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Production of lipospheres as carriers for bioactive compounds
Biomaterials 23 (2002) 2283–2294
Production of lipospheres as carriers for bioactive compounds Rita Cortesia, Elisabetta Espositoa, Giovanni Lucab, Claudio Nastruzzib,* b
a Department of Pharmaceutical Sciences, University of Ferrara, 1-44100 Ferrara, Italy Department of Pharmaceutical Chemistry and Technology, Istituto di Chimica e Tecnologia del Farmaco, via del Liceo, University of Perugia, I-06100 Perugia, Italy
Received 1 March 2001; accepted 8 October 2001
Abstract Aim of the present paper was to investigate the influence of preparation parameters on the production of lipospheres (LS) for drug delivery. LS composed of triglycerides and monoglycerides were alternatively produced by melt dispersion technique, solvent evaporation or w/o/w double emulsion method. The influence of preparation parameters, such as (a) type and amount of lipids, (b) presence and concentration of surfactants, (c) stirring speed and (d) type of stirrer was studied. In the case of LS prepared by melt dispersion, the use of a lipid composition of cetyl alcohol/cholesterol (2:1, w/w), a 5% (w/w) gelatin solution (50 bloom grades) and 1000 rpm stirring speed resulted in the production of spherical particles, with high percentage of recovery (82%, w/w) a mean diameter of 80 mm and a narrow size distribution. In the case of LS prepared by solvent evaporation, the best results in terms of LS morphology, recovery and size distribution were obtained by the use of a lipid composition of tristearin/monostearate (66:34, w/w), a 1% (w/w) PVA solution, a 750 rpm stirring speed and a 55 mm three-blade turbine rotor. The solvent evaporation method resulted in the production of LS characterised by a smaller size (20 mm mean diameter) but poor mechanical properties with respect to particles with the same composition obtained by the melt dispersion technique (170 mm mean diameter). The use of a combination of lipids and a methacrylic polymer (Eudragit RS 100) overcame this problem, resulting in the production of spherical particles, with a narrower size distribution and good mechanical properties. Two lipophilic drugs, such as retinyl acetate and progesterone, and one hydrophilic drug, sodium cromoglycate (SCG), were encapsulated in LS as model compounds. Lypophilic drugs displayed satisfactory encapsulation efficiencies (over 70% w/w), while SCG was very scarcely encapsulated (about 2% w/w). To solve this drawback, the use of a w/o/w double emulsion strategy was proposed, enabling to increase the encapsulation of SCG up to 50% w/w. Finally, in vitro drug release studies were performed, showing that all drugs were released in a control manner. In particular, the retinyl acetate release efficacy within the first 8 h was 27% of the total amount of the drug, while in the same period, the amount of progesterone released was 63%. With regard to SCG containing LS, the release of the drug was largely influenced by the type of stabiliser of the primary emulsion, in any case the SCG release reached the 100% of the total amount of drug after 5 h from the beginning of the experiment. r 2002 Elsevier Science Ltd. All rights reserved. Keywords: Lipospheres; Drug delivery; Solid lipid nanoparticles (SLN); Solvent evaporation; Melt dispersion
1. Introduction One approach for increasing the beneficial action of drugs and decreasing systemic adverse effects is to deliver the necessary amount of drugs to the diseased sites, where they are most needed, for the appropriate period of time [1–3].
*Corresponding author. Tel.: +39-075-5855158; fax: +39-0755847469. E-mail address: [email protected] (C. Nastruzzi).
Although the drug delivery system concept is not new, great progress has recently been made in the treatment of a variety of diseases. Particulate carriers (e.g. polymeric nano- and microparticles, fat emulsion, liposomes) possess specific advantages and disadvantages. For instance, in the case of polymeric microparticles the degradation of the polymer might possibly cause systemic toxic effects by impairment of the reticuloendothelial system [4] or by accumulation at the injection site [5]; cytotoxic effects have been indeed observed in vitro after phagocytosis of particles by human macrophages and granulocytes [6]. In addition, organic solvents residues deriving from the
0142-9612/02/$ - see front matter r 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 1 4 2 - 9 6 1 2 ( 0 1 ) 0 0 3 6 2 - 3
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preparation procedures such as the solvent evaporation technique often used for liposome [23] and polyester microparticles [24] can be present in the delivery system and could result in severe acceptability and toxicity problems [7]. In order to solve these adverse effects, lipid microspheres, often called lipospheres (LS), have been proposed as new type of fat-based encapsulation system for drug delivery of bioactive compounds (especially lipophilic compounds). LS consist of solid microparticles with a mean diameter usually comprised between 0.2 and 500 mm, composed of a solid hydrophobic fat matrix, where the bioactive compound(s) is dissolved or dispersed [8–10]. LS have some advantages over other delivery systems, such as (a) good physical stability, (b) low cost of ingredients, (c) ease of preparation and scaleup and (d) high entrapment yields for hydrophobic drugs. Due to their large particle size range, LS can be administered by different routes, such as oral, subcutaneous, intramuscular, topic or used for cells encapsulation being thus proposed for the treatment of a number of diseases [11–13]. For instance, the in vivo distribution of LS demonstrated a high affinity to vascular wells (including capillaries), to inflamed tissues and to granulocytes [14,15]. LS have been used for the controlled delivery of various types of drugs, including vasodilator and antiplatelet drugs, anti-inflammatory compounds, local anaesthetics, antibiotics and anti-cancer agents; they have also been used successfully as carriers of vaccines and adjuvants [14,16,17]. This paper will discuss (a) the production and characterisation of LS formed by emulsion-melt dispersion technique, by solvent evaporation method, and by w/o/w double emulsion method, (b) the influence of preparation parameters on LS morphology, (c) the encapsulation efficiency and the release characteristics of two lipophilic model drugs, such as retinyl acetate and progesterone, and one hydrophilic drug, sodium cromoglycate (SCG), from the prepared LS. To obtain a biocompatible formulation suitable for human administration, triglycerides and monoglycerides have been chosen as LS’ biomaterials, in reason of their high biocompatibility, high physico-chemical stability and drug delivery release.
monostearate and glyceryl monooleate were from Eastman Chemical Company the used gelatines (250, 200, 150, 50 Bloom) were from Fluka and the polyvinyl alcohol (PVA) was from Air Products and Chemicals (Utrecht, Netherlands). All the other materials and solvents were from Fluka and were of the highest purity available. 2.2. Preparation of LS The preparation of LS was performed using two alternative techniques, as following described (see Fig. 1A and Table 1). 2.2.1. Melt dispersion technique Five grams of a lipidic mixture, with/without 200 mg of lipophilic model drug, were melted at 701C then emulsified into 150 ml of an external aqueous phase containing a suitable surfactant. Eight natural or synthetic emulsifiers were employed, namely gelatin (200 bloom), pectin, carrageenan l; k; i; PVA, polyoxyethylene 20 sorbitan trioleate (polysorbate 85, tween 85) and lauryl sarcosine. The emulsion was stirred using
2. Materials and methods 2.1. Materials Retinyl acetate and progesterone were obtained from Fluka Chemical Co. (Buchs, Switzerland), glyceryl tristearate (tristearin), glyceryl tribehenate (tribehenin) and glyceryl tripalmitate (tripalmitin) were from Gattefosse! (Saint-Priest Cedex, France), the glyceryl
Fig. 1. (A) Schematic representation of LS’ methods of production: melt dispersion and solvent evaporation method. (B) Impellers employed for microsphere production: (a) a three-blade rotor with a diameter of 55 mm (taken as reference impeller); (b) a four-blade helicoidal rotor with a diameter of 50 mm; (c) a two-blade rotor with a diameter of 50 mm and finally (d) a double truncated cone rotor with a diameter of 50 mm.
