Lincomycin hydrochloride loaded albumin microspheres for controlled drug release, produced by Supercritical Assisted Atomization

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Lincomycin hydrochloride loaded albumin microspheres for controlled drug release, produced by Supercritical Assisted Atomization Renata Adami a,∗ , Sara Liparoti a , Giovanna Della Porta a , Pasquale Del Gaudio b , Ernesto Reverchon a a b

Department of Industrial Engineering, University of Salerno Via Giovanni Paolo II, 132-84084 Fisciano, SA, Italy1 Department of Pharmacy, University of Salerno Via Giovanni Paolo II, 132-84084 Fisciano, SA, Italy

a r t i c l e

i n f o

Article history: Received 5 August 2016 Received in revised form 20 September 2016 Accepted 20 September 2016 Available online xxx Keywords: Supercritical Assisted Atomization Lincomycin hydrochloride BSA Nanostructured microparticles Controlled release

a b s t r a c t Supercritical Assisted Atomization (SAA) is proposed for the production of nanostructured microparticles of Bovine Serum Albumin (BSA) loaded with lincomycin hydrochloride (lincoHCl). The process is used to coprecipitate BSA and lincoHCl, producing thermal denaturated BSA microparticles, entrapping the drug in the protein matrix. Several lincoHCl/BSA ratios in water solutions were processed, to produce protein microspheres with different size and drug content. SAA precipitation temperature was set as 100 ◦ C to obtain BSA coagulation and efficient entrapping of lincoHCl. Spherical microparticles showed no coalescence and were produced in all cases studied, with a mean particle size in the range 1–2 ␮m and loading efficiencies between 87 and 90%. The microspheres produced by SAA showed a controlled release of the drug over about 6 days. © 2016 Published by Elsevier B.V.

1. Introduction Lincomycin hydrochloride (lincoHCl) is an antibiotic of the group of licosamides and is used for human and veterinary medical applications. The active principle works against most gram positive bacteria, like Staphylococcus sp., Streptococcus sp., Clostridium sp., Bacillus anthracis and Corynebacterium sp., which usually can also be treated with penicillin or erythromycin. It is effective for the treatment of infectious diseases, like acne, forunculosis, burns and wounds [1–4]. LincoHCl is mainly used in injection and capsule formulations, although its efficacy in topical applications as gel, paste or aqueous spray has been proved [3,5]. The topical administration has the advantage of delivering the drug directly to the site of action, whereas a prolonged release of the drug can be tailored in order to improve the drug efficacy [6]. Polymeric microparticles that swell in presence of wound fluid, forming in situ hydrogels, can naturally adapt to the shape of the wound, therefore the probability of the formation of compartments, easily attacked by bacteria, is reduced [7–10]. If the polymeric carrier is adequately chosen, the

∗ Corresponding author. E-mail address: [email protected] (R. Adami). 1 www.supercriticalfluidgroup.unisa.it.

pharmacokinetic profile of the loaded drug can be improved and its efficacy prolonged [11–14]. Among all the available carriers, albumin is an ideal material to produce particles for drug delivery due to its nontoxic, nonimmunogenic, biocompatible and biodegradable properties [15]. Additionally, albumin particles show high binding capacity and no serious side effect are evidenced [16–18]. Moreover, the protein shows the properties of a natural polymer: when used as a carrier, allows a controlled and sustained drug release [19,20]. Bovine Serum Albumin (BSA) is able to form complexes and has been extensively used as a matrix for the encapsulation of several drugs [15,16,21–26]. It has a great affinity with hydrophilic compounds and, since it fast solubilizes in water, cannot be used for the controlled release without modifications. Heat treatments of BSA at high temperatures for long time generate intramolecular crosslinks and the new bonds give to the protein properties of hydrolytic resistance [27]. Thermal denaturation concerns the secondary structure of BSA, modifying the content of ␣-helix and ␤-sheet. Increasing the temperature, at 50 ◦ C the ␣-helices have a reversible unfolding and above 65 ◦ C the unfolding induces the formation of irreversible disulphide bridges [28]. Drug/BSA formulations can be prepared using several techniques: emulsion polymerization and stabilization using glu-

http://dx.doi.org/10.1016/j.supflu.2016.09.017 0896-8446/© 2016 Published by Elsevier B.V.

