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Setting Precise Temperature for Triggered Release from Nanostructured Lipid Carriers
10th IFAC Symposium on Biological and Medical Systems 10th IFAC Symposium on Biological and Medical Systems São Paulo, Brazil, September 3-5, 2018 São Paulo, Brazil, September 3-5, 2018 Available online at www.sciencedirect.com 10th IFAC Symposium on Biological and Medical Systems São Paulo, Brazil, September 3-5, 2018
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Setting Precise Temperature for Triggered Release from Nanostructured Lipid IFAC PapersOnLine 51-27 (2018) 1–6 Setting Precise Temperature for Triggered Release from Nanostructured Lipid Setting Precise Temperature for Triggered CarriersRelease from Nanostructured Lipid Carriers Setting Temperature forValle**, Triggered Release Nanostructured Milena Santos*,Precise Ana Beatriz Caribé dos Santos Ana Cristina Mourafrom Gualberto***, Fernanda BritoLipid Leite****, Carriers Milena Santos*, Ana Beatriz Caribé dos Santos Valle**, Ana Cristina Moura Gualberto***, Fernanda Brito Leite****, Frederico Pittella***** Carriers Milena Santos*, Ana Beatriz Caribé dos Santos Valle**, Ana Cristina Moura Gualberto***, Fernanda Brito Leite****, Frederico Pittella***** FredericoAna Pittella***** Milena Santos*, Ana Beatriz Caribé dos Santos Valle**, Cristina Moura Gualberto***, Fernanda Brito Leite****, * Federal University of Juiz de Fora, Juiz de Fora, Brazil (e-mail: [email protected]) Frederico Pittella***** * ** Federal University of of Juiz de de Fora, Juiz de de Fora, Brazil (e-mail: [email protected]) Federal University Juiz Fora, Juiz Fora, Brazil (e-mail: [email protected]) * Federal University of Juiz de Fora, Juiz de Fora, Brazil (e-mail: [email protected]) ** Federal University of Juiz de Fora, de Fora, Brazil (e-mail: [email protected]) *** Federal University of Juiz de Fora, JuizJuiz de Fora, Brazil (e-mail: [email protected]) Federal University Juiz Fora, Juiz Fora, Brazil (e-mail: [email protected]) * ** Federal University of of Juiz de de Fora, Juiz de de Fora, Brazil (e-mail: [email protected]) *** Juiz Fora, Fora, (e-mail: [email protected]) ****Federal FederalUniversity Universityof Juizde Fora,Juiz Juizde Fora,Brazil Brazil(e-mail: (e-mail:[email protected]) [email protected]) *** Federal University ofofJuiz dedeFora, Juiz dedeFora, Brazil ** Federal University of Juiz de Fora, Juiz de Fora, Brazil (e-mail: [email protected]) **** Federal University of Juiz deScience, Fora, Juiz de Fora, Brazil (e-mail: [email protected]) ***** Department of Pharmaceutical Federal University of Juiz [email protected]) Fora, Juiz de Fora, Brazil (e-mail: **** Federal University of Juiz de Fora, Juiz de Fora, Brazil (e-mail: *** Federal University of Juiz de Fora, Juiz de Fora, Brazil (e-mail: [email protected]) ***** Department of Pharmaceutical Science, Federal University of Juiz de Fora, Juiz de Fora, Brazil (e-mail: [email protected]) ***** Department of Pharmaceutical Federal University of Juiz [email protected]) Fora, Juiz de Fora, Brazil (e-mail: **** Federal University of Juiz deScience, Fora, Juiz de Fora, Brazil (e-mail: [email protected]) [email protected]) ***** Department of Pharmaceutical Science, Federal University of Juiz de Fora, Juiz de Fora, Brazil (e-mail: [email protected]) _______________________________________________________________________________________ _______________________________________________________________________________________ _______________________________________________________________________________________ Abstract: The preparation and use of nanostructured lipid carriers (NLCs) as drug delivery system is constantly growing. NLCs are from synthetic lipid or natural lipids with as known, not controlled, Abstract: The preparation andprepared use of nanostructured carriers (NLCs) drug but delivery system is _______________________________________________________________________________________ Abstract: TheHere preparation andprepared usepreparation of nanostructured carriers (NLCs) as drug delivery system is melting point. we propose the of NLCslipid combining natural lipids to have selected melting constantly growing. NLCs are from synthetic or natural lipids with known, but not controlled, constantly growing. NLCs are prepared from synthetic or natural lipids with known, but not controlled, temperature. Vegetable butters were subjected to gas chromatography to determine and define each melting point. we propose of NLCslipid combining lipids to have selected melting Abstract: TheHere preparation and the usepreparation of nanostructured carriersnatural (NLCs) as drug delivery system is melting point. Here we propose the preparation oftoNLCs combining natural lipids to have selected melting constituent and melting point. Three different NLCs suspensions were prepared from a specific blend of temperature. Vegetable butters were subjected gas chromatography to determine and define each constantly growing. NLCs are prepared from synthetic or natural lipids with known, but not controlled, temperature. Vegetable butters were subjected to or gas chromatography to determine and define vegetable butters, adjusted to start melting at 29, 37 42°C, and characterized. Preciseaengineering ofeach the constituent and melting point. Three different NLCs suspensions prepared specific of melting point. Here we propose the preparation of NLCs combiningwere natural lipids from to have selectedblend melting constituent and melting point. Three different NLCs suspensions were prepared from a specific blend of delivery system will contribute to efficient temperature triggered release. vegetable butters, adjusted to startwere melting at 29, 37 or 42°C, and characterized. Precise engineering ofeach the temperature. Vegetable butters subjected to gas chromatography to determine and define vegetable butters, adjusted to start meltingtemperature at 29, 37 ortriggered 42°C, and characterized. Precise