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Table 1 Overview of LS preparation methods Method
Disperse phase
Dispersion medium
Melt dispersion
Melted lipid (5 g) at 701C
Water (150 ml) plus stabilisera
o/w Solvent evaporation
w/o/w Double emulsion
Dissolved lipids (5 g) in ethyl acetate (10 ml) at 501C
w/o Emulsion of melted lipids (5 g) at 701C stabilised with gelatin or poloxamer 407
Stirring speed (rpm) 500
Water (150 ml) plus PVA as stabiliser
Water (150 ml) plus 0.25% (w/v) PVA as stabiliser
750 1000 500 750 1000 500
Process duration (h)
Particle isolation
1 with rapid cool up to 201C
Filtration through a glass filterb
6–8 at room temperature
Filtration through a glass filterb
3–5 at room temperature
Filtration through a glass filterb
750 1000 a b
See Table 6. Glass filters with maximum nominal pore size 10–16 mm.
a mechanical stirrer Eurostar Digital (IKA Labortechnik, Germany) equipped with alternative impellers, namely: (a) a three-blade rotor with a diameter of 55 mm (taken as reference impeller), (b) a four-blade helicoidal rotor with a diameter of 50 mm, (c) a double truncated cone rotor with a diameter of 50 mm and finally (d) a two-blade rotor with a diameter of 50 mm (see Fig. 1B). Afterwards the emulsion was heated to the same temperature as the melted lipidic phase. The milky formulation was then rapidly cooled to about 201C by immersing the formulation flask in a cool water bath without stopping the agitation to yield a uniform dispersion of LS. The obtained LS were then washed with water and isolated by filtration through a paper filter. 2.2.2. Solvent evaporation technique Five grams of the lipid matrix dissolved in 10 ml of an organic solvent such as ethyl acetate at 501C were emulsified in 150 ml of an external aqueous phase containing the surfactant agent using a mechanical stirrer Eurostar Digital (IKA Labortechnick Germany). The resulting oil-in-water emulsion was stirred for 6–8 h under ambient conditions to allow the solvent evaporation. LS, after water rise, were collected by filtration through a paper filter. 2.2.3. Water-in-oil-in-water double emulsion (w/o/w) method This method consists in the solubilization of the drug to be encapsulated in the internal aqueous phase of a w/ o/w double emulsion, along with a stabiliser able to prevent loss of drug to the external phase during solvent evaporation [18]. One millititer of an aqueous solution of 100 mg of drug was emulsified in 5 g of melted lipids at 701C by using an ultra-turrax (IKA, Labortechnik,
Germany, EU). This primary emulsion was stabilised by adding as stabilisers, gelatin (250 bloom grades) or the polyoxyethylene–polyoxypropylene block copolymer, poloxamer 407, solubilized in the aqueous phase. The primary emulsion was then dispersed, at 701C, into 150 ml of an aqueous phase containing 0.25% (w/v) of PVA. The obtained double emulsion was stirred at 300 rpm by a four-blade turbine impeller. After 3–5 h microparticles were isolated by filtration. 2.3. LS morphological analysis The morphology of LS was evaluated by optical microscopy (Nikon Diaphot inverted microscope, Tokyo, Japan) and scanning electron microscopy (SEM) observations (360 Stereoscan Cambridge Instruments Ltd, Cambridge, UK). Microsphere size distributions were determined by photomicrograph analyses, analysing at least 300 microparticle/sample. 2.4. Drug content of LS The amount of encapsulated model drug (retinyl acetate or progesterone) per mg of dried LS was determined by solubilisation of 50 mg of LS in 2 ml of ethyl acetate at 601C. Following filtration the solution was analysed by reverse phase HPLC to find the drug content. The HPLC determinations were performed using a HPLC system consisting of a Bruker apparatus (Bremer, FRG) consisting of a three plungers alternative pump, a variable wavelength UV-detector, operating at 215 nm, and a Rheodine Inc. injection valve with a 100 ml loop. Samples were chromatographed on a stainless steel C18 reverse-phase column (15 0.76 cm) (Supelco) packed with 5 mm particles (Model LC-18-DB, Supelco, USA),
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eluted isocratically at room temperature, at a flow rate of 1 ml/min. The mobile phase used for the drugs were 180 mm ammonium acetate (pH 3.0)/methanol (4:96, v/v) for retinyl acetate [19], methanol/water (70:30, v/v) for progesterone [20] and phosphate buffer (pH 2.3)/ methanol (50:50, v/v) for cromoglycate [21]. Drug detection was monitored at the lmax characteristic of each compound. 2.5. In vitro drug release In vitro drug release profiles were obtained placing 50 mg of drug containing LS in 10 ml of buffer under magnetic stirring at 150 rpm. The buffer was ethanol/ water, 30:70 with addition of 0.5% (w/w) of polysorbate 20. Following different length of time (0–8 h), 200 ml of receiving buffer, were filtered and analysed for drug content by reverse phase HPLC as previously described.
3. Results and discussion 3.1. LS preparation LS were prepared by two alternative approaches, namely the emulsion-melt dispersion or the solvent evaporation technique, as described in the experimental section.
Fig. 2. Effect of gelatin bloom on morphology and particle size of cetyl alcohol:cholesterol 2:1 (w/w) LS. SEM (A) and optical micrographs (B) of microspheres produced with gelatin 200 bloom grades (#7). Bar corresponds to 50 and 250 mm in panel A and B, respectively.
3.2. LS prepared by melt dispersion technique The choice of the lipid matrix plays an important role on the morphology of the particles and on the possible formation of aggregates. In a first set of experiments, LS were prepared by the emulsion melt dispersion technique (see Fig. 1A and Table 1) using a lipid mixture constituted of cetyl alcohol and cholesterol (2:1, w/w) and gelatin as stabiliser. The resulting microparticles (see Fig. 2 and Table 2) were characterised by an irregular surface, in addition some aggregates caused by the fusion of lipid droplets before solidification, were present. An improvement of LS features was obtained in term of recovery, mean diameter and aggregate formation by decreasing the molecular weight of the gelatin used as stabiliser. The viscosity of the dispersing phase was in fact progressively reduced passing from gelatin 50 to 250 bloom grades (batches 3, 4, 6, 7). By adjusting the stirring speed during the emulsification process, it was possible to modify the size of the particles. Increasing the stirring speed from 500 to 1000 rpm, the mean diameter of particles progressively decreased (see Table 3) (batches 11, 13, 14). With the aim to further improve the characteristics of LS, alternative lipid compositions were considered. For
instance, LS were prepared with apolar triglycerides, such as tristearin, tripalmitin or tribehenin in combination with other polar (more hydrophilic) lipids, including glyceryl monostearate, glyceryl monooleate, cetyl alcohol and cholesterol (see Table 4). As general consideration all formulations, apart from those including cholesterol and glyceryl monooleate, were satisfactory in term of shape, recovery and size (Figs. 3 and 4). Other experiments were undertaken to evaluate the effect of glyceryl monostearate concentration on LS characteristics. Glyceryl monostearate was used in mixture with tristearin (batches 57, 62, 55, 52, 54) or tripalmitin (batches 11, 64, 60, 59, 24) at different weight ratios. In Table 5 are reported the results of such experiments in which the percentage of glyceryl monostearate was varied from 0 up to 33% (w/w). In the case of tripalmitin, was impossible to produce LS without the presence of at least 1% of glyceryl monostearate (due to the formation of large blobs), while pure tristearin particles were obtained even if they are of poor quality. By increasing the content of glyceryl monostearate a progressive decrease of LS size was evident. No effect was on the contrary detectable on LS recovery.
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Table 2 Effect of gelatin type on LS characteristics Batch
Lipid composition (w/w ratio)
#6 #7 #4 #3
Cetyl Cetyl Cetyl Cetyl
alcohol/cholesterol alcohol/cholesterol alcohol/cholesterol alcohol/cholesterol
(2:1) (2:1) (2:1) (2:1)
Gelatin bloom grades
Mean diameter (mm)
Recovery (%)a
250 200 150 50
250712 205715 19778 150721
52 60 61 82
a Percentage (w/w) of LS production with respect to the total amount of lipids used for the preparation. Experimental parameters were: a 55 mm three-blade turbine rotor, a 5% gelatin solution and 750 rpm stirring speed. Data represent the average of three independent experiments on different microsphere batches 7SD.
Table 3 Effect of stirring speed on characteristics of cetyl alcohol/cholesterol LS Batch
Stirring speed (rpm)
Gelatin bloom grades
Mean diameter (mm)
Recovery (%)a
#13 #11 #14
500 750 1000
50 50 50
250714 150716 80724
92 82 82
a Percentage (w/w) of LS production with respect to the total amount of lipids used for the preparation. Experimental parameters were: a 55 mm three-blade turbine rotor and a 5% gelatin solution. Data represent the average of three independent experiments on different microsphere batches 7SD.
Table 4 Effect of lipid composition on the production of LS Batch
Lipid composition (w/w ratio)
Mean diameter (mm)
Recovery (%)a
#25 #26 #27 #24 #21 #22 #23 #51
Tristearin:monostearate (2:1) Tristearin:cetyl alcohol (2:1) Tristearin:cholesterol (2:1) Tripalmitin:monostearate (2:1) Tripalmitin:monooleate (2:1) Tripalmitin:cholesterol (2:1) Tripalmitin:cetyl alcohol (2:1) Tribehenin:monostearate (2:1)
170719 200726 250716 25078
90 92 98 82 Fused mass Fused mass
300715 200722
96 75
a Percentage (w/w) of LS production with respect to the total amount of lipids used for the preparation. Experimental parameters were: a 55 mm three-blade turbine rotor, a 1% PVA solution and 750 rpm stirring speed. Data represent the average of three independent experiments on different microspheres batches 7SD.