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taraldehyde, emulsion formulation with thermal crosslinking, pHcoacervation coupled with glutaraldehyde-ethanol cross-linking and emulsification-dispersion with high pressure homogenization, in which the shear forces induce oxidation and crosslinking [29–32]. Spray drying has been used to produce composite particles of active principles/BSA, obtaining thermal stabilization of the protein during particle drying and using glutaraldehyde as crosslinker [22,24,25]. All these techniques require a second process step for the formation of crosslinking, some of them use organic solvents and need a post processing for solvents and cross-linker removal; furthermore, it is difficult to control the particle size distribution. To overcome the limitations of traditional techniques, supercritical fluid based processes have been developed for several applications [33–35]. In particular, Supercritical Antisolvent (SAS), Supercritical Assisted Atomization (SAA) and Supercritical Extraction from Emulsion (SEE) have been developed to produce micro and nanoparticles [36–39]. SAA has been successfully used for the production of coprecipitates, as nanosctructured microparticles formed by a carrier in which the active principle is uniformly distributed [40–43] or as polymeric microparticles loaded with nanoparticles [44,45]. In SAA process, supercritical carbon dioxide (SC-CO2 ) is intimately mixed with the liquid solution containing the compound to be micronized in a high pressure vessel, in which the conditions of temperature and pressure are selected to get an expanded liquid solution. This solution, characterized by reduced viscosity and surface tension, is sprayed in a precipitator, obtaining an effective atomization. The droplets are dried using nitrogen at adequate temperature, that continuously flows in the precipitator during the injection of the solution [46–49]. SAA equipped with a vacuum system has been previously applied to produce BSA microparticles using precipitation temperatures of 70 ◦ C and 60 ◦ C. At the used conditions, no modification or denaturation of the protein was detected [50,51]. Using higher precipitation temperatures, it is possible to denaturate the BSA, with the consequent possibility to use it as a carrier for controlled and delayed release of hydrophilic drugs, as in the case of gentamicin/BSA [40]. SAA technique defeats the difficulties of the traditional techniques in controlling particle size and distribution of the coprecipitate and induces, during atomization, the denaturation of BSA with no use of chemical crosslinkers and post processing. In this work, we want to obtain nanostructured microparticles of BSA loaded with lincoHCl for the controlled release of this drug for topical applications, using SAA technique. Optimizing the operating conditions of the process, it is possible to have the stabilization of BSA due to the decrease of pH in the mixer during the solubilisation of SC-CO2 in the aqueous solution and by the thermal effect during precipitation and particle drying [52]. Particle size can be in the appropriate range for transdermal controlled release formulations, that can be obtained by direct spray or spreading the microparticulate powder directly on the wounds or incorporating the particles into gels, foams or pastes. Therefore, we will try to produce the composite microparticles in the range 1-5 ␮m, suitable for transdermal controlled release to be used for direct spraying on the wounds.

2. Materials and methods Bovine Serum Albumin (BSA) and lincomycin hydrochloride (lincoHCl) were supplied by Sigma-Aldrich (Milan, Italy). Water (HPLC grade) with a purity of 99.5% was supplied by Sigma-Aldrich (Milan, Italy). Carbon dioxide (CO2 ; purity 99.9%) and Nitrogen (N2 ; purity 99.9%) were purchased from SON (Naples, Italy). SAA laboratory apparatus used is composed by two highpressure pumps (mod. 305, Gilson, Villiers Le Bel France) to deliver the water solution and the liquid CO2 to the saturator. The satura-