engineering of the delivery system will contribute to efficient release. constituent and(International melting point. Three different NLCs suspensions were prepared a specific blend of © 2018, IFAC Federation of Automatic Control) Hosting byRelease, Elsevier Ltd. from All rights reserved. delivery system will contribute to efficient temperature triggered release. Keywords: Nanostructured lipid carriers, Melting point, Triggered Thermoresponsive, Vegetable vegetable butters, adjusted to start melting at 29, 37 or 42°C, and characterized. Precise engineering of the butter. Keywords: Nanostructured lipid carriers, Melting point, Triggered Release, Thermoresponsive, Vegetable delivery system will contribute to efficient temperature triggered release. Keywords: Nanostructured lipid carriers, Melting point, Triggered Release, Thermoresponsive, Vegetable butter. butter. ____________________________________________________________________________________ Keywords: Nanostructured lipid carriers, Melting point, Triggered Release, Thermoresponsive, Vegetable ____________________________________________________________________________________ butter. ____________________________________________________________________________________ ____________________________________________________________________________________ 1. INTRODUCTION In fact, the choice of the lipid in a NLC formulation might trigger. 1. INTRODUCTION In fact,bethecritical choicefor of the the precise lipid in temperature a NLC formulation The use of nanoparticles as drug delivery systems allows 1. INTRODUCTION In fact, the choice of the lipid in a NLC formulation Vegetable butters have as main constituent naturally might be critical for the precise temperature trigger. therapeutic such asas:drug i) delivery stability systems of the allows active might be fatty critical for the precise temperature trigger. The use of benefits, nanoparticles balanced acids. These are constituent long carbonic chain Vegetable main naturally 1. INTRODUCTION In fact, thebutters choice have of theas lipid inconstituent a NLC formulation The use ofii)benefits, nanoparticles asas:drug delivery systems substance; reductionsuch of toxicity; increased Vegetable butters have as main naturally therapeutic i) iii) stability of therapeutic the allows active with a carboxyl group at the end of the molecule. The balanced acids. These are long carbonictrigger. chain might be fatty critical for the precise temperature therapeutic such as: i) iii) stability of therapeutic the et active efficacy; and iv) increased bioavailability (Pallaerla al., balanced fatty acids. These areorof long carbonic chain substance; ii)benefits, reduction of toxicity; increased carbonic chain can be saturated unsaturated (Drevon The use of nanoparticles as drug delivery systems allows with a carboxyl group at the end the molecule. The Vegetable butters haveatasthemain constituent naturally substance; ii) reduction toxicity; iii) increased 2013). Among the of existing drug delivery therapeutic systems, with carboxyl group endorof theacids molecule. The efficacy; and iv) increased bioavailability al., et al.,a2005). Typically, saturated fatty have high therapeutic benefits, such as: i) stability(Pallaerla of the et active carbonic chain can be saturated unsaturated (Drevon balanced fatty acids. These are long carbonic chain efficacy; and iv) increased bioavailability (Pallaerla et al., nanostructured lipid carriers (NLCs) aredelivery characterized for carbonic chain can be saturated or unsaturated (Drevon 2013). Among the existing drug systems, melting point while unsaturated present low melting substance; ii) reduction of toxicity;drug iii) increased therapeutic et al.,a2005). Typically, fatty have high with carboxyl group atsaturated the end of theacids molecule. The 2013). Among the existing systems, having amorphous solid lipid (NLCs) core at room temperature that et al., 2005). Typically, saturated fatty acids have high nanostructured lipid carriers aredelivery characterized for temperature (Salas et al., 2009). The naturally balanced efficacy; and iv) increased bioavailability (Pallaerla et al., melting point while unsaturated present low melting carbonic chain can be saturated or unsaturated (Drevon nanostructured lipid carriers (NLCs) are characterized for accommodates hydrophobic compounds (Abdelaziz et al., melting point while unsaturated present low melting having amorphous solid lipid core drug at room temperature that amount of fatty acids define melting point of a 2013). Among the delivery systems, temperature et al., 2009).the The naturally balanced et al., 2005).(Salas Typically, saturated fatty acids have high having amorphous solid existing core room temperature that 2017). It is considered alipid type of at solid lipid nanoparticles temperature (Salas et al., 2009). The naturally balanced accommodates hydrophobic compounds (Abdelaziz et al., vegetable butter. Understanding the melting nanostructured lipid carriers compounds (NLCs) are (Abdelaziz characterized for amount of fatty acidsunsaturated define thepresent meltinglow point of a melting point while melting accommodates hydrophobic et al., (SLN) by blend aoftype lipidsoftosolid resultlipid in non-crystalline amount of fatty acidsconstituents define the ofmelting point of a 2017). prepared It is considered nanoparticles temperature and the the the butter allows having amorphous solid lipid core at room temperature that vegetable butter. Understanding melting temperature (Salas et al., 2009). The naturally balanced 2017). It islipid considered aoftype ofto solid nanoparticles amorphous coreblend (Muller et al., 2002). vegetable butter. Understanding the melting (SLN) prepared by lipids resultlipid in non-crystalline the elaboration of the exact melting point of the lipid to accommodates hydrophobic compounds (Abdelaziz et al., temperature and the the butter amount of fatty acidsconstituents define the of pointallows of a (SLN) prepared by of lipids to result in non-crystalline temperature andofthe constituents ofmelting theetof butter allows amorphous lipid coreblend (Muller et al., 2002). be used to construct the NLCs (Rehman al., 2017). 