3.3. Effect of the stirring speed
3.4. Effect of impeller geometry
The effect of the stirring speed was considered on the production of LS (see Table 6 and Fig. 5). LS with dimensions comprised between 170 and 90 mm were obtained by changing the stirring speed from 500 to 1000 rpm (batches 25, 28, 29). In particular, particles obtained at 1000 rpm presented a spherical geometry with a narrow size distribution; in addition, aggregation phenomena were almost absent. The recovery efficiency of particles produced at the higher stirring speed was 69%; whilst for those produced at 500 rpm was over 90%.
LS (batches 28, 34, 35, 36), were produced by means of different impellers as described in the experimental section and displayed in Fig. 1B. The use of rotors (b) and (c) allowed obtaining smaller particles with mean diameters of 50 and 55 mm and a recovery of 64% and 77%, respectively (Table 7). On the other hand, the use of the rotor (d) did not allow the production of lipid particles, in fact this particular impeller caused the formation of elliptical particles and filaments.
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Fig. 3. Effect of lipid composition on the morphology of LS. LS were prepared with cetyl alcohol:cholesterol 2:1 (w/w) (#3) (A), tripalmitin:glyceryl monostearate 2:1 (w/w) (#24) (B) tristearin:glyceryl monostearate 2:1 (w/w) (#25) (C) and tripalmitin:glyceryl monostearate 9:1 (w/w) (#59) (D). Bar corresponds to 300, 600, 200 and 375 mm in panel A, B, C and D, respectively.
Fig. 4. SEM photographs showing the effect of lipid composition on the morphology of LS. LS were prepared with tristearin:glyceryl monostearate 2:1 (w/w) (#25) (A), tristearin:cetyl alcohol 2:1 (w/w) (#26) (B), tripalmitin:glyceryl monostearate 2:1 (w/w) (#24) (C), and tripalmitin:cetyl alcohol 2:1 (w/w) (#23) (D). Bar corresponds to 125, 160, 200 and 150 mm in panel A, B, C and D, respectively.
3.5. Effect of the stabiliser type and concentration The effect of different stabilisers (see list in the Materials and Method section) was tested on particle morphology and recovery (see Table 8). As clearly appreciable from the obtained results, the addition of
emulsifiers, leads to variable effects on the size of LS droplets during the emulsification step thus influencing the final microspheres size. In particular, LS obtained with natural polymers, such as gelatin, pectin and carrageenans k and i (batches 32, 40, 42, 43) allowed the production of spherical particles with an irregular
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Table 5 Effect of monostearate on the production of LS Batch
Lipid composition (w/w ratio)
Mean diameter (mm)
Recovery (%)a
#57 #62 #55 #54 #52 #11 #0 #64 #60 #59 #24
Tristearin:glyceryl monostearate 100:0 Tristearin:glyceryl monostearate 98:2 Tristearin:glyceryl monostearate 95:5 Tristearin:glyceryl monostearate 90:10 Tristearin:glyceryl monostearate 80:20 Tristearin:glyceryl monostearate 66:33 Tripalmitin:glyceryl monostearate 100:0 Tripalmitin:glyceryl monostearate 99:1 Tripalmitin:glyceryl monostearate 95:5 Tripalmitin:glyceryl monostearate 90:10 Tripalmitin:glyceryl monostearate 66:33
220731 200723 170718 160715 158728 170719
76 93 90 89 73 90 Fused mass
30079.0 300716 300731 250721
96 75 72 82
a Percentage (w/w) of LS production with respect to the total amount of lipids used for the preparation. Experimental parameters were: a 55 mm three-blade turbine rotor, 750 rpm stirring speed, 1% PVA solution as disperding phase. Data represent the average of three independent experiments on different microsphere batches 7SD.
Table 6 Effect of stirring speed on tristearin LS characteristics Batch
Stirring speed (rpm)
Mean diameter (mm)
Recovery (%)a
#25 #28 #29
500 750 1000
170735 120724 90719
90 78 69
a
Percentage (w/w) of LS production with respect to the total amount of lipids used for the preparation. Experimental parameters were: a 55 mm three-blade turbine rotor and a 1% PVA solution. Data represent the average of three independent experiments on different microsphere batches 7SD.
surface and a mean diameter comprised between 150 and 250 mm. In the case of carrageenan l (batch 41) it was not possible to isolate the particles since the high viscosity of the suspension did not allow the filtration process. On the contrary, in the cases of gelatin and others carrageenans the high viscosity was compatible with the separation process even if caused by a lower recovery efficiency (comprised between 62% and 69%) with respect to pectin (80%). The use of synthetic emulsifiers gave different results (batches 28, 30, 39, 49); for instance the use of 1% (w/w) of the polyoxyethylene–polyoxypropylene block copolymer Pluronic PE 8100 did not allow the stabilisation of the o/w emulsion during the preparation, thus resulting in the formation of large lipid aggregates. The use of 1% of polyoxyethylene sorbitan trioleate or PVA allowed the production of spherical particles with a mean diameter of 190 and 120 mm and a recovery of 54% and 78%, respectively. Finally, lauryl sarcosine caused the formation of a very fine o/w emulsion resulting in the final formation of very small particles (mean diameter 1074.1 mm) that were isolated by centrifuga-
tion. In this respect further studies are in progress to evaluate the experimental parameters for the production of lipid nanoparticles. 3.6. LS prepared by solvent evaporation method As alternative to the melt dispersion technique, a solvent evaporation method was also tested for the production of LS (Fig. 1B). This approach was considered with the aim to possibly reduce the exposure to high temperature of thermolabile compounds, such as proteins and nucleic acids. The solvent evaporation method is based on the evaporation of the organic solvent in which lipids are dissolved, allowing the formation of solid microparticles. By this technique were produced LS constituted of tristearin:glyceryl monostearate 2:1 w/w, in the presence of 1% PVA as emulsifier agent. The obtained particles (see Fig. 6) were spherical and characterised by a smaller size with respect to the particles with the same composition produced by the melt-fusion technique. Unfortunately, the produced LS showed some poor mechanical properties being fragile together with a higher proportion of interparticellar bridges as compared to the melt dispersion technique. With the aim to improve the mechanical properties of LS produced by solvent evaporation, as well as to obtain prolonged release profiles, the possibility to produce particles with a mixed matrix, was considered (see Table 9). LS were produced using lipids in combination with different polymers, in ratio up to 20% with respect to the lipid components. Both biodegradable, such as polylactic acid (PLA) and nonbiodegradable polymers, such as Eudragit RS 100, were used. The different polymers allowed the improvement of the mechanical characteristics of the LS and, particularly in the case of Eudragit RS 100, allowed
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Fig. 5. Optical microscopy photographs showing the effect of stirring speed on morphology and particle size of tristearin:monostearate 2:1 (w/w) LS. Optical micrographs and frequency distribution plot of microspheres produced at 500 rpm (#25) (A–B), 750 rpm (#28) (C–D) and 1000 rpm (#29) (E– F). Bar corresponds to 350, 600, and 800 mm in panel A, C and E, respectively. Frequency distribution data are the mean of three different microsphere batches.
Table 7 Effect of impeller on tristearin LS characteristics Batch
Impeller type
Mean diameter (mm)
Recovery (%)a
#28 #34 #35 #36
Three-blade rotor Four-blade helical rotor Turbine rotor Two-blade helical rotor
120715 5078.0 55719 Elliptical particles and filaments
78 64 77
a Percentage (w/w) of LS production with respect to the total amount of lipids used for the preparation. Experimental parameters were: a 1% PVA solution and 750 rpm stirring speed. Data represent the average of three independent experiments on different microsphere batches 7SD.
the production of more spherical particles with a narrow size distribution and a mean diameter of 15 mm, in addition interparticle fusion phenomena were almost absent.
3.7. Drug encapsulation efficiency As model drugs two hydrophobic compounds, such as retinyl acetate and progesterone, and one hydrophilic
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Table 8 Effect of stabiliser on tristearin LS characteristics Batch
Stabiliser (%, w/v)
Mean diameter (mm)
Recovery (%)a
#32 #40 #42 #43 #41 #28 #30 #39 #49
Gelatin 200 bloom (8) Pectin (0.5) Carrageenan k (0.5) Carrageenan i (0.5) Carrageenan l (0.5) PVA (1) Polyoxyethylene sorbitan trioleate (1) Pluronic PE 8100 (1) Lauryl sarcosine (1)
150733 250717 200725 200731
69 80 62 65 Compromised separation
120726 190718
78 54 Lipid aggregation
10741
F
a
Percentage (w/w) of LS production with respect to the total amount of lipids used for the preparation. Experimental parameters were: a 55 mm three-blade turbine rotor and 750 rpm stirring speed. Data represent the average of three independent experiments on different microsphere batches 7SD.
