1/2. For a correct assignment, the plot passes through the origin, and the slope is 1/a. 2.10. In Vitro Release Studies. For GMs alone, 5 mg weighed amount LGAGM and LGluGM was put in a test tube along with 5 mL of release media (PBS, pH 7.4.) Then, all samples were maintained at 37 °C in an incubator with shaking (100 rpm). An aliquot of a 2 mL sample of the supernatant solution after a brief centrifugation was withdrawn at predetermined time points and replaced with 2 mL of fresh phosphate buffer. A membrane-less diffusion system was used to study the release of lysozyme from LGAGM and LGluGM in PF127 gel.39 Accurately weighed 1 g sample of each type was put in a flat-bottomed vial (internal diameter about 18 mm), followed by gentle addition of 1 mL of phosphate buffer (pH 7.4) on the surface of gels; then, the vial was shaken in a thermostatic shaker at 37 °C and 100 rpm. Supernatant solution aliquots were withdrawn at predetermined time points and replaced with equal amount of fresh phosphate buffer. The concentration of bioactive lysozyme in the supernatant solution was determined using the method described in Section 2.3. To estimate the lysozyme release from lysozyme-loaded GMs, we subjected unloaded microspheres to same release conditions to obtain the actual release.

3. RESULTS AND DISCUSSION 3.1. Characterization of the GMs. GluGMs and GAGMs were prepared by thermal gelation method, as first described by Tabata and Ikada, and later modified by several authors.8,36,37 There are several different mechanisms proposed for crosslinking of gelatin by glutaraldehyde as well as D-glucose. In the case of cross-linking by glutaraldehyde, because of aldehyde functional groups, it is suggested that reaction occurs mainly with the ε-amino groups of gelatin lysyl residues, ultimately forming cross-linked product, as shown in Scheme 1a. In the case of D-glucose, a possible mechanism of reaction is that the sugar aldehyde group reacts with the free ε-amino groups of gelatin molecule producing aminoglycoside, which further reacts with another amine group producing a cross-linked structure, as shown in Scheme 1b. Lysozyme is a cationic antibacterial enzyme having isoelectric pH of ∼11; therefore, when adsorbed on the gelatin surface, it can react through the mechanism of polyion complexation. Gelatin B (isoelectric pH 4.7 to 5.2) was preferred over gelatin A (isoelectric pH 7.0
9.0) because of its higher anionic character. 3188

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The loading and encapsulation efficiencies of the two types of GMs, viz. LGAGM and LGluGM, used in this study differed significantly from each other, as displayed by data reported in Table 1. These differences are mainly due to the different methods used for loading the lysozyme into the microspheres. In the case of LGAGMs, the loading of lysozyme was done after preparing the empty GAGMs. In the case of LGluGMs, lysozyme was added before the emulsification in the aqueous gelatin and glucose solution. In the latter case, almost all the lysozyme was loaded successfully with encapsulation ratio ED (cf. eq 3) equal to 98 ( 2%. For LGAGMs, the ED was much lower, namely, 7.0 ( Scheme 1. Crosslinking Reaction Mechanism of Gelatin with (a) Glutaraldehyde and (b) D-Glucose

0.2%, because an excess amount of lysozyme was used for the loading procedure. Conversely, the loading capacity LD (cf. eq 2) of GAGMs was significantly higher (7.0 ( 0.2%) than that of GluGMs (1.96 ( 0.04%). It is noteworthy that in both cases the lysozyme did not lose its biological activity under the harsh conditions of formulation, as demonstrated by the assay used to evaluate the loading (cf. Section 2.3). The lysozyme-loaded GMs obtained through the two different cross-linking procedures described in Section 2.2 were dried under the vacuum and then further characterized by SEM technique. Figure 1 and Table 2 summarize significant SEM images and data obtained from image analysis. The images of both types of microspheres revealed that they were nearly spherical in shape with some differences in size and external surface texture. The LGAGMs are larger than LGluGMs as shown in Figure 1a,e, and the LGAGM surface is smoother than that of the LGluGMs, the latter showing a wavy surface with the presence of small plaques, as displayed in Figure 1b,f. These observations about the surface morphology of glucose and glutaraldehyde cross-linked microspheres are consistent with the literature.8,36 These differences in surface properties are again related to the different cross-linking agents. The formulation parameters that can significantly affect the particle size, surface morphology, and dispersion of gelatin microspheres are the concentration of the gelatin solution, the concentration of the emulsifier, the w/o ratio, the emulsifying time, and the stirring speed.40 Therefore, a concentration of gelatin solution of 20% w/v, a concentration of emulsifier (Span 80) of 1% v/v, a w/o ratio of 0.05, an emulsifying time of 10 min, and a stirring speed of 1000 rpm were kept constant to evaluate the Table 2. Size Distribution Parameters for LGluGM and LGAGM