2017). It is considered a type of solid lipid nanoparticles the elaboration the exact melting point the lipid to The NLCs are after et theal., homogenization of the lipid vegetable butter. Understanding melting amorphous lipidobtained core (Muller 2002). theused elaboration of thethe exact melting pointetthe ofal., the2017). lipid to (SLN) prepared by blend ofhigh lipids to result in non-crystalline be to construct NLCs (Rehman and NLCs aqueous phase atafter followed by temperature and the constituents of the butter allows The are obtained the temperatures homogenization of the lipid Several works use synthetic lipids (Brezaniova et al., be used to construct the NLCs (Rehman et al., 2017). amorphous lipid core (Muller al., 2002). The NLCs are theallow homogenization of theoflipid decreasing the obtained temperature toet recrystallization the the elaboration of the exact melting point of the lipid to and aqueous phase atafter high temperatures followed by 2016; Pandiya et al., 2017; Stella et al., 2018), natural Several works use synthetic lipids (Brezaniova et al., and core. aqueous phase at of high temperatures by lipid The production a less ordered solidfollowed lipid core is be used to construct the NLCs (Rehman et al., 2017). Several works use synthetic lipids (Brezaniova et al., decreasing the temperature to allow recrystallization of the lipids (Mandawgade and Patravale, 2008) or even The NLCs are obtained aftertotheallow homogenization of theoflipid 2016; Pandiya et al., 2017; Stella et al., 2018), natural decreasing the temperature recrystallization essential for loading drugsof into the ordered core. The drug is usually 2016; et al., lipids 2017; Stella et al., 2018), lipid core. The production a less solid lipid corethe is mixture of synthetic et 2008) al., 2017) with and aqueous phase at high temperatures followed by lipids Pandiya (Mandawgade and (Zoubari Patravale, ornatural even Several works use synthetic lipids (Brezaniova eteven al., lipid core. The production of a less ordered solid lipid core located infor between the chains of the fatty acids, thus it is lipids (Mandawgade and Patravale, 2008) or essential loading drugs into the core. The drug is usually known melting points to produce SLNs. However the decreasing the temperature to allow recrystallization of the mixturePandiya of synthetic lipids (Zoubari et al., 2017) with 2016; et al., 2017; Stella et al., 2018), natural essential for loading drugs into the core. The drug is usually important to have fatty acids with different chain length (Dal of synthetic (Zoubari et al.,However 2017) with located in The between the chains of the fattysolid acids, thus it is is mixture of lipid core. production of a less ordered lipid core known (Mandawgade melting pointslipids to produce SLNs. the lipids Patravale, 2008) or even located the acids chains of different the fattychain acids, thus it is Pizzol etinal., 2014; Muller et al., 2002). known melting points and to produce SLNs. However the important tobetween have fatty with length (Dal essential for loading drugs into the core. The drug is usually mixture of mixture important to 2014; have fatty acids with different chain length (Dal mixture of synthetic lipids (Zoubari et al., 2017) with of Pizzol etinal., Muller et al., 2002). located between the chains of the fatty acids, thus it is known melting points to produce SLNs. However the Pizzol et al., 2014; Muller et al., 2002). Copyright IFAC important©to2018 have fatty acids with different chain length (Dal 1 mixture of Pizzol et al., 2014; Muller et al., 2002). Copyright 2018 IFAC 1 Hosting by Elsevier Ltd. All rights reserved. 2405-8963 © 2018, IFAC (International Federation of Automatic Control) Copyright 2018 responsibility IFAC 1 Control. Peer review©under of International Federation of Automatic 10.1016/j.ifacol.2018.11.598 Copyright © 2018 IFAC 1
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vegetable lipids in order to reach a new melting temperature is a little explored area.
NLCs were prepared through the high shear homogenization method based on Zoubari et al. (2017). Briefly, the butter associations (1% w/v) were melted in water bath at 10°C above respective melting points. Sudan Red III (0.1% w/v) was used as hydrophobic active and was added to the lipid phase. The aqueous phase was obtained by dissolving Pluronic F127 (0.7% w/v) in distilled water and heated with the lipid phase. The last phase was poured over the first one and high shear homogenization was carried out at 17,000 rpm for 15 minutes by using a FSH-2 High Speed Homogenizer. Finally, the nanoparticles were maintained at room temperature for solidification of the lipid core.
Thus, in this work, the lipid phase was set to melt in defined temperature combining distinct vegetable butters based on its melting point and constituents. Three formulation of NLCs were prepared with lipids to melt at the defined temperatures of 29°C, 37°C and 42°C, generating the drug release triggered by precise temperature. 2. MATERIAL AND METHODS 2.1 Materials The vegetable butters extracted from Theobroma cacao (Lot 08DS07), Passiflora incarnata (Lot PFB018/03) and Butyrospermum parkii (Lot OLV012546) were produced by Ebpm Comercial Ltda and purchased from Armazém Peter Paiva Exclusividades Artesanais Ltda (Brazil); the lipid extract from Olea europaea was purchased from Sovena Group (Portugal). Pluronic F127 was obtained from SigmaAldrich. Sudan Red III and Bromothymol Blue were generously donated by the Laboratorio de Farmacognosia (UFJF, Brazil). Amicon Ultra 0.5mL (Centrifugal Filters) 10K were purchased from Millipore (Germany). Distilled water was prepared freshly whenever required. All other chemicals and solvents were of analytical grade.