tor is a heated high pressure packed vessel (volume: 25 cm3 ) which assures a large contact surface between liquid solution and CO2 . The expanded liquid obtained in the saturator is sprayed through a nozzle into the precipitator (volume: 3 dm3 ) that operates at atmospheric pressure. A controlled heated flow of N2 (about 1200 nL/h) is flown to the precipitator to enhance the evaporation of water from the droplets. A sintered filter at the bottom of the precipitator, with a porosity of 0.5 ␮m, allows the collection of the powder and the flowing through of the gases. SAA apparatus layout and further details can be found in previous papers [51,53]. The morphology of BSA and lincoHCl-BSA loaded particles has been analysed by a Field Emission Scanning Electron Microscope (FESEM, mod. LEO 1525, Carl Zeiss SMT AG, Oberkochen, Germany). Powders were dispersed on a carbon tab previously stuck to an aluminium stub (Agar Scientific, United Kingdom). Samples were coated with gold (layer thickness 250 A) using a sputter coater (mod. 108 Å, Agar Scientific, Stansted, United Kingdom). At least 20 SEM images were taken for each batch to verify the powder uniformity. Particle size (PS) and particle size distribution (PSD) were measured from FESEM images using the Sigma Scan Pro Software (rel. 5.0, Jandel Scientific, Erkrath, Germany). Histograms representing PSDs in terms of particles number and volumetric cumulative were best fitted using Microcal Origin Software (rel. 8.0, Microcal Software, Inc., Northampton, MA). SAA coprecipitates were also characterized by microanalysis to investigate their chemical structure. Elemental analysis and element mapping were performed using the FESEM equipped with an energy dispersive X-ray spectroscopy (EDX, INCA Energy 350, Oxford Instruments, Witney, United Kingdom). Diffraction patterns of co-precipitated powders were obtained using an X-ray diffractometer (mod. D8 Discover, Bruker AXS, Inc., Madison, USA). The measuring conditions were as follows: Nifiltered CuK radiation, ␭ = 1.54 Å, 2␪ angle ranging between 5◦ and 60◦ with a scan rate of 3 s/step and a step size of 0.2◦ . Thermograms of powder samples were obtained using a differential scanning calorimeter (DSC mod. TC11, Mettler Toledo, Inc., Columbus, USA). The samples (about 4 mg) were heated from 25 to 200 ◦ C at 5 ◦ C/min, under a nitrogen purge of 50 mL/min. Fourier Transform Infrared (FT-IR) spectra were obtained via M2000 FTIR (MIDAC Co), at a resolution of 0.5 cm−1 . The scan wavenumber range was 4000–400 cm−1 , and 16 scan signals were averaged to reduce the noise. Powder samples were ground and mixed thoroughly with potassium bromide (KBr) as infrared transparent matrix. Drug loading was studied using Pharmacopoeia HPLC (High Performance Liquid Chromatography) method (USP 29) [54] with some modifications for the measurement of the lincoHCl drug content. Samples of lincoHCl/BSA powder were dissolved under vigorous stirring in a solution water-acetic acid 1% v/v (pH 4) at 37 ◦ C. The solution was sonicated for 30 min and stored for 24 h to obtain the complete release of the drug from the carrier. The obtained solution was filtered to eliminate the BSA residue and diluted with water to increase the pH and analysed by HPLC-UV/vis (HewlettPackard model G131-132, USA). The column used is a reverse phase C18 column (4.6 mm × 250 mm; 5 mm particle size; 80 Å pore size; Hypersil BDS RP-C18); it was equilibrated at a flow rate of 1 mL/min with a mobile phase consisting of phosphate buffer pH 7.0 and acetonitrile (ratio 75:25 v/v). LincoHCl was monitored at 204 nm with a retention time of 10 min. Loading efficiency was calculated as the ratio of the drug content in the produced particles over the drug loaded at the beginning of the process. The drug release rate was performed using a 0.1 M Phosphate Buffer Solution (PBS) at pH 7.0 as dissolution medium. 100 mg of powder was suspended in 2 mL of PBS with 0.5% w/w of tween 80

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Fig. 1. SEM photomicrographs of BSA precipitated from water at 100 ◦ C, 20 mg/mL (a), 5 mg/mL (b).