Table 1. Loading and Entrapment Ratio of Lysozyme Loaded Gelatin Microspheres LD (%)

ED (%)

LGAGM

7.0 ( 0.2

7.0 ( 0.2

LGluGM

1.96 ( 0.04

98 ( 2

parameter

LGluGM (μm)

LGAGM (μm)

mean standard deviation

15.91 6.63

22.06 13.68

minimum median

a

4.80

2.72

14.50

17.39

maximum

48.02

70.49

D90a

25.23

42.65

90% of particles are of size less than this value.

Figure 1. SEM images of two types of microspheres at different magnifications: LGAGM (a
c) and LGluGM (e
g). Also shown are the size distribution histograms for LGAGM (d) and LGluGM (h). 3189

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Biomacromolecules effect of the two different cross-linking agents on the release properties. The image analysis of SEM micrographs leads us to obtain size distribution and other interesting features related to the different cross-linkers. Figure 1d,h shows the size distribution histograms for LGAGM and LGluGM, respectively. From these, the different particle size distributions between the two types of gelatin microspheres here compared clearly come out. Both types of gelatin microspheres showed a log-normal size distribution. Table 2 summarizes the various size distribution parameters for the two types of GMs. For LGluGM and LGAGM, the mean particle size was ∼16 and 22 μm, respectively. In the case of LGluGM, the D90 value (i.e., 90% of GMs are smaller than this value) was ∼25 μm, whereas in the case of LGAGM it was ∼43 μm. This clearly shows that the microspheres formed with glucose as cross-linking agent were relatively smaller in size than those obtained with glutaraldehyde. The formation of bigger LGAGM particles with respect to LGluGMs may be explained taking into account the different cross-linking procedure used (cf. Section 2.2). Specifically, for LGluGM, preparation glucose was added before the emulsification step, whereas for LGAGM preparation, glutaraldehyde was added after the formation of the un-cross-linked GMs. The latter procedure involves suspension of the microspheres in the glutaraldehyde ethanol solution for 4 h. During this time, it is likely that some microspheres swell because of the absorption of a considerable amount of water and ethanol inside them. In turn, the absorption of ethanol may be responsible for the formation of cavities inside the microspheres, as identified in SEM micrographs. An example can be seen in Figure 1c, where the cross-section of a large particle gives the clear evidence of a cavity. Nevertheless, it is not clear why only a huge inner cavity is observed rather than a homogeneous pore structure. As an alternative rationale, it can be suggested that the core of the bigger GMs may collapse because of an incomplete cross-linking process: As cross-linking is determined by diffusion, smaller particles may be completely cross-linked and therefore stable during drying. Differently, the bigger particle core might be un-cross-linked and therefore collapse during drying, originating the observed cavities. These microspheres did not fracture or shrink even after freeze-drying. It should also be remarked that when these microspheres are suspended in aqueous medium, some of them float over the surface because of the hollow space inside them. Conversely, the cross-section of LGluGMs did not reveal the presence of any cavity in the inner part of the microspheres, as shown in Figure 1g. To obtain qualitative information on the degree of crosslinking of gelatin microspheres prepared with the two different cross-linking agents, we used the ATR-FTIR technique. The infrared graphs of un-cross-linked gelatin microspheres and lysozyme-loaded gelatin microspheres cross-linked by glucose and glutaraldehyde are shown in Figure 2. In the IR spectrum of the three types of GMs, the characteristic absorption peaks of protein due to NH and CO stretching are observed. The NH stretching referred to as amide A peak is observed at ∼3300 cm
1 and CO stretching peaks referred to as amides I, II and III were found at ∼1650, 1550, and 1240 cm
1, respectively. Remarkably, the intensity of the CO stretching peaks was highest when glutaraldehyde was used for cross-linking compared with that with glucose, indicating that more carboxyl groups are activated for LGAGM than for LGluGM. An additional strong absorption peak, characteristic of an aldimine stretching vibration, is observed at ∼1450 cm
1,