2.6 Characterization of NLCs For the determination of the hydrodynamic diameter of the particles a Laser Diffraction Particle Size Analyzer LS 13320 (Beckman Coulter, USA) was used. The PdI (Polydispersity Index) was calculated according to the following formula (1): (1)
PdI:
(𝑑𝑑²) (𝑑𝑑′ )²
Where d is the average value of squares of measured diameters, and d’ is the arithmetic average of measured diameters.
2.2 Gas chromatography An aliquot of each vegetable lipid was subjected to analysis by high-resolution gas chromatography (HR-GC) (HP7820A, Agilent, USA) equipped with flame ionization detector. A SUPELCOWAX-10 15mm x 0.2mm x 0.2µm column was used, with a temperature gradient of (A) 120°C, (0 min), 10°C/min to 1240°C (B) 60°C, (0 min), 10°C/min to 240°C; injector (split of 1/50) at 250°C and detector at 260 °C. The carrier gas used was hydrogen (4 mL/min) and the injection volume was of 1 µL. Identification of peaks was made by comparison with standard methylated fatty acids Supelco37 Fame mix (Supelco cat no 47885-U).
2.7 Encapsulation efficiency The encapsulation efficiency (EE) was measured based on Kushwaha et al. (2013) and Zoubari et al. (2017) using bromothymol blue (0.1% w/v). Briefly, the formulation was added in a 10K MWCO Amicon Ultra Centrifugal Filters and centrifuged for 20 minutes at 15,000 rpm. The filtrate was then analyzed in Nanodrop 2000 (Thermo Fisher Scientific Inc., USA) at a wavelength of 433 nm (Shimada and Hasegawa 2017). Absorbance was fitted to the calibration curve to find the amount of dye that was incorporated. The percentage of encapsulation efficiency (%EE) was calculated according to the formula 2:
2.3 Analysis of melting point To determine the melting point of the lipid, an aliquot of each butter was analyzed in the Digital Apparatus of Melting Point MQAPF-301 (Microquímica Ind. And Com. LTDA, Brazil), in triplicate.
(2) % EE =
𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇 𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑 𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐− 𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝐹 𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑 𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇 𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑 𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐
x 100
2.8 Release assay
2.4 Preparation of defined melting temperature lipid blend
To determine the release efficiency (RE) of the drug from NLCs, aqueous solution containing ethanol (10% v/v) was preheated at the aimed melting temperature. Then, NLCs were added to the solution (1:6 v/v) and the tube was incubated at the aimed melting temperature for 10, 30 and 60 minutes. The solution was placed in 30K MWCO Amicon Ultra centrifugal filters and centrifuged for 10 minutes at 8000 rpm. The filtrate volume was measured, analyzed in Nanodrop 2000 at 433 nm (Shimada and Hasegawa 2017) and fitted to the bromothymol blue calibration curve. The amount of released active was adjusted to percentage of mass in the flow through. As control, the
The lipid associations were chosen based on the melting point of each vegetable butter. The aimed melting temperatures to be produced were 29, 37 and 42°C. Predetermined amount of each fat was weighted, based on the melting point and proportion of constituents. Thereafter, the butters were pooled, heated 10 degrees above aimed melting temperature and homogenized for 2 minutes to form the blend of lipids. The analysis of the melting point of the butter mixture was made as described in Section 2.3. 2.5 Preparation of Nanostructured Lipid Carriers (NLC)
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Table 2. Melting point of vegetable butters
nanoparticle formulated to melt at 37°C was incubated at 35°C, and the release was evaluated. 2.9 Statistical analysis All assays were performed in triplicate. The results presented were expressed as mean values ± SD (standard deviation). Data were analyzed by one-way ANOVA using the GraphPad Prism software. P-values less than 0.05 were considered statistically significant.
Vegetable butter
Melting temperature (°C)
1
35.6
2
42.5
3
39.9
4
10
3. RESULTS AND DISCUSSION 3.1 Characterization of vegetable lipids In order to characterize the chemical composition of the vegetable butters, HR-GC analysis was carried out. Table 1 shows the fatty acids identified by HR-GC in each butter (1T. cacao; 2- P. incarnate; 3- B. parkii; 4- O. europaea). Interestingly, the major constituent present in all of the vegetable butter was oleic acid (C18:1) (over 30%) followed by stearic acid (C18:0); except for O. europaea that had the second major constituent as palmitic acid (C16:0). The third major constituent was palmitic acid for T. cacao and P. incarnata butter, while it was linoleic acid (C18:2) for B. parkii and O. europaea.
The melting point of the butters can be influenced by number of unsaturation in carbon chain of the fatty acids, which causes it to decrease its melting point. This is due to the fact that double bonds make the system more unstable when compared to a saturated chain. In addition, the length of the fatty acid chain may also influence the melting point of the butter. In this way, the greater number of carbons in the chain causes the melting point to increase. Since a large chain requires more energy to be broken, a higher temperature to break the bonds and melt the butter is necessary (Rustan and Drevon, 2005). This can be observed in butter number 4 that has a lower melting point since it contains a greater number of unsaturations and also fatty acids with shorter carbon chain length when compared to other butters.