Fig. 6. Effect of the type of the method of preparation on morphology and particle size of LS. Optical micrographs and frequency distribution plot of microspheres produced by melt dispersion (#25) (A–B) and solvent evaporation (#58) (C–D) technique. LS were constituted of tristearin:glyceryl monostearate 2:1 (w/w) and prepared in the presence of 1% PVA. Bar corresponds to 350, and 100 mm in panel A and C, respectively. Frequency distribution data are the mean of three different microsphere batches.
drug, SCG, were considered. LS were produced by meltfusion technique. LS containing hydrophobic retinyl acetate were yellow (due to the colour of the drug), spherical with a slightly waved surface (see Fig. 7) and with a narrow size distribution (18476.6 mm); particles containing progesterone were very similar in shape (data not shown) and white (mean diameter 192711.6 mm). As reported in Table 10, both LS types are characterised by a high encapsulation and recovery efficiencies. In the case of
the hydrophilic drug SCG, again the microparticles were morphologically almost identical (data not shown) (mean diameter 234714.8 mm), but the encapsulation efficiency was on the contrary unsatisfactory, being only 2%. This result was partially expected, since hydrophilic drugs can be less efficiently incorporated in LS with respect to hydrophobic ones. In fact, if the drug is too hydrophilic to be soluble in organic solvents, microcrystalline fragments of the drug could be incorporated in the microparticle. The water-soluble drug could then
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Table 9 Effect of synthetic polymers on the production of LS by solvent evaporationa Batch
Microparticle composition (w/w ratio)
Stabiliser (%, w/v)
Stirring speed (rpm)
Mean diameter (mm)
#58 #70 #69 #68 #66 #73 #65 #75 #71 #72
Tristearin:monostearate (66:34) Tristearin:monostearate (66:34) Tristearin:monostearate (66:34) Tristearin:monostearate (66:34) Tristearin:monostearate:PLA (52:28:20) Tristearin:monostearate:PLA (52:28:20) Tristearin:monostearate:eudragit RS (52:28:20) Tristearin:monostearate:eudragit RS (52:28:20) Tristearin:monostearate:eudragit RS (52:28:20) Tristearin:monostearate:eudragit RS (52:28:20)
PVA PVA PVA PVA PVA PVA PVA PVA PVA PVA
750 500 250 250 750 250 750 250 500 250
2074.4 16576.2 n.d. n.d. 50711 n.d. 1573.6 n.d. 50712 10079
(1) (1) (0.5) (0.25) (1) (0.1) (1) (1) (0.1) (0.1)
a
Percentage (w/w) of LS production with respect to the total amount of lipids used for the preparation. Common experimental parameter was a 55 mm three-blade turbine rotor. Data represent the average of three independent experiments on different microsphere batches 7SD. n.d.: not determined.
resulted in a final encapsulation of the drug of 50% (see Table 9). 3.8. In vitro release of drug from LS
Fig. 7. Scanning electron micrographs of tristearin:glyceryl monostearate 66:33 (w/w) LS containing retinyl acetate (#80). Bar corresponds to 60 mm.
diffuse into the outer continuous aqueous phase, resulting in low trapping of the compound and inducing an initial rapid release of the drug known as ‘‘burst effect’’. In order to improve the encapsulation of SCG, LS were produced using a w/o/w double emulsion strategy. In particular, we studied the effect of various stabilisers of the primary emulsion on the encapsulation of SCG. Different hydrophilic polymers were employed, namely gelatin (250 bloom grades) or the polyoxyethylene–polyoxypropylene block copolymer, poloxamer 407. In order to further optimise the encapsulation yield, some experimental contrivances have been performed: (a) dispersion by turbine of the drug within the lipidic matrix, (b) rapid emulsion cooling using a ice bath and (c) rapid separation of LS by filtration. The optimised procedure
In order to obtain quantitative and qualitative informations on drug release from the LS and possibly to correlate the experimental data with the release mechanism, the complete release profile of LS encapsulated drugs, was determined. In Fig. 8 are reported the release kinetics of retinyl acetate and progesterone (panel A) and of SCG (panel B). As appreciable, the release of both hydrophobic drugs was much slower with respect to SCG, especially in the case of retinyl acetate containing LS. For these particles, indeed, the drug release efficacy within the first 8 h of release, was 27% of the total amount of the drug. In the same period, the amount of progesterone released was 63%. This behaviour could be ascribed to the physico-chemical characteristics of the drugs, which, as expected, showed a high affinity for the oil phase instead of the aqueous one. Concerning SCG containing LS, the release of the drug was largely influenced by the type of stabiliser of the primary emulsion used. In the case of LS produced in the presence of gelatin the shape of the release has a sigmoid form and after 8 h the release reaches the 80% of the total amount of the drug. Conversely, LS produced in presence of poloxamer 407, showed a drug release typified by a biphasic profile. The first part, characterised by a rapid drug release, is followed by a slower release rate, during which the drug is released in an approximately linear mode. In addition, it is to be underlined that the release of cromoglycate reaches the 100% of the total amount of the drug after 5 h from the beginning of the experiment.
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Table 10 Drug encapsulation efficiency and recovery of LS Batches
Drug
Encapsulation yield (%)
Recovery (%)a
#79–84 #85–90 #91 #92–93 #94–95 #96 #97
Retinyl acetate Progesterone SCG (o/w) SCG (o/w) SCG (w/o/w, gelatin) SCG (w/o/w, gelatin) SCG (w/o/w, poloxamer)
87.471.5 70.772.1 2.070.6 3.071.6 22.072.4 50.078.1 12.073.2
85.4 93.0 67.0 63.4 72.7 81.0 77.0
a Percentage (w/w) of LS production with respect to the total amount of lipids used for the preparation. Experimental parameters were: a 55 mm three-blade turbine rotor and 500 rpm stirring speed. Data represent the average of three independent experiments on different microsphere batches 7SD.
Fig. 8. Release profiles of drugs encapsulated in LS. (A) Lipophilic compounds, such as retinyl acetate () and progesterone (J). (B) Hydrophilic compounds, such as SCG encapsulated using as stabiliser gelatin (&) or poloxamer 407 (J). As reference the release of free SCG is also reported. The releases were determined by dialysis method. Data represent the average of five independent experiments on different microsphere batches.
In particular, in the case of LS prepared by melt dispersion, the use of different lipid mixtures, types of stabiliser and stirring speeds affected both microparticles shape and size distribution. The use of lauryl sarcosine as stabilizer allowed the formation of very small LS (SLN), in this respect further experiments will be performed to better investigate the experimental parameters involved in the production of SLN. LS, under appropriate experimental conditions, can entrap both hydrophobic and hydrophilic drugs and control the release of the encapsulated drug. The encouraging results obtained in this study could propose LS for future in vivo studies, especially in the vehiculation of anti-infectives and hormones. In this respect in a previous paper we described the production and characterization of biodegradable microparticles containing tetracycline, designed for periodontal diseases therapy. Microparticles were made by using different preparation procedures and different polyesters, namely poly(l-lactide), L-PLA, poly(dllactide), DL-PLA and poly(dl-lactide-co-glycolide) 50:50, DL-PLG. Selection of the appropriate preparation method and polyester enabled to obtain biodegradable microparticle, intended for sustained delivery of tetracycline to the periodontal pocket [22]. Lipid-based microspheres appear as ideal candidates to administer anti-bacterial agent for periodontal therapy since they are biodegradable, they do not have to be removed after the treatment period and possess mucoadhesive properties.
References 4. Conclusions Melt dispersion, solvent evaporation and w/o/w double emulsion methods enabled to produce LS whose morphology and size were influenced by the experimental parameters employed.
[1] Domb A, Amselem S, Shah J, Maniar M. Degradable polymers for site-specific drug delivery. Polym Adv Technol 1992;3:279–92. [2] Barker SA, Khossravi D. Drug delivery strategies for the new millennium. DDT 2001;6:75–7. [3] Mizushima Y. Lipid microspheres (lipid emulsions) as a drug carrier. An overview. Adv Drug Del Rev 1996;20:113–5. [4] Ravi Kumar MNV. Nano and microparticles as controlled drug delivery devices. J Pharm Pharmaceut Sci 2000;3:234–58.