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Figure 2. ATR-FTIR spectra of (a) LGAGM, (b) LGluGM, and (c) GM.

Figure 3. Change of elastic modulus as a function of temperature for 20% w/v PF127, 20% w/v PF127 + 0.5% w/v LGluGM, and 20% w/v PF127 + 0.5% w/v LGAGM. Phase transition processes of PF127 gel formulations characterized by the sudden increase in elastic modulus as a function of temperature.

thus proving the occurrence of the cross-linking reaction. (See Scheme 1.) Moreover, the significant differences found in the intensities of such an absorption peak for the two crosslinkers represent an additional confirmation that the use of glutaraldehyde favors the cross-linking process. 3.2. Characterization of the PF127 Gel Embedding the GMs. At first, a rheological characterization was performed to investigate the sol
gel transition process and the viscoelastic behavior of Pluronic F127 thermosensitive gels.41
44 Figure 3 shows that the sol
gel transition temperature of 20% PF127 alone is not affected by the presence of LGAGMs and LGluGMs, as reported in Table 3. The presence of the GMs becomes critical in the rheological properties of the system only when, above the sol
gel transition temperature, they can perturb the close-packed arrangement of the PF127 micelles with the consequent weakening of the elastic modulus of the gel systems. Viscosity data at increased temperatures were obtained through the oscillatory measurements to ascertain the practical application of final formulations. Table 3 summarizes the relevant parameters. It can be noted that the viscosity at 20 °C was very low in all cases. This implies that these formulations have the desired characteristic of being liquid at temperatures around or below 20 °C. Viscosity increases sharply at 22 °C and further at 35 °C. Similarly to what reported for the elastic modulus, complex viscosity (η*) decreases significantly at 3190

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all investigated temperatures when either LGAGMs or LGluGMs are added to the PF127 solutions. Whereas this finding is expected in the gel state because of the inclusion within the PF127 gel matrix of defects represented by the GMs themselves, it appears quite interesting that such an effect is also observed in the sol state at 20 °C, where the micelles are known to be wellseparated and the solution is an isotropic Newtonian fluid.45 It also deserves noticing that such a decrease in both the elastic modulus and η* may easily be related to the size of the gelatin microspheres: the bigger LGAGM microspheres provoke larger defects in the continuum of the PF127 micelles, thus inducing a higher decrease in the elastic modulus and η* with respect to LGluGMs. Clearly, this is more evident for a closer micellar packing that is at the highest temperature. Above the sol
gel transition temperature, the PF127-water systems display peculiar features that are not typical for gels. Indeed, the close micellar packing produces a long-range ordered nanostructure that is a discrete cubic liquid crystal. These features were investigates through SAXS analysis. The PF127 gel microstructure was determined by the relative positions of the Bragg peaks in the diffraction patterns shown in Figure 4. The three patterns are strikingly similar and show least√five √ weak √ at √ Bragg peaks with relative positions in ratios 3:2: 8: 11: 12, which can be indexed as hkl = 111, 200, 220, 311, 222 reflections of a face-centered cubic phase of Fm3m space group (Q225).24 Some not easily indexable weak peaks can be seen; however, the gel microstructure is essentially dominated by a clear Fm3m pattern. Two parameters are characteristic of the microstructure of the liquid crystalline phases based on EOnPOmEOn copolymers:23,24 the lattice parameter, a, and the interfacial area per EO block, ap. In the normal micellar cubic structure, the interfacial area per EO block can be calculated according to the following eq 4 ap ¼ ð36πnu f 2 Þ1=3

vp 2Φp a

ð4Þ

Table 3. Phase Transition Temperature (TT) and Complex Viscosity (η*) Data Recorded at Different Temperatures for PF127 Gel and PF127 Gel with Inclusion of LGluGM or LGAGM η* (Pa 3 s) at different T (°C) sample