and charged into dialysis tube (cut off 9–10 kDa). The tube was immersed in 200 mL of PBS continuously stirred at 200 rpm and 37 ◦ C. At stated time ranges, 2 mL of PBS solution was taken from the bottle, filtered and injected in the HPLC, following the method previously described. To keep constant the volume of the release medium, 2 mL of PBS was added to the system after each sample taking. The drug release conditions simulated the transdermal delivery in a wound tissue environment. 3. Results and discussion Preliminary studies were performed processing separately BSA and lincoHCl aqueous solutions to verify the feasibility of the SAA technique for these materials and to find suitable operating conditions to produce coprecipitates. BSA micronization was performed using a mass flow ratio between CO2 and water (GLR = Gas to Liquid Ratio) of 1.8, a saturator temperature of 85 ◦ C and pressure of 105 bar and a precipitator temperature of 100 ◦ C. GLR = 1.8 was chosen in to obtain in the saturator the right proportion of CO2 and solution [44–47]. The effect of the concentration of BSA in water from 5 to 40 mg/mL was studied. The particles obtained during these experiments showed a spherical morphology and were well separated. An example of these results is shown in Fig. 1a, where a SEM photomicrograph of BSA microparticles obtained at a concentration of 20 mg/mL is reported. At the lowest BSA concentrations tested (5 mg/mL), the majority of particles collapsed,

Fig. 2. PSD of BSA powders precipitated by SAA at different concentrations of BSA in water, in terms of number of particle (a) and volumetric cumulative (b) percentage. 10 mg/ml, 20 mg/ml, 40 mg/ml.

as can be observed in the photomicrograph in Fig. 1b, suggesting that the particles are like empty balloons formed by shell of solute. To obtain an explanation, we can refer to the theory of droplet formation in spray drying and spray pyrolysis, due to the similarity of these processes with SAA. In this process, when solutions with low concentration are processed, the precipitation of the solid starts on the droplet surface and a thin layer is formed. As a consequence, hollow particles are produced, with a shell thickness depending on the concentration of the starting solution. If the solid content is low, the shell is flexible and fragile and when the drying temperature is higher than the vapour pressure inside the particle, the structure can collapse, forming inflated particles [55–57]. In Fig. 2a the particle size distributions in terms of number of particle percentage are reported. The distributions of the particles at different concentrations show very similar mean diameters. However, BSA concentration modifies the maximum diameter of particles: in the calculations performed in terms of cumulative volumetric percentages, it is clearly shown that the percentage of particles with a larger diameter increases with the BSA concentration. At a concentration of 5 mg/mL of BSA in water, D90 is of 1.09 ␮m, whereas at 40 mg/mL it is 1.95 ␮m; therefore, particle size distribution enlarges as BSA concentration increases.

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Table 1 Operating conditions of the SAA experiments varying the concentration and the corresponding PSD data. conc = solute concentration, Pm = mixer pressure, Tm = mixer temperature, Pp = precipitator pressure, Tp = precipitator temperature. conc mg/mL