Table 1. Analysis of fatty acids in each butter by HR-GC
Compound
1
2
3
4
Percentage (%) C8:0 - Caprilic acid
0.0
0.7
0.0
0.0
C10:0 - Capric acid
0.0
0.9
0.0
0.0
C12:0 - Lauric acid
0.0
12.2
0.0
0.0
C14:0 - Myristic acid
0.1
4.9
0.2
0.0
C16:0 - Palmitic acid
26.5
13.6
4.0
10.6
C16:1 - Palmitoleic acid
0.6
0.3
0.1
1.1
C18:0 - Stearic acid
32.8
15.8
40.0
3.7
C18:1 - Oleic acid
33.3
38.7
45.1
76.8
C18:2 - Linoleic acid
4.1
10.6
6.7
5.7
C18:3 - Linolenic acid
0.4
0.2
0.2
0.7
C20:0 - Arachidic acid
1.0
0.3
1.2
0.3
Others
1.2
1.7
2.4
1.0
Next, the samples of the lipid associations were subjected to melting temperature analysis. Table 3 summarizes the results and indicate the proportion of mass used to obtain the exact melting point.
Table 3. Melting temperature obtained by lipid associations and their mass proportions Lipid association
Melting point obtained (°C)
Proportion of Mass (%)
1+4
29
75.00 + 25.00
1+3
37
98.03 + 1.96
1+2
42
92.60 + 7.40
These fat samples constitute the solid lipid core of the nanoparticle. As mentioned by Siafaka et al. (2016), intelligent nanocarriers can transport the drug into the target tissue and, once in the presence of a stimulus such as temperature, release the encapsulated active.
In addition to the percentage of constituents, the melting point of each lipid was determined as shown in Table 2.
3.2 Characterization of Solid Lipid Nanoparticle
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The lipids used in this paper have similar composition of saturated and unsaturated fatty acids, but distinct on proportions of each one. This fact allows easy homogenization by giving them individual characteristics, such as their melting temperature. In this work, the combination of vegetable butters at right proportions was used to form specific melting points. Pluronic block copolymer was used to stabilize the lipid core. Hypothetically, the hydrophobic poly (propylene oxide) block connected to the solid lipidic core, and the hydrophilic PEG block surround the core providing size control to the nanoparticles (Kabanov; Batrakova; Alakhov, 2002; Betrakova and Kabanov, 2008). The Figures 1A, 1B and 1C, shows the frequency curve of the prepared NLCs. The average diameter of nanoparticles were 124 nm ± 39 nm, 146 nm ± 51 nm and 127 nm ± 54 nm, for NCLs made of lipids with melting points on 29°C, 37°C and 42°C, respectively. The calculated PdI was 0,197, 0,192 and 0,184 for NLCs with melting point at 29°C, 37°C and 42°C, respectively. The results indicated that the proportion of lipids did not significantly change the diameter distribution of the nanoparticles, using the same method of preparation.
Figure 1. Particle size distribution of NLCs prepared with lipid with melting point of (A) 29°C, (B) 37°C and (C) 42°C.
The size of a lipid particle depends on many variants. By mixing natural fatty acids, Rehman et al. (2017) found similar results of size and PdI for their thermosensitive SLNs. In addition, Dal Pizzol et al. (2014) tested not only different lipids, but also different surfactants to study the influence of the components in the formulation. Yet, the amount of active to be loaded is also important and may interfere in the formation of the nanoparticle. 3.3 Encapsulation efficiency and release assay Encapsulation efficiency was found to be 82.07%, 88.63%, and 91.63% for the 29°C, 37°C and 42°C, respectively. This is in accordance to other authors as seen by Kushwaha et al. (2013) (70% EE), Zoubari et al. (2017) and Stella et al. (2018) (above 80% EE), Venkateswarlu e Manjunath (2004) and Padiya et al. (2017) (above 90% EE). In fact, the non-organized lipid core formed from fatty acids with different carbon chain length allows the accommodation of drugs in the NLCs (Muller et al, 2002). The release assay was carried out to test the release after submitting the formulations to the defined temperature. As observed in Figure 2, there is increased release of the active after 10 minutes of incubation in defined temperature, followed to a continued reduction in the content of drug in the nanoparticles. This indicate that the NCLs prepared having defined melting point are indeed releasing the drug in the specific temperatures.
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Brezaniova, I., Hruby, M., Kralova, J., Kral, V., Cernochova, Z., Cernoch, P., Slouf, M., Kredatusova, J., Stepanek, P. (2016). Temoporfin-loaded 1tetradecanol-based thermoresponsive solid lipid nanoparticles for photodynamic therapy. Journal of Controlled Release, 241, 34-44.
D ru g c o n te n t (% )
100
80
60
2 9 °C 3 7 °C
40
4 2 °C
Chilkoti, A., Dreher, M.R., Meyer, D. E., Raucher, D. (2002). Targeted drug delivery by thermally responsive polymers. Advanced Drug Delivery Reviews, 54, 613– 630.
C o n tr o l 3 5 ° C
20
0 0
20
40
5
60
M in u te s
Dal Pizzol. C., Filippin-Monteiro, F. B., Restrepo, J. A. S., Pittella, F., Silva, A. H., Souza, P. A., Campos, A. M., Creczynski-Pasa, T. B. (2014). Influence of Surfactant and Lipid Type on the Physicochemical Properties and Biocompatibility of Solid Lipid Nanoparticles. International Journal of Environmental Research and Public Health, 11, 8581-8596.
Figure 2. Percentage of drug content in the NCLs, after individual incubation at 29°C (open circle), 37°C (triangle), 42°C (diamond), control 35°C (open square).