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[5] Gombotz W, Pettit D, Pankey S, Lawter JR, Huang WJ. Prolonged release of GM-CSF United States Patent 1995; 5,942,253 Appl. No.: 542445. [6] Wake MC, Gerecht PD, Lu L, Mikos AG. Effects of biodegradable polymer particles on rat marrow-derived stromal osteoblasts in vitro. Biomaterials 1998;19:1255–68. [7] Cortesi R, Esposito E, Gambarin S, Telloli P, Menegatti E, Nastruzzi C. Preparation of liposomes by reverse-phase evaporation using alternative organic solvents. J Microencapsulation 1999;16:251–6. [8] Vyas SP, Singh R, Dimitrijevic D. Development and characterization of nifedipine lipospheres. Pharmazie 1997;52:403–4. [9] Jenning V, Thunemann AF, Gohla SH. Characterization of a novel solid lipid nanoparticle carrier system based on binary mixtures of liquid and solid lipids. Int J Pharm 2000;199:167–77. [10] Schwarz C, Mehnert W. Solid lipid nanoparticles (SLN) for controlled drug delivery. II. Drug incorporation and physicochemical characterization. J Microencapsulation 1999;16:205–13. [11] Muller . RH, Mader K, Gohla S. Solid lipid nanoparticles (SLN) for controlled drug deliveryFa review of the state of the art. Eur J Pharm Biopharm 2000;50:161–77. [12] Takenaga M. Application of lipid microspheres for the treatment of cancer. Adv Drug Del Rev 1996;20:209–19. [13] Yang S, Zhu J, Lu Y, Liang B, Yang C. Body distribution of camptothecin solid lipid nanoparticles after oral administration. Pharm Res 1999;16:751–7. [14] Inoue K, Aoki Y, Hayashi M, Kitahara S, Tanabe H, Kiyoki M, Araki H. Ex vivo anti-platelet effects of isocarbacyclin methyl
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Production of lipospheres as carriers for bioactive compounds Rita Cortesia, Elisabetta Espositoa, Giovanni Lucab, Claudio Nastruzzib,* b
a Department of Pharmaceutical Sciences, University of Ferrara, 1-44100 Ferrara, Italy Department of Pharmaceutical Chemistry and Technology, Istituto di Chimica e Tecnologia del Farmaco, via del Liceo, University of Perugia, I-06100 Perugia, Italy
Received 1 March 2001; accepted 8 October 2001
Abstract Aim of the present paper was to investigate the influence of preparation parameters on the production of lipospheres (LS) for drug delivery. LS composed of triglycerides and monoglycerides were alternatively produced by melt dispersion technique, solvent evaporation or w/o/w double emulsion method. The influence of preparation parameters, such as (a) type and amount of lipids, (b) presence and concentration of surfactants, (c) stirring speed and (d) type of stirrer was studied. In the case of LS prepared by melt dispersion, the use of a lipid composition of cetyl alcohol/cholesterol (2:1, w/w), a 5% (w/w) gelatin solution (50 bloom grades) and 1000 rpm stirring speed resulted in the production of spherical particles, with high percentage of recovery (82%, w/w) a mean diameter of 80 mm and a narrow size distribution. In the case of LS prepared by solvent evaporation, the best results in terms of LS morphology, recovery and size distribution were obtained by the use of a lipid composition of tristearin/monostearate (66:34, w/w), a 1% (w/w) PVA solution, a 750 rpm stirring speed and a 55 mm three-blade turbine rotor. The solvent evaporation method resulted in the production of LS characterised by a smaller size (20 mm mean diameter) but poor mechanical properties with respect to particles with the same composition obtained by the melt dispersion technique (170 mm mean diameter). The use of a combination of lipids and a methacrylic polymer (Eudragit RS 100) overcame this problem, resulting in the production of spherical particles, with a narrower size distribution and good mechanical properties. Two lipophilic drugs, such as retinyl acetate and progesterone, and one hydrophilic drug, sodium cromoglycate (SCG), were encapsulated in LS as model compounds. Lypophilic drugs displayed satisfactory encapsulation efficiencies (over 70% w/w), while SCG was very scarcely encapsulated (about 2% w/w). To solve this drawback, the use of a w/o/w double emulsion strategy was proposed, enabling to increase the encapsulation of SCG up to 50% w/w. Finally, in vitro drug release studies were performed, showing that all drugs were released in a control manner. In particular, the retinyl acetate release efficacy within the first 8 h was 27% of the total amount of the drug, while in the same period, the amount of progesterone released was 63%. With regard to SCG containing LS, the release of the drug was largely influenced by the type of stabiliser of the primary emulsion, in any case the SCG release reached the 100% of the total amount of drug after 5 h from the beginning of the experiment. r 2002 Elsevier Science Ltd. All rights reserved. Keywords: Lipospheres; Drug delivery; Solid lipid nanoparticles (SLN); Solvent evaporation; Melt dispersion
1. Introduction One approach for increasing the beneficial action of drugs and decreasing systemic adverse effects is to deliver the necessary amount of drugs to the diseased sites, where they are most needed, for the appropriate period of time [1–3].
*Corresponding author. Tel.: +39-075-5855158; fax: +39-0755847469. E-mail address: [email protected] (C. Nastruzzi).
Although the drug delivery system concept is not new, great progress has recently been made in the treatment of a variety of diseases. Particulate carriers (e.g. polymeric nano- and microparticles, fat emulsion, liposomes) possess specific advantages and disadvantages. For instance, in the case of polymeric microparticles the degradation of the polymer might possibly cause systemic toxic effects by impairment of the reticuloendothelial system [4] or by accumulation at the injection site [5]; cytotoxic effects have been indeed observed in vitro after phagocytosis of particles by human macrophages and granulocytes [6]. In addition, organic solvents residues deriving from the
0142-9612/02/$ - see front matter r 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 1 4 2 - 9 6 1 2 ( 0 1 ) 0 0 3 6 2 - 3
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preparation procedures such as the solvent evaporation technique often used for liposome [23] and polyester microparticles [24] can be present in the delivery system and could result in severe acceptability and toxicity problems [7]. In order to solve these adverse effects, lipid microspheres, often called lipospheres (LS), have been proposed as new type of fat-based encapsulation system for drug delivery of bioactive compounds (especially lipophilic compounds). LS consist of solid microparticles with a mean diameter usually comprised between 0.2 and 500 mm, composed of a solid hydrophobic fat matrix, where the bioactive compound(s) is dissolved or dispersed [8–10]. LS have some advantages over other delivery systems, such as (a) good physical stability, (b) low cost of ingredients, (c) ease of preparation and scaleup and (d) high entrapment yields for hydrophobic drugs. Due to their large particle size range, LS can be administered by different routes, such as oral, subcutaneous, intramuscular, topic or used for cells encapsulation being thus proposed for the treatment of a number of diseases [11–13]. For instance, the in vivo distribution of LS demonstrated a high affinity to vascular wells (including capillaries), to inflamed tissues and to granulocytes [14,15]. LS have been used for the controlled delivery of various types of drugs, including vasodilator and antiplatelet drugs, anti-inflammatory compounds, local anaesthetics, antibiotics and anti-cancer agents; they have also been used successfully as carriers of vaccines and adjuvants [14,16,17]. This paper will discuss (a) the production and characterisation of LS formed by emulsion-melt dispersion technique, by solvent evaporation method, and by w/o/w double emulsion method, (b) the influence of preparation parameters on LS morphology, (c) the encapsulation efficiency and the release characteristics of two lipophilic model drugs, such as retinyl acetate and progesterone, and one hydrophilic drug, sodium cromoglycate (SCG), from the prepared LS. To obtain a biocompatible formulation suitable for human administration, triglycerides and monoglycerides have been chosen as LS’ biomaterials, in reason of their high biocompatibility, high physico-chemical stability and drug delivery release.
monostearate and glyceryl monooleate were from Eastman Chemical Company the used gelatines (250, 200, 150, 50 Bloom) were from Fluka and the polyvinyl alcohol (PVA) was from Air Products and Chemicals (Utrecht, Netherlands). All the other materials and solvents were from Fluka and were of the highest purity available. 2.2. Preparation of LS The preparation of LS was performed using two alternative techniques, as following described (see Fig. 1A and Table 1). 2.2.1. Melt dispersion technique Five grams of a lipidic mixture, with/without 200 mg of lipophilic model drug, were melted at 701C then emulsified into 150 ml of an external aqueous phase containing a suitable surfactant. Eight natural or synthetic emulsifiers were employed, namely gelatin (200 bloom), pectin, carrageenan l; k; i; PVA, polyoxyethylene 20 sorbitan trioleate (polysorbate 85, tween 85) and lauryl sarcosine. The emulsion was stirred using
2. Materials and methods 2.1. Materials Retinyl acetate and progesterone were obtained from Fluka Chemical Co. (Buchs, Switzerland), glyceryl tristearate (tristearin), glyceryl tribehenate (tribehenin) and glyceryl tripalmitate (tripalmitin) were from Gattefosse! (Saint-Priest Cedex, France), the glyceryl
Fig. 1. (A) Schematic representation of LS’ methods of production: melt dispersion and solvent evaporation method. (B) Impellers employed for microsphere production: (a) a three-blade rotor with a diameter of 55 mm (taken as reference impeller); (b) a four-blade helicoidal rotor with a diameter of 50 mm; (c) a two-blade rotor with a diameter of 50 mm and finally (d) a double truncated cone rotor with a diameter of 50 mm.