Pm bar

Tm ◦ C

Pp bar

Tp ◦ C

D10 ␮m

D50 ␮m

D90 ␮m

SD ␮m

150 20

96 107

86 83

1.5 2.0

94 90

0.86 0.42

1.98 1.00

3.04 1.60

1.07 0.61

In a previous work, it was found that SAA process can influence the structure of BSA: at precipitation temperatures higher than 90 ◦ C, relevant modifications can occur in its molecular structure [40]. The solubility of untreated BSA in water at room temperature was about 50 mg/mL; after SAA micronization at a precipitation temperature of 100 ◦ C, it decreased since the microparticles showed a tendency to produce hydrophobic aggregates in water, clearly indicating that the process caused the coagulation of the protein. Modification of the properties of BSA are an advantage for the use as drug carrier; indeed, the larger hydrolytic resistance and the cross-linked structure can lead to a slower dissolution rate of the carried drug. SAA experiments on lincoHCl alone were not successful; indeed during SAA processing the pores of the filter in the precipitation vessel were blocked and almost no material was collected at the end of the experiment. Probably, the precipitation temperature used (100 ◦ C) and the water used as solvent induced some modification, leading to the sublimation of the compound, or to a beginning of degradation of lincoHCl [58]. Therefore, there is a good reason to try to coprecipitate lincoHCl with BSA: the denaturated protein may entrap lincoHCl during particle formation and protect it from thermal effects, obtaining a stable coprecipitate. According to the previous considerations, BSA and lincoHCl were dissolved in water, forming an homogenous solution, that was mixed with SC-CO2 in the saturator and, then, was sprayed through the injector. The droplets obtained were dried in the precipitator, forming particles. These tests were performed at a GLR of 1.8, saturator temperature 85 ◦ C, saturator pressure 96–110 bar, precipitation temperature 100 ◦ C. First, the effect of the total concentration of the solutes in the solution was studied. As a rule, the concentration of solute is a relevant parameter in SAA processing: it influences viscosity, density and boiling point of the starting solution and, as a consequence, can modify the size of the droplets at the exit of the injector and the final particle size. Coprecipitation experiments were performed at lincoHCl/BSA ratio wt/wt R = 1/2 and at two different concentrations of total solute in water, 20 mg/mL and 150 mg/mL. In both cases spherical microparticles were obtained, as reported in Fig. 3, where examples of SEM photomicrographs are shown. As expected, increasing the concentration of the solution, the mean particle size increases (Table 1) and the size distribution enlarges (Fig. 4). Coprecipitated microparticles obtained at 20 mg/mL showed also a sharper PSD; therefore, this concentration was used for all the further coprecipitation experiments performed in this work. Keeping constant the total concentration of solutes in the starting solution and varying only R at 1/1, 1/2, 1/4, 1/6, several other experiments were performed. The powder obtained was again formed by regular spherical particles; in Table 2 PSD value are reported. It can be observed that, increasing the amount of BSA in the starting solution, there is a slight increase of particles size and an enlargement of the particle size distribution. All the experiments allowed to efficiently recover the precipitates, excluding the test at lincoHCl/BSA ratio 1/1 that produced a recovery lower than 1%. This result is related to the low amount of BSA into the feed solution, that did not properly entrap the drug during the formation of the microparticles and, therefore,

Fig. 3. SEM photomicrographs of BSA/lincoHCl coprecipitates obtained at concentration of 20 mg/mL (a), 150 mg/mL (b).

Fig. 4. PSD of lincoHCl/BSA micronized by SAA at different concentrations, in terms 150 mg/ml, 20 mg/ml. of number of particle percentage.

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Table 2 Operating conditions of SAA experiments at different global concentration, R = lincoHCl/BSA, conc = solute concentration, Pm = mixer pressure, Tm = mixer temperature, Pp = precipitator pressure, Tp = precipitator temperature, SD = standard deviation. R wt/wt

conc mg/mL

Pm bar

Tm ◦ C

Pcam bar

Tcam ◦ C

D10 ␮m

D50 ␮m

D90 ␮m

SD ␮m

1/6 1/4 1/2 1/1

20 20 20 20

105 106 107 110

84 85 83 85

1.7 1.9 2.0 5.0

100 100 100 100

0.80 0.65 0.42 2.18

2.09 1.70 1.00 3.46

3.58 3.10 1.60 4.64

1.19 1.07 0.61 1.60

Fig. 6. DSC thermograms of lincoHCl/BSA: comparison among untreated materials and microspheres produced by SAA. R = lincoHCl/BSA ratio, wt/wt.

Fig. 5. PSD of lincoHCl/BSA micronized by SAA at different R (lincoHCl/BSA ratio, R = 1/2, R = 1/4, wt/wt), in terms of cumulative volumetric percentage. R = 1/6.

a situation similar to the one of pure lincoHCl precipitation was reproposed. Furthermore, the powder obtained at R = 1/1 had the tendency to form aggregates, as can be noted from the data in Table 2, that show the largest PS for these particles. Although the total concentration of solutes in the starting solution was the same, it is interesting to point out that decreasing R and, therefore, increasing the BSA concentration in feed, leads to an increase of PS and an enlargement of PSD (Fig. 5). This is particularly evident in the cumulative volumetric PSD, in which the D50 clearly moves from 1.0 ␮m to 2.1 ␮m. DSC thermograms showed that lincoHCl has a melting point at 150 ◦ C and starts the degradation process at a temperature of about 200 ◦ C, BSA shows a melting point at about 220 ◦ C. The thermograms of their physical mixture show the melting peaks of both BSA and lincoHCl, with their intensity varying with R. In the thermograms of coprecipitates, reported in Fig. 6, the melting peak of lincoHCl is not present and the intensity of the peak related to BSA increases when R decreases, indicating that lincoHCl eventually entrapped in the microparticles is in an amorphous state. The thermograms also show the presence of an enlarged peak at around 70 ◦ C, that represents the evaporation of residual water in the SAA coprecipitates. This peak is especially evident in case of R = 1/4 and 1/2, in which there is the lower BSA content. This hypothesis of amorphous structure of coprecipitates is confirmed by XRPD analyses. They show for untreated lincoHCl a spectrum typical of crystalline form, as well as for the physical mixture, with the characteristic peaks of lincoHCl. For all the samples of SAA coprecipitates the spectra show the halo typical of the amorphous form, in particular the spectra are similar to the one of BSA. As an example, in Fig. 7 the spectra of untreated BSA and lincoHCl, the physical mixture and the coprecipitate at R = 1/4 are reported.