Approximately 15% of the drug was released from the control nanoparticles incubated at 35°C. In fact, the direct addition of the nanoparticles to the water phase containing ethanol allows some of the active to migrate to water phase. This was possible mainly because of the characteristic of bromothymol that is soluble in water but also in oil. In addition, the incubation at precise temperature started melting the lipid core and allowed the bromothymol to partition from the melted lipid to the water phase, increasing its concentration in the flow through.
El-Housiny, S., Eldeen, M. A. S., El-Attar, Y. A., Salem, H. A., Attia, D. Bendas, E. R., El-Nabarawi, M. A. (2018). Fluconazole-loaded solid lipid nanoparticles topical gel for treatment of pityriasis versicolor: formulation and clinical study. Drug delivery, 25:1, 7890. Garcês, A., Amaral, M. H., Sousa Lobo J. M., Silva, A. C. (2018). Formulations based on solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC) for cutaneous use: A review. European Journal of Pharmaceutical Sciences, 112, 159-167.
4. CONCLUSIONS In this work we prepared NLCs using blend of lipids from vegetable origin to adjust the melting temperature of the lipid phase. The vegetable butters were characterized by gas chromatography and the melting point was determined. Based on the proportion of the constituents, it was possible to set precise melting points for the blend of lipids with which the nanoparticles were prepared. The particles size and PdI of NLCs were not affected by the lipid constitution. Drug incorporation and release assay confirmed that the NLCs could accommodate the drug in the core and were responsive to the defined temperature. The method to set precise temperature for triggered release from NLCs presented here might be applicable to a broad range of temperatures.
Hatakeyama, H. (2017). Recent advances in Endogenous and Exogenous Stimuli-Responsive Nanocarriers for Drug Delivery and Therapeutics. Chemical and Pharmaceutical Bulletin, 65:7, 612-617. Hou, D., Xie, C., Huang, K., Zhu, C. (2003). The production and characteristics of solid lipid nanoparticles (SLNs). Biomaterials, 24, 1781-1785. Kabanov, A. V., Batrakova, E. V., Alakhov, V. Y. (2002). Pluronic ® block copolymers as novel polymer therapeutics for drug and gene delivery. Journal of Controlled Release, 82, 182-212. Kushwaha, A. K., Vuddanda, P. R., Karunanidhi, P., Singh, S. K., Singh, S. (2013). Development and evaluation of solid lipid nanoparticles of raloxifene hydrochloride for enhanced bioavailability. BioMed Research International, 1-9.
5. REFERENCES Abdelaziz, H. M., Gaber, M., Abd-Elwakil, M.M., Mabrouk, M.T., Elgohary M.M., Kamel, N.M., Kabary, D.M., Freag, M.S., Samaha, M.W., Mortada, S.M., Elkhodairy, K.A., Fang, J.Y., Elzoghby, A.O. (2017). Inhalable particulate drug delivery systems for lung cancer therapy: Nanoparticles, microparticles, nanocomposites and nanoaggregates. Journal of Controlled Release, 269, 374-392.
Lavrador, P., Gaspar, V. M., Mano, J. F. (2018). Stimuli-responsive nanocarriers for delivery of boné therapeutics – Barriers and progress. Journal of Controlled Release, 273, 51-67. Mandawgade, S., Patravale, V. (2008). Development of SNLs from natural lipids: application to topical delivery of tretinoin. International Journal of Pharmaceutics, 363, 132-138.
Batrakova, E. V., Kabanov, A. V. (2008). Pluronic Block Copolymers: evolution of drug delivery concept from inert nanocarriers to biological response modifiers. Journal of Controlled Release, 130 (2), 98-106. 5
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Miyata, K., Nishiyama, N., Kataoka, K. (2012). Rational design of smart supramolecular assemblies for gene delivery: chemical challenges in the creation of artificial viruses. Chemical Society Reviews, 41, 2562-2574.
lipid nanoparticles. Journal of Controlled Release, 95, 627-638. Yadav, N., Khatak, S., Sara, U. V. S. (2013). Solid lipid nanoparticles: a review. International Journal of Applied Pharmaceutics, 5 (2), 8-18.
Muller, R. H., Radtke, M., Wissing, S. A. (2002). Solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC) in cosmetic and dermatological preparations. Advanced Drug Delivery Reviews, 54, 131-155.
Zoubari, G., Staufenbiel, S., Volz, P., Alexiev, U., Bodmeier, R. (2017). Effect of drug solubility and lipid carrier on drug release from lipid nanoparticles for dermal delivery. European Journal of Pharmaceutics and Biopharmaceutics, 110, 39-46.