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Table 1 Overview of LS preparation methods Method
Disperse phase
Dispersion medium
Melt dispersion
Melted lipid (5 g) at 701C
Water (150 ml) plus stabilisera
o/w Solvent evaporation
w/o/w Double emulsion
Dissolved lipids (5 g) in ethyl acetate (10 ml) at 501C
w/o Emulsion of melted lipids (5 g) at 701C stabilised with gelatin or poloxamer 407
Stirring speed (rpm) 500
Water (150 ml) plus PVA as stabiliser
Water (150 ml) plus 0.25% (w/v) PVA as stabiliser
750 1000 500 750 1000 500
Process duration (h)
Particle isolation
1 with rapid cool up to 201C
Filtration through a glass filterb
6–8 at room temperature
Filtration through a glass filterb
3–5 at room temperature
Filtration through a glass filterb
750 1000 a b
See Table 6. Glass filters with maximum nominal pore size 10–16 mm.
a mechanical stirrer Eurostar Digital (IKA Labortechnik, Germany) equipped with alternative impellers, namely: (a) a three-blade rotor with a diameter of 55 mm (taken as reference impeller), (b) a four-blade helicoidal rotor with a diameter of 50 mm, (c) a double truncated cone rotor with a diameter of 50 mm and finally (d) a two-blade rotor with a diameter of 50 mm (see Fig. 1B). Afterwards the emulsion was heated to the same temperature as the melted lipidic phase. The milky formulation was then rapidly cooled to about 201C by immersing the formulation flask in a cool water bath without stopping the agitation to yield a uniform dispersion of LS. The obtained LS were then washed with water and isolated by filtration through a paper filter. 2.2.2. Solvent evaporation technique Five grams of the lipid matrix dissolved in 10 ml of an organic solvent such as ethyl acetate at 501C were emulsified in 150 ml of an external aqueous phase containing the surfactant agent using a mechanical stirrer Eurostar Digital (IKA Labortechnick Germany). The resulting oil-in-water emulsion was stirred for 6–8 h under ambient conditions to allow the solvent evaporation. LS, after water rise, were collected by filtration through a paper filter. 2.2.3. Water-in-oil-in-water double emulsion (w/o/w) method This method consists in the solubilization of the drug to be encapsulated in the internal aqueous phase of a w/ o/w double emulsion, along with a stabiliser able to prevent loss of drug to the external phase during solvent evaporation [18]. One millititer of an aqueous solution of 100 mg of drug was emulsified in 5 g of melted lipids at 701C by using an ultra-turrax (IKA, Labortechnik,
Germany, EU). This primary emulsion was stabilised by adding as stabilisers, gelatin (250 bloom grades) or the polyoxyethylene–polyoxypropylene block copolymer, poloxamer 407, solubilized in the aqueous phase. The primary emulsion was then dispersed, at 701C, into 150 ml of an aqueous phase containing 0.25% (w/v) of PVA. The obtained double emulsion was stirred at 300 rpm by a four-blade turbine impeller. After 3–5 h microparticles were isolated by filtration. 2.3. LS morphological analysis The morphology of LS was evaluated by optical microscopy (Nikon Diaphot inverted microscope, Tokyo, Japan) and scanning electron microscopy (SEM) observations (360 Stereoscan Cambridge Instruments Ltd, Cambridge, UK). Microsphere size distributions were determined by photomicrograph analyses, analysing at least 300 microparticle/sample. 2.4. Drug content of LS The amount of encapsulated model drug (retinyl acetate or progesterone) per mg of dried LS was determined by solubilisation of 50 mg of LS in 2 ml of ethyl acetate at 601C. Following filtration the solution was analysed by reverse phase HPLC to find the drug content. The HPLC determinations were performed using a HPLC system consisting of a Bruker apparatus (Bremer, FRG) consisting of a three plungers alternative pump, a variable wavelength UV-detector, operating at 215 nm, and a Rheodine Inc. injection valve with a 100 ml loop. Samples were chromatographed on a stainless steel C18 reverse-phase column (15 0.76 cm) (Supelco) packed with 5 mm particles (Model LC-18-DB, Supelco, USA),
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eluted isocratically at room temperature, at a flow rate of 1 ml/min. The mobile phase used for the drugs were 180 mm ammonium acetate (pH 3.0)/methanol (4:96, v/v) for retinyl acetate [19], methanol/water (70:30, v/v) for progesterone [20] and phosphate buffer (pH 2.3)/ methanol (50:50, v/v) for cromoglycate [21]. Drug detection was monitored at the lmax characteristic of each compound. 2.5. In vitro drug release In vitro drug release profiles were obtained placing 50 mg of drug containing LS in 10 ml of buffer under magnetic stirring at 150 rpm. The buffer was ethanol/ water, 30:70 with addition of 0.5% (w/w) of polysorbate 20. Following different length of time (0–8 h), 200 ml of receiving buffer, were filtered and analysed for drug content by reverse phase HPLC as previously described.
3. Results and discussion 3.1. LS preparation LS were prepared by two alternative approaches, namely the emulsion-melt dispersion or the solvent evaporation technique, as described in the experimental section.
Fig. 2. Effect of gelatin bloom on morphology and particle size of cetyl alcohol:cholesterol 2:1 (w/w) LS. SEM (A) and optical micrographs (B) of microspheres produced with gelatin 200 bloom grades (#7). Bar corresponds to 50 and 250 mm in panel A and B, respectively.
3.2. LS prepared by melt dispersion technique The choice of the lipid matrix plays an important role on the morphology of the particles and on the possible formation of aggregates. In a first set of experiments, LS were prepared by the emulsion melt dispersion technique (see Fig. 1A and Table 1) using a lipid mixture constituted of cetyl alcohol and cholesterol (2:1, w/w) and gelatin as stabiliser. The resulting microparticles (see Fig. 2 and Table 2) were characterised by an irregular surface, in addition some aggregates caused by the fusion of lipid droplets before solidification, were present. An improvement of LS features was obtained in term of recovery, mean diameter and aggregate formation by decreasing the molecular weight of the gelatin used as stabiliser. The viscosity of the dispersing phase was in fact progressively reduced passing from gelatin 50 to 250 bloom grades (batches 3, 4, 6, 7). By adjusting the stirring speed during the emulsification process, it was possible to modify the size of the particles. Increasing the stirring speed from 500 to 1000 rpm, the mean diameter of particles progressively decreased (see Table 3) (batches 11, 13, 14). With the aim to further improve the characteristics of LS, alternative lipid compositions were considered. For
instance, LS were prepared with apolar triglycerides, such as tristearin, tripalmitin or tribehenin in combination with other polar (more hydrophilic) lipids, including glyceryl monostearate, glyceryl monooleate, cetyl alcohol and cholesterol (see Table 4). As general consideration all formulations, apart from those including cholesterol and glyceryl monooleate, were satisfactory in term of shape, recovery and size (Figs. 3 and 4). Other experiments were undertaken to evaluate the effect of glyceryl monostearate concentration on LS characteristics. Glyceryl monostearate was used in mixture with tristearin (batches 57, 62, 55, 52, 54) or tripalmitin (batches 11, 64, 60, 59, 24) at different weight ratios. In Table 5 are reported the results of such experiments in which the percentage of glyceryl monostearate was varied from 0 up to 33% (w/w). In the case of tripalmitin, was impossible to produce LS without the presence of at least 1% of glyceryl monostearate (due to the formation of large blobs), while pure tristearin particles were obtained even if they are of poor quality. By increasing the content of glyceryl monostearate a progressive decrease of LS size was evident. No effect was on the contrary detectable on LS recovery.
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Table 2 Effect of gelatin type on LS characteristics Batch
Lipid composition (w/w ratio)
#6 #7 #4 #3
Cetyl Cetyl Cetyl Cetyl
alcohol/cholesterol alcohol/cholesterol alcohol/cholesterol alcohol/cholesterol
(2:1) (2:1) (2:1) (2:1)
Gelatin bloom grades
Mean diameter (mm)
Recovery (%)a
250 200 150 50
250712 205715 19778 150721
52 60 61 82
a Percentage (w/w) of LS production with respect to the total amount of lipids used for the preparation. Experimental parameters were: a 55 mm three-blade turbine rotor, a 5% gelatin solution and 750 rpm stirring speed. Data represent the average of three independent experiments on different microsphere batches 7SD.
Table 3 Effect of stirring speed on characteristics of cetyl alcohol/cholesterol LS Batch
Stirring speed (rpm)
Gelatin bloom grades
Mean diameter (mm)
Recovery (%)a
#13 #11 #14
500 750 1000
50 50 50
250714 150716 80724
92 82 82
a Percentage (w/w) of LS production with respect to the total amount of lipids used for the preparation. Experimental parameters were: a 55 mm three-blade turbine rotor and a 5% gelatin solution. Data represent the average of three independent experiments on different microsphere batches 7SD.