TT (°C)

20

25

35

PF

21.90

0.105

15 544

21 650

PF+LGAGM

21.87

0.078

10 393

14 524

PF+LGluGM

21.86

0.083

11 023

16 551

where nu is the number of spherical micelles in the cubic cell, f is the apolar volume fraction, υp is the volume of one PF127 molecule, Φp is the copolymer volume fraction in the system, and a is the lattice parameter. The apolar volume fraction, f, is given by eq 5 f ¼ 0:34Φp

ð5Þ

where 0.34 is the volume fraction of the PO block in the pluronic F127 molecule (PO weight fraction = 0.30). To convert the weight fractions of the components into volume fractions, we use the bulk densities of the copolymer (1.05 g/cm3) and water. The volume of one molecule is 20 000 Å3. The aggregation number of the micelles, Nagg, defined as the number of P127 molecules per micelle can be determined using Nagg ¼

Φ p a3 n u vp

ð6Þ

In these equations nu = 4 for the unit cell of the face-centered cubic lattice consisting of four micelles. Mortensen et al have shown that 20% w/v PF127 gel may have face-centered cubic (fcc) or body-centered cubic (bcc) ordering depending on the proportion of diblock copolymer in the commercial triblock copolymer, with as-received samples forming fcc-structured gels and purified samples forming bcc gels.22 Recently, Chaibundit et al found that as-received PF127 forms an fcc structure that changes to a bcc structure upon the addition of 10
30% ethanol.46 Our results are in agreement with these reported findings; indeed, all samples used in this study were prepared with as-received PF127 and fcc nanostructures were obtained. No significant variations in the nanostructure of PF127 gel were found after inclusion of GMs; only small decreases, within 5% of the estimated error, in the lattice parameter are determined in the presence of GMs, as reported in Table 4. This result implies that lysozyme diffusion from GMs or diffusion of GMs themselves is not significantly affecting the discrete cubic LC structure of PF127 gel. 3.3. In Vitro Cumulative Release Studies. As demonstrated by the drug release profiles (Figure 5), the two microspheres showed a rather different behavior. Indeed, lysozyme release from LGluGM was quite rapid during the first hour (89%), reaching a plateau. Therefore, results seem to indicate that the degree of glucose cross-linking was too low to control GM swelling, thus leading to a very loose network matrix through which lysozyme could easily diffuse to be released. The lysozyme release from the LGAGM showed two well recognizable steps. Indeed, after the initial burst release (51% lysozyme released within the first hour), these microspheres

Figure 4. SAXS patterns of (a) PF127 20% w/v, (b) PF127 20% w/v + 0.5% w/v LGluGM, and (c) PF127 20% w/v + 0.5% w/v LGAGM. 3191

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Table 4. PF127 Volume Fraction (Φp), Apolar Volume Fraction (f), Lattice Parameter (a), Interfacial Area (ap), and Aggregation Number (Nagg) Obtained from SAXS Data for PF127 Gel and PF127 Gel with LGAGM and LGluGM Included sample name

PF127/W/GM

Φp

f

space group

a (Å)

ap (Å2)

Nagg

PF

20/80/0

0.192

0.065

Fm3m (fcc)

294 ( 9

238

56

PF+LGAGM PF+LGluGM

19.9/79.6/0.5 19.9/79.6/0.5

0.183 0.183

0.062 0.062

Fm3m (fcc) Fm3m (fcc)