Pallerla, S. M., Prabhakar, B. (2013). A Review on Solid Lipid Nanoparticles. International Journal of Pharmaceutical Sciences: Review and Research, 20, 196-206. Pandiya, N. T., Jani, P., Vanza, J., Tandel, H. (2018). Solid lipid nanoparticles as an efficient drug delivery system of olmesartan medoxomil for the treatment of hypertension. Colloids and Surfaces B: Biointerfaces, 165. Pardeike, J., Hommoss, A., Muller, R. H. (2009). Lipid nanoparticles (SLN, NLC) in cosmetic and pharmaceutical dermal products. International Journal of Pharmaceutics, 366, 170-184. Rehman, M., Ihsan, A., Madni, A., Bajwa, S. Z., Shi, D., Webster, T. J., Khan, W. S. (2017). Solid lipid nanoparticles for the thermoresponsive targeting: evidence from spectrophotometry, electrochemical and cytotoxicity studies. International Journal of Nanomedicine, 12, 8325-8336. Rehman, M., Madni, A., Ihsan, A., Khan, W. S., Khan, M. I., Ahmad, M., Mahmood, Ashfaq, M., Bajwa, A. Z., Shakir, I. (2015). Solid and liquid lipid-based binary solid lipid nanoparticles of diacerein: in vitro evaluation of sustained release, simultaneous loading of gold nanoparticles, and potential thermoresponsive behavior. International Journal of Nanomedicine, 10, 2805-2814. Rustan, A. C., Drevon, C. A. (2005). Fatty Acids: Structures and Properties. Encyclopedia of Life Sciences, 1-7. Salas, J., Bootello, M., Martinez-Force, E., Garcés, R. (2009). Tropical vegetable fats and butters: properties and new alternatives. OCL Journal, 16 (4), 254-258. Shimada, T., Hasegawa, T. (2017). Determination of equilibrium structures of bromothymol blue revealed by using quantum chemistry with an aid of multivariate analysis of electronic absorption spectra. Spectrochmica Acta Part A: Molecular and Biomolecular Spectroscopy, 185, 104-110. Siafaka, P. I., Okur, N. U., Karavas, E., Bikiaris, D. N. (2016). Surface Modified Multifunctional and Stimuli Responsive. International Journal of Molecular Sciences, 17:9, 1-40. Stella, B., Peira, E., Dianzani, C., Gallarate, M., Battaglia, L., Gigliotti, C. L., Boggio, E., Dianzani, U., Dosio, F. (2018). Development and characterization of solid lipid nanoaprticles loaded with a highly active doxorubicin derivative. Nanomaterials, 8:110, 1-16. Venkateswarlu, V., Manjunath, K. (2004). Preparation, characterization and in vitro release kinetics of clozapine solid
6
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Setting Precise Temperature for Triggered Release from Nanostructured Lipid IFAC PapersOnLine 51-27 (2018) 1–6 Setting Precise Temperature for Triggered Release from Nanostructured Lipid Setting Precise Temperature for Triggered CarriersRelease from Nanostructured Lipid Carriers Setting Temperature forValle**, Triggered Release Nanostructured Milena Santos*,Precise Ana Beatriz Caribé dos Santos Ana Cristina Mourafrom Gualberto***, Fernanda BritoLipid Leite****, Carriers Milena Santos*, Ana Beatriz Caribé dos Santos Valle**, Ana Cristina Moura Gualberto***, Fernanda Brito Leite****, Frederico Pittella***** Carriers Milena Santos*, Ana Beatriz Caribé dos Santos Valle**, Ana Cristina Moura Gualberto***, Fernanda Brito Leite****, Frederico Pittella***** FredericoAna Pittella***** Milena Santos*, Ana Beatriz Caribé dos Santos Valle**, Cristina Moura Gualberto***, Fernanda Brito Leite****, * Federal University of Juiz de Fora, Juiz de Fora, Brazil (e-mail: [email protected]) Frederico Pittella***** * ** Federal University of of Juiz de de Fora, Juiz de de Fora, Brazil (e-mail: [email protected]) Federal University Juiz Fora, Juiz Fora, Brazil (e-mail: [email protected]) * Federal University of Juiz de Fora, Juiz de Fora, Brazil (e-mail: [email protected]) ** Federal University of Juiz de Fora, de Fora, Brazil (e-mail: [email protected]) *** Federal University of Juiz de Fora, JuizJuiz de Fora, Brazil (e-mail: [email protected]) Federal University Juiz Fora, Juiz Fora, Brazil (e-mail: [email protected]) * ** Federal University of of Juiz de de Fora, Juiz de de Fora, Brazil (e-mail: [email protected]) *** Juiz Fora, Fora, (e-mail: [email protected]) ****Federal FederalUniversity Universityof Juizde Fora,Juiz Juizde Fora,Brazil Brazil(e-mail: (e-mail:[email protected]) [email protected]) *** Federal University ofofJuiz dedeFora, Juiz dedeFora, Brazil ** Federal University of Juiz de Fora, Juiz de Fora, Brazil (e-mail: [email protected]) **** Federal University of Juiz deScience, Fora, Juiz de Fora, Brazil (e-mail: [email protected]) ***** Department of Pharmaceutical Federal University of Juiz [email protected]) Fora, Juiz de Fora, Brazil (e-mail: **** Federal University of Juiz de Fora, Juiz de Fora, Brazil (e-mail: *** Federal University of Juiz de Fora, Juiz de Fora, Brazil (e-mail: [email protected]) ***** Department of Pharmaceutical Science, Federal University of Juiz de Fora, Juiz de Fora, Brazil (e-mail: [email protected]) ***** Department of Pharmaceutical Federal University of Juiz [email protected]) Fora, Juiz de Fora, Brazil (e-mail: **** Federal University of Juiz deScience, Fora, Juiz de Fora, Brazil (e-mail: [email protected]) [email protected]) ***** Department of Pharmaceutical Science, Federal University of Juiz de Fora, Juiz de Fora, Brazil (e-mail: [email protected]) _______________________________________________________________________________________ _______________________________________________________________________________________ _______________________________________________________________________________________ Abstract: The preparation and use of nanostructured lipid carriers (NLCs) as drug delivery system is constantly growing. NLCs are from synthetic lipid or natural lipids with as known, not controlled, Abstract: The preparation andprepared use of nanostructured carriers (NLCs) drug but delivery system is _______________________________________________________________________________________ Abstract: TheHere preparation andprepared usepreparation of nanostructured carriers (NLCs) as drug delivery system is melting point. we propose the of NLCslipid combining natural lipids to have selected melting constantly growing. NLCs are from synthetic or natural lipids with known, but not controlled, constantly growing. NLCs are prepared