Table 4 Effect of lipid composition on the production of LS Batch
Lipid composition (w/w ratio)
Mean diameter (mm)
Recovery (%)a
#25 #26 #27 #24 #21 #22 #23 #51
Tristearin:monostearate (2:1) Tristearin:cetyl alcohol (2:1) Tristearin:cholesterol (2:1) Tripalmitin:monostearate (2:1) Tripalmitin:monooleate (2:1) Tripalmitin:cholesterol (2:1) Tripalmitin:cetyl alcohol (2:1) Tribehenin:monostearate (2:1)
170719 200726 250716 25078
90 92 98 82 Fused mass Fused mass
300715 200722
96 75
a Percentage (w/w) of LS production with respect to the total amount of lipids used for the preparation. Experimental parameters were: a 55 mm three-blade turbine rotor, a 1% PVA solution and 750 rpm stirring speed. Data represent the average of three independent experiments on different microspheres batches 7SD.
3.3. Effect of the stirring speed
3.4. Effect of impeller geometry
The effect of the stirring speed was considered on the production of LS (see Table 6 and Fig. 5). LS with dimensions comprised between 170 and 90 mm were obtained by changing the stirring speed from 500 to 1000 rpm (batches 25, 28, 29). In particular, particles obtained at 1000 rpm presented a spherical geometry with a narrow size distribution; in addition, aggregation phenomena were almost absent. The recovery efficiency of particles produced at the higher stirring speed was 69%; whilst for those produced at 500 rpm was over 90%.
LS (batches 28, 34, 35, 36), were produced by means of different impellers as described in the experimental section and displayed in Fig. 1B. The use of rotors (b) and (c) allowed obtaining smaller particles with mean diameters of 50 and 55 mm and a recovery of 64% and 77%, respectively (Table 7). On the other hand, the use of the rotor (d) did not allow the production of lipid particles, in fact this particular impeller caused the formation of elliptical particles and filaments.
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Fig. 3. Effect of lipid composition on the morphology of LS. LS were prepared with cetyl alcohol:cholesterol 2:1 (w/w) (#3) (A), tripalmitin:glyceryl monostearate 2:1 (w/w) (#24) (B) tristearin:glyceryl monostearate 2:1 (w/w) (#25) (C) and tripalmitin:glyceryl monostearate 9:1 (w/w) (#59) (D). Bar corresponds to 300, 600, 200 and 375 mm in panel A, B, C and D, respectively.
Fig. 4. SEM photographs showing the effect of lipid composition on the morphology of LS. LS were prepared with tristearin:glyceryl monostearate 2:1 (w/w) (#25) (A), tristearin:cetyl alcohol 2:1 (w/w) (#26) (B), tripalmitin:glyceryl monostearate 2:1 (w/w) (#24) (C), and tripalmitin:cetyl alcohol 2:1 (w/w) (#23) (D). Bar corresponds to 125, 160, 200 and 150 mm in panel A, B, C and D, respectively.
3.5. Effect of the stabiliser type and concentration The effect of different stabilisers (see list in the Materials and Method section) was tested on particle morphology and recovery (see Table 8). As clearly appreciable from the obtained results, the addition of
emulsifiers, leads to variable effects on the size of LS droplets during the emulsification step thus influencing the final microspheres size. In particular, LS obtained with natural polymers, such as gelatin, pectin and carrageenans k and i (batches 32, 40, 42, 43) allowed the production of spherical particles with an irregular
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Table 5 Effect of monostearate on the production of LS Batch
Lipid composition (w/w ratio)
Mean diameter (mm)
Recovery (%)a
#57 #62 #55 #54 #52 #11 #0 #64 #60 #59 #24
Tristearin:glyceryl monostearate 100:0 Tristearin:glyceryl monostearate 98:2 Tristearin:glyceryl monostearate 95:5 Tristearin:glyceryl monostearate 90:10 Tristearin:glyceryl monostearate 80:20 Tristearin:glyceryl monostearate 66:33 Tripalmitin:glyceryl monostearate 100:0 Tripalmitin:glyceryl monostearate 99:1 Tripalmitin:glyceryl monostearate 95:5 Tripalmitin:glyceryl monostearate 90:10 Tripalmitin:glyceryl monostearate 66:33
220731 200723 170718 160715 158728 170719
76 93 90 89 73 90 Fused mass
30079.0 300716 300731 250721
96 75 72 82
a Percentage (w/w) of LS production with respect to the total amount of lipids used for the preparation. Experimental parameters were: a 55 mm three-blade turbine rotor, 750 rpm stirring speed, 1% PVA solution as disperding phase. Data represent the average of three independent experiments on different microsphere batches 7SD.
Table 6 Effect of stirring speed on tristearin LS characteristics Batch
Stirring speed (rpm)
Mean diameter (mm)
Recovery (%)a
#25 #28 #29
500 750 1000
170735 120724 90719
90 78 69
a
Percentage (w/w) of LS production with respect to the total amount of lipids used for the preparation. Experimental parameters were: a 55 mm three-blade turbine rotor and a 1% PVA solution. Data represent the average of three independent experiments on different microsphere batches 7SD.
surface and a mean diameter comprised between 150 and 250 mm. In the case of carrageenan l (batch 41) it was not possible to isolate the particles since the high viscosity of the suspension did not allow the filtration process. On the contrary, in the cases of gelatin and others carrageenans the high viscosity was compatible with the separation process even if caused by a lower recovery efficiency (comprised between 62% and 69%) with respect to pectin (80%). The use of synthetic emulsifiers gave different results (batches 28, 30, 39, 49); for instance the use of 1% (w/w) of the polyoxyethylene–polyoxypropylene block copolymer Pluronic PE 8100 did not allow the stabilisation of the o/w emulsion during the preparation, thus resulting in the formation of large lipid aggregates. The use of 1% of polyoxyethylene sorbitan trioleate or PVA allowed the production of spherical particles with a mean diameter of 190 and 120 mm and a recovery of 54% and 78%, respectively. Finally, lauryl sarcosine caused the formation of a very fine o/w emulsion resulting in the final formation of very small particles (mean diameter 1074.1 mm) that were isolated by centrifuga-
tion. In this respect further studies are in progress to evaluate the experimental parameters for the production of lipid nanoparticles. 3.6. LS prepared by solvent evaporation method As alternative to the melt dispersion technique, a solvent evaporation method was also tested for the production of LS (Fig. 1B). This approach was considered with the aim to possibly reduce the exposure to high temperature of thermolabile compounds, such as proteins and nucleic acids. The solvent evaporation method is based on the evaporation of the organic solvent in which lipids are dissolved, allowing the formation of solid microparticles. By this technique were produced LS constituted of tristearin:glyceryl monostearate 2:1 w/w, in the presence of 1% PVA as emulsifier agent. The obtained particles (see Fig. 6) were spherical and characterised by a smaller size with respect to the particles with the same composition produced by the melt-fusion technique. Unfortunately, the produced LS showed some poor mechanical properties being fragile together with a higher proportion of interparticellar bridges as compared to the melt dispersion technique. With the aim to improve the mechanical properties of LS produced by solvent evaporation, as well as to obtain prolonged release profiles, the possibility to produce particles with a mixed matrix, was considered (see Table 9). LS were produced using lipids in combination with different polymers, in ratio up to 20% with respect to the lipid components. Both biodegradable, such as polylactic acid (PLA) and nonbiodegradable polymers, such as Eudragit RS 100, were used. The different polymers allowed the improvement of the mechanical characteristics of the LS and, particularly in the case of Eudragit RS 100, allowed
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Fig. 5. Optical microscopy photographs showing the effect of stirring speed on morphology and particle size of tristearin:monostearate 2:1 (w/w) LS. Optical micrographs and frequency distribution plot of microspheres produced at 500 rpm (#25) (A–B), 750 rpm (#28) (C–D) and 1000 rpm (#29) (E– F). Bar corresponds to 350, 600, and 800 mm in panel A, C and E, respectively. Frequency distribution data are the mean of three different microsphere batches.
Table 7 Effect of impeller on tristearin LS characteristics Batch
Impeller type
Mean diameter (mm)
Recovery (%)a
#28 #34 #35 #36
Three-blade rotor Four-blade helical rotor Turbine rotor Two-blade helical rotor
120715 5078.0 55719 Elliptical particles and filaments
78 64 77
a Percentage (w/w) of LS production with respect to the total amount of lipids used for the preparation. Experimental parameters were: a 1% PVA solution and 750 rpm stirring speed. Data represent the average of three independent experiments on different microsphere batches 7SD.
the production of more spherical particles with a narrow size distribution and a mean diameter of 15 mm, in addition interparticle fusion phenomena were almost absent.