281 ( 12 281 ( 2

244 235

45 50

lysozyme release. Indeed, as can be seen in Figure 6, release profile from both microspheres was very similar and showed a slow but constant enzyme release during the entire experiment. The same profile was shown by the lysozyme solution that, as expected, showed a quicker release. Remarkably, the PF127 LC matrix controls both the rate of the GM swelling and the rate of lysozyme release, leading to almost linear release profiles from both aqueous solution and GMs and reducing the differences between the two types of GMs. The use of a formulation based on GMs suspended in the liquid-crystalline phase of a polymer provides an interesting choice to attain sustained release and to reduce unwanted phenomena such as the initial burst release. Figure 5. In vitro release of lysozyme from LGAGM and LGluGM.

Figure 6. In vitro release of lysozyme from PF127 20% w/v and PF127 20% w/v loaded with LGAGM and LGluGM.

showed a slow sustained release of lysozyme up to the end of the experiments. Therefore, release experiments show the influence of both type and degree of cross-linking of the prepared microspheres. Gelatin is known to swell and solubilize in aqueous medium because of hydration. To improve release control, gelatin has to be cross-linked by reacting with glutaraldehyde and glucose that form bridges between the polymeric chains. However, the type of the bridges as well as the degree of crosslinking is fundamental in affecting microsphere swelling and therefore the lysozyme release. Swelling of GMs increases the mobility of the encapsulated lysozyme and hence a quick diffusion from the microspheres into the release medium occurs. Because the use of glutaraldehyde leads to higher cross-linking density than glucose, lysozyme is released at a higher rate from GluGMs than from GAGMs. Figure 6 shows the results of the lysozyme release experiments from LGluGM and LGAGM suspended in PF127 LC gel in comparison with lysozyme dissolved in PF127 LC gel. Suspension of the microspheres in the PF127 LC gel strongly affected

4. CONCLUSIONS The results of this study allow us to point out that the crosslinking of gelatin with glutaraldehyde and D-glucose produced two different cross-linked structures. The shape of microspheres was spherical in both cases, but the surface texture and the size distribution was clearly distinct. The infrared spectroscopy analysis showed that the degree of cross-linking density as well as lysozyme loading and encapsulation were significantly different for the two types of GMs. Despite the relatively harsh conditions of the preparation, particularly in the case of LGluGMs, it is worth noticing that lysozyme without losing its biological activity was successfully loaded onto both types of GMs. The 20% w/v PF127 gel is a discrete cubic LC phase having an Fm3m lattice structure that was not significantly affected from GMs presence. In addition, the presence of GMs did not affect the sol
gel transition temperature. Conversely, the elastic modulus and the complex viscosity of the PF127 LC gel were significantly reduced upon the addition of the GMs. The suspension of GMs in the PF127 LC gel allowed for a more sustained release of lysozyme from both LGluGM and LGAGM systems. Finally, it should be remarked that the combination of these two different formulation systems into one can give more options to control the drug release. Indeed, this new formulation might be used as a depot system for protein delivery provided that full biocompatibility is proved. ’ AUTHOR INFORMATION Corresponding Author

*Tel: +390706754453 (S.M.), +390706754385 (M.M.). Fax: +390706754388. E-mail: [email protected], [email protected].

’ ACKNOWLEDGMENT Alessandro Riva (University of Cagliari) is thanked for the use of SEM and useful suggestions. P.H. acknowledges MIUR for “Young Indian Scientist fellowships” for years 2008 and 2009. MIUR Prin 2008 and DM28142 are acknowledged for funding support. The Scientific Park “Sardegna Ricerche” (Pula, CA, 3192

dx.doi.org/10.1021/bm200679w |Biomacromolecules 2011, 12, 3186–3193

Biomacromolecules Italy) is acknowledged for free access to rheometer, FTIR, and SAXS. S.L. acknowledges the Scientific Park “Sardegna Ricerche” (Pula, CA, Italy) for her fellowship. C.C. was financed by Regione Autonoma della Sardegna under the Master and Back Program, reference code: PR1-MAB-A2008-433. We thank anonymous reviewers for very useful comments.

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