Fig. 7. XRPD spectra of lincoHCl/BSA: comparison among untreated materials and microspheres produced by SAA.

The physical mixture lincoHCl/BSA and SAA coprecipitates have been analysed by FTIR to get information about the presence of specific interactions. These spectra are reported in Fig. 8. In the physical mixture the characteristic groups of both compounds are present, indicating that no chemical or physical links have been formed between BSA and lincoHCl. In the case of SAA coprecipitates, the peaks related to the characteristic groups are overlapped and less defined compared to the ones in the physical mixtures, indicating that no new bonds are formed by lincoHCl and BSA that are however intimately mixed in the matrix structure. In particular, at wavelengths of 3290 and 1650 cm−1 , characteristic of the carboxylic acids, and of the primary amine, it is possible to observe an increase in intensity of the BSA characteristic peaks due to the presence of lincoHCl overlapping peaks. EDX analyses showed the presence of the elements characteristic of the two compounds (Fig. 9). In particular, oxygen (white in Fig. 9) is present in both BSA and lincoHCl molecules, sulphur (red in Fig. 9) is present only in the BSA molecule and chlorine (cyan in Fig. 9) is present only in the lincoHCl molecule. Fig. 9 shows that in the coprecipitates all three elements are present, confirming the presence of both BSA and lincoHCl in the particles; furthermore, the key chemical elements are uniformly distributed in the microparticles, confirming the hypothesis that lincoHCl and BSA are intimately mixed in each particle at nanometric level, forming microspheres. EDX analysis does not give a clear indication of the quantity of elements and, as a consequence, of the quantity of each compound in the samples. This information was given by HPLC analysis used to detect lincoHCl loading in the powder. The loading efficiency, expressed as the amount of lincoHCl in the coprecipitate

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Fig. 10. Release profiles from lincoHCl/BSA microspheres produced by SAA at different drug loadings. Symbols: experimental points (䊉 untreated,  R = 1/2, 䊐 R = 1/4, ♦ R = 1/6), lines: fitted by the relationship proposed by Korsmeyer-Peppas [59]. R = lincoHCl/BSA ratio, wt/wt.

Fig. 8. FT-IR spectra of untreated lincoHCl/BSA physical mixture (a) and lincoHCl/BSA/microspheres produced by SAA (b) at different R (lincoHCl/BSA ratio, wt/wt).

vs its amount in the starting solution, ranges between 87.5% and 98.6% with the highest value of the case R = 1/6, confirming the role of BSA in the entrapment of lincoHCl. Drug release studies have been performed using a dialysis bag in PBS solution pH 7.0, simulating the wet environment of a tissue, with the transdermal mass transfer simulated by the bag pores. As it can be noted in Fig. 10, lincoHCl raw material completely dissolves in about 24 h. The release of lincoHCl from BSA coprecipitates is slower, even if particle size is considerably smaller than the 100 ␮m needles of untreated lincoHCl, and it is controlled by lincoHCl/BSA

ratio. The higher is the amount of BSA, the slower is the release. Indeed, coprecipitates obtained at R = 1/2 and R = 1/4 released lincoHCl in about 2 days (48 and 52 h, respectively) whereas for R = 1/6 coprecipitates, drug was released in more than 6 days. Particularly, 97% of the drug was released after about 45 h for R = 1/2, 95% after about 52 h for R = 1/4 and 77% after about 153 h for R = 1/6. An observation of the morphology of the microparticles after the release tests showed that the particles were dissolved and formed gel-like structures, suggesting a swelling of BSA during the release of the drug. Such observations confirmed that thermal coagulation of BSA obtained during SAA micronization entraps lincoHCl and has a barrier effect, delaying the release of the drug. In the release curves reported in Fig. 10, it can be observed that there is no burst effect, that is typical of the presence of active principle on the surface of microspheres, and the mechanism, at least in the starting