3.7. Drug encapsulation efficiency As model drugs two hydrophobic compounds, such as retinyl acetate and progesterone, and one hydrophilic
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Table 8 Effect of stabiliser on tristearin LS characteristics Batch
Stabiliser (%, w/v)
Mean diameter (mm)
Recovery (%)a
#32 #40 #42 #43 #41 #28 #30 #39 #49
Gelatin 200 bloom (8) Pectin (0.5) Carrageenan k (0.5) Carrageenan i (0.5) Carrageenan l (0.5) PVA (1) Polyoxyethylene sorbitan trioleate (1) Pluronic PE 8100 (1) Lauryl sarcosine (1)
150733 250717 200725 200731
69 80 62 65 Compromised separation
120726 190718
78 54 Lipid aggregation
10741
F
a
Percentage (w/w) of LS production with respect to the total amount of lipids used for the preparation. Experimental parameters were: a 55 mm three-blade turbine rotor and 750 rpm stirring speed. Data represent the average of three independent experiments on different microsphere batches 7SD.
Fig. 6. Effect of the type of the method of preparation on morphology and particle size of LS. Optical micrographs and frequency distribution plot of microspheres produced by melt dispersion (#25) (A–B) and solvent evaporation (#58) (C–D) technique. LS were constituted of tristearin:glyceryl monostearate 2:1 (w/w) and prepared in the presence of 1% PVA. Bar corresponds to 350, and 100 mm in panel A and C, respectively. Frequency distribution data are the mean of three different microsphere batches.
drug, SCG, were considered. LS were produced by meltfusion technique. LS containing hydrophobic retinyl acetate were yellow (due to the colour of the drug), spherical with a slightly waved surface (see Fig. 7) and with a narrow size distribution (18476.6 mm); particles containing progesterone were very similar in shape (data not shown) and white (mean diameter 192711.6 mm). As reported in Table 10, both LS types are characterised by a high encapsulation and recovery efficiencies. In the case of
the hydrophilic drug SCG, again the microparticles were morphologically almost identical (data not shown) (mean diameter 234714.8 mm), but the encapsulation efficiency was on the contrary unsatisfactory, being only 2%. This result was partially expected, since hydrophilic drugs can be less efficiently incorporated in LS with respect to hydrophobic ones. In fact, if the drug is too hydrophilic to be soluble in organic solvents, microcrystalline fragments of the drug could be incorporated in the microparticle. The water-soluble drug could then
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Table 9 Effect of synthetic polymers on the production of LS by solvent evaporationa Batch
Microparticle composition (w/w ratio)
Stabiliser (%, w/v)
Stirring speed (rpm)
Mean diameter (mm)
#58 #70 #69 #68 #66 #73 #65 #75 #71 #72
Tristearin:monostearate (66:34) Tristearin:monostearate (66:34) Tristearin:monostearate (66:34) Tristearin:monostearate (66:34) Tristearin:monostearate:PLA (52:28:20) Tristearin:monostearate:PLA (52:28:20) Tristearin:monostearate:eudragit RS (52:28:20) Tristearin:monostearate:eudragit RS (52:28:20) Tristearin:monostearate:eudragit RS (52:28:20) Tristearin:monostearate:eudragit RS (52:28:20)
PVA PVA PVA PVA PVA PVA PVA PVA PVA PVA
750 500 250 250 750 250 750 250 500 250
2074.4 16576.2 n.d. n.d. 50711 n.d. 1573.6 n.d. 50712 10079
(1) (1) (0.5) (0.25) (1) (0.1) (1) (1) (0.1) (0.1)
a
Percentage (w/w) of LS production with respect to the total amount of lipids used for the preparation. Common experimental parameter was a 55 mm three-blade turbine rotor. Data represent the average of three independent experiments on different microsphere batches 7SD. n.d.: not determined.
resulted in a final encapsulation of the drug of 50% (see Table 9). 3.8. In vitro release of drug from LS
Fig. 7. Scanning electron micrographs of tristearin:glyceryl monostearate 66:33 (w/w) LS containing retinyl acetate (#80). Bar corresponds to 60 mm.
diffuse into the outer continuous aqueous phase, resulting in low trapping of the compound and inducing an initial rapid release of the drug known as ‘‘burst effect’’. In order to improve the encapsulation of SCG, LS were produced using a w/o/w double emulsion strategy. In particular, we studied the effect of various stabilisers of the primary emulsion on the encapsulation of SCG. Different hydrophilic polymers were employed, namely gelatin (250 bloom grades) or the polyoxyethylene–polyoxypropylene block copolymer, poloxamer 407. In order to further optimise the encapsulation yield, some experimental contrivances have been performed: (a) dispersion by turbine of the drug within the lipidic matrix, (b) rapid emulsion cooling using a ice bath and (c) rapid separation of LS by filtration. The optimised procedure
In order to obtain quantitative and qualitative informations on drug release from the LS and possibly to correlate the experimental data with the release mechanism, the complete release profile of LS encapsulated drugs, was determined. In Fig. 8 are reported the release kinetics of retinyl acetate and progesterone (panel A) and of SCG (panel B). As appreciable, the release of both hydrophobic drugs was much slower with respect to SCG, especially in the case of retinyl acetate containing LS. For these particles, indeed, the drug release efficacy within the first 8 h of release, was 27% of the total amount of the drug. In the same period, the amount of progesterone released was 63%. This behaviour could be ascribed to the physico-chemical characteristics of the drugs, which, as expected, showed a high affinity for the oil phase instead of the aqueous one. Concerning SCG containing LS, the release of the drug was largely influenced by the type of stabiliser of the primary emulsion used. In the case of LS produced in the presence of gelatin the shape of the release has a sigmoid form and after 8 h the release reaches the 80% of the total amount of the drug. Conversely, LS produced in presence of poloxamer 407, showed a drug release typified by a biphasic profile. The first part, characterised by a rapid drug release, is followed by a slower release rate, during which the drug is released in an approximately linear mode. In addition, it is to be underlined that the release of cromoglycate reaches the 100% of the total amount of the drug after 5 h from the beginning of the experiment.
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Table 10 Drug encapsulation efficiency and recovery of LS Batches
Drug
Encapsulation yield (%)
Recovery (%)a
#79–84 #85–90 #91 #92–93 #94–95 #96 #97
Retinyl acetate Progesterone SCG (o/w) SCG (o/w) SCG (w/o/w, gelatin) SCG (w/o/w, gelatin) SCG (w/o/w, poloxamer)
87.471.5 70.772.1 2.070.6 3.071.6 22.072.4 50.078.1 12.073.2
85.4 93.0 67.0 63.4 72.7 81.0 77.0
a Percentage (w/w) of LS production with respect to the total amount of lipids used for the preparation. Experimental parameters were: a 55 mm three-blade turbine rotor and 500 rpm stirring speed. Data represent the average of three independent experiments on different microsphere batches 7SD.
Fig. 8. Release profiles of drugs encapsulated in LS. (A) Lipophilic compounds, such as retinyl acetate () and progesterone (J). (B) Hydrophilic compounds, such as SCG encapsulated using as stabiliser gelatin (&) or poloxamer 407 (J). As reference the release of free SCG is also reported. The releases were determined by dialysis method. Data represent the average of five independent experiments on different microsphere batches.
In particular, in the case of LS prepared by melt dispersion, the use of different lipid mixtures, types of stabiliser and stirring speeds affected both microparticles shape and size distribution. The use of lauryl sarcosine as stabilizer allowed the formation of very small LS (SLN), in this respect further experiments will be performed to better investigate the experimental parameters involved in the production of SLN. LS, under appropriate experimental conditions, can entrap both hydrophobic and hydrophilic drugs and control the release of the encapsulated drug. The encouraging results obtained in this study could propose LS for future in vivo studies, especially in the vehiculation of anti-infectives and hormones. In this respect in a previous paper we described the production and characterization of biodegradable microparticles containing tetracycline, designed for periodontal diseases therapy. Microparticles were made by using different preparation procedures and different polyesters, namely poly(l-lactide), L-PLA, poly(dllactide), DL-PLA and poly(dl-lactide-co-glycolide) 50:50, DL-PLG. Selection of the appropriate preparation method and polyester enabled to obtain biodegradable microparticle, intended for sustained delivery of tetracycline to the periodontal pocket [22]. Lipid-based microspheres appear as ideal candidates to administer anti-bacterial agent for periodontal therapy since they are biodegradable, they do not have to be removed after the treatment period and possess mucoadhesive properties.
References 4. Conclusions Melt dispersion, solvent evaporation and w/o/w double emulsion methods enabled to produce LS whose morphology and size were influenced by the experimental parameters employed.
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