Fig. 9. EDX microanalysis of SAA coprecipitated lincoHCl/BSA microparticles. Particulars of: SEM image of the analysed areas; oxygen maps (white); sulphur maps (red); chlorine maps (cyan). R = lincoHCl/BSA ratio, wt/wt. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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part of the release, can be fitted by the simple relationship proposed by Korsmeyer-Peppas [59]: Mt = kt n M0

(1)

where Mt /M0 is the drug released at time t, k is the release rate constant and n is the release exponent, which value characterizes the release mechanism of drug. The exponent of n has to be calculated using the portion of the release curve where Mt /M0 < 0.6 and in this case, for all the R, n values are in the range 0.47–0.49 for all R used, corresponding to a Fickian diffusion mechanism. Denaturated BSA, acting as a barrier to the drug diffusion, is able to strongly prolong the release. Moreover, when its concentration into the microparticles is relatively high, as in the case of R = 1/6 formulation, its hydrophobicity also reduces water diffusion into the particle matrix slowing down polymer chain relaxation time and a further reduction of release rate of lincoHCl over 6 days. Such properties can be used to tune particle drug release behavior in accordance to their administration as antibacterial formulations; furthermore, these observed behaviors suggest the use of lincoHCl/BSA nanostructured microparticles for topical applications on wounds. 4. Conclusions SAA technique demonstrated to be effective for the production of microparticles of lincoHCl/BSA with mean diameters from 1 to 2 ␮m. The particle size and distribution change with the ratio between the active principle and the carrier. During the atomization process the modification of the protein secondary structure of BSA and the entrapment of lincoHCl in the microparticle matrix take place. LincoHCl and BSA are uniformly distributed in the co-precipitate microparticle structure. BSA stabilizes and protects the drug during the microparticle formation, and the denaturation of the protein increases its hydrolytic resistance and gives the property of barrier for the drug diffusion. The dissolution rate of lincoHCl is reduced and its release is prolonged, showing a diffusion mechanism. Varying the lincoHCl/BSA ratio, the drug delivery can be controlled and the drug release rate modulated; moreover, increasing the amount of BSA in the microparticles, it is possible that, some other phenomena, like for example swelling mechanism, couple with the water diffusion inside the carrier matrix and the release can be prolonged over 6 days. The characteristics of nanostructured microparticles produced suggest their use for topical applications on wounds. Acknowledgements The authors gratefully acknowledge Dr. Valentina Gregori and Dr. Filomena De Rosa for the help in performing the micronization experiments and Dr. Mariarosa Scognamiglio for the expertise in chemical analyses. MIUR (Ministero dell’Istruzione, dell’Università e della Ricerca) is acknowledged for the financial support. References [1] M. Rajeswaran, T. Srikrishnan, Crystal and molecular structure and absolute configuration of lincomycin hydrochloride monohydrate, Carbohydr. Res. 339 (2004) 2111–2115. ˇ [2] J. Spíˇzek, T. Rezanka, Lincomycin, clindamycin and their applications, Appl. Microbiol. Biot. 64 (2004) 455–464. [3] L. Panigrahi, S. Ghosal, S. Pattnaik, L. Maharana, B. Barik, Effect of permeation enhancers on the release and permeation kinetics of Lincomycin hydrochloride gel formulations through mouse skin, Indian J. Pharm. Sci. 68 (2006) 205–211. [4] P. Rajeevkumar, N. Subramanian, Spectrophotometric method for the determination of lincomycin hydrochloride in pure form and pharmaceutical formulations, Int. J. Chem. Tech. Res. 2 (2010) 2052–2055.

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Please cite this article in press as: R. Adami, et al., Lincomycin hydrochloride loaded albumin microspheres for controlled drug release, produced by Supercritical Assisted Atomization, J. Supercrit. Fluids (2016), http://dx.doi.org/10.1016/j.supflu.2016.09.017