from synthetic or natural lipids with known, but not controlled, temperature. Vegetable butters were subjected to gas chromatography to determine and define each melting point. we propose of NLCslipid combining lipids to have selected melting Abstract: TheHere preparation and the usepreparation of nanostructured carriersnatural (NLCs) as drug delivery system is melting point. Here we propose the preparation oftoNLCs combining natural lipids to have selected melting constituent and melting point. Three different NLCs suspensions were prepared from a specific blend of temperature. Vegetable butters were subjected gas chromatography to determine and define each constantly growing. NLCs are prepared from synthetic or natural lipids with known, but not controlled, temperature. Vegetable butters were subjected to or gas chromatography to determine and define vegetable butters, adjusted to start melting at 29, 37 42°C, and characterized. Preciseaengineering ofeach the constituent and melting point. Three different NLCs suspensions prepared specific of melting point. Here we propose the preparation of NLCs combiningwere natural lipids from to have selectedblend melting constituent and melting point. Three different NLCs suspensions were prepared from a specific blend of delivery system will contribute to efficient temperature triggered release. vegetable butters, adjusted to startwere melting at 29, 37 or 42°C, and characterized. Precise engineering ofeach the temperature. Vegetable butters subjected to gas chromatography to determine and define vegetable butters, adjusted to start meltingtemperature at 29, 37 ortriggered 42°C, and characterized. Precise engineering of the delivery system will contribute to efficient release. constituent and(International melting point. Three different NLCs suspensions were prepared a specific blend of © 2018, IFAC Federation of Automatic Control) Hosting byRelease, Elsevier Ltd. from All rights reserved. delivery system will contribute to efficient temperature triggered release. Keywords: Nanostructured lipid carriers, Melting point, Triggered Thermoresponsive, Vegetable vegetable butters, adjusted to start melting at 29, 37 or 42°C, and characterized. Precise engineering of the butter. Keywords: Nanostructured lipid carriers, Melting point, Triggered Release, Thermoresponsive, Vegetable delivery system will contribute to efficient temperature triggered release. Keywords: Nanostructured lipid carriers, Melting point, Triggered Release, Thermoresponsive, Vegetable butter. butter. ____________________________________________________________________________________ Keywords: Nanostructured lipid carriers, Melting point, Triggered Release, Thermoresponsive, Vegetable ____________________________________________________________________________________ butter. ____________________________________________________________________________________ ____________________________________________________________________________________ 1. INTRODUCTION In fact, the choice of the lipid in a NLC formulation might trigger. 1. INTRODUCTION In fact,bethecritical choicefor of the the precise lipid in temperature a NLC formulation The use of nanoparticles as drug delivery systems allows 1. INTRODUCTION In fact, the choice of the lipid in a NLC formulation Vegetable butters have as main constituent naturally might be critical for the precise temperature trigger. therapeutic such asas:drug i) delivery stability systems of the allows active might be fatty critical for the precise temperature trigger. The use of benefits, nanoparticles balanced acids. These are constituent long carbonic chain Vegetable main naturally 1. INTRODUCTION In fact, thebutters choice have of theas lipid inconstituent a NLC formulation The use ofii)benefits, nanoparticles asas:drug delivery systems substance; reductionsuch of toxicity; increased Vegetable butters have as main naturally therapeutic i) iii) stability of therapeutic the allows active with a carboxyl group at the end of the molecule. The balanced acids. These are long carbonictrigger. chain might be fatty critical for the precise temperature therapeutic such as: i) iii) stability of therapeutic the et active efficacy; and iv) increased bioavailability (Pallaerla al., balanced fatty acids. These areorof long carbonic chain substance; ii)benefits, reduction of toxicity; increased carbonic chain can be saturated unsaturated (Drevon The use of nanoparticles as drug delivery systems allows with a carboxyl group at the end the molecule. The Vegetable butters haveatasthemain constituent naturally substance; ii) reduction toxicity; iii) increased 2013). Among the of existing drug delivery therapeutic systems, with carboxyl group endorof theacids molecule. The efficacy; and iv) increased bioavailability al., et al.,a2005). Typically, saturated fatty have high therapeutic benefits, such as: i) stability(Pallaerla of the et active carbonic chain can be saturated unsaturated (Drevon balanced fatty acids. These are long carbonic chain efficacy; and iv) increased bioavailability (Pallaerla et al., nanostructured lipid carriers (NLCs) aredelivery characterized for carbonic chain can be saturated or unsaturated (Drevon 2013). Among the existing drug systems, melting point while unsaturated present low melting substance; ii) reduction of toxicity;drug iii) increased therapeutic et al.,a2005). Typically, fatty have high with carboxyl group atsaturated the end of theacids molecule. The 2013). Among the existing systems, having amorphous solid lipid (NLCs) core at room temperature that et al., 2005). Typically, saturated fatty acids have high nanostructured lipid carriers aredelivery characterized for temperature (Salas et al., 2009). The naturally balanced efficacy; and iv) increased bioavailability (Pallaerla et al., melting point while unsaturated present low melting carbonic chain can be saturated or unsaturated (Drevon nanostructured lipid carriers (NLCs) are characterized for accommodates hydrophobic compounds (Abdelaziz et al., melting point while unsaturated present low melting having amorphous solid lipid core drug at room temperature that amount of fatty acids define melting point of a 2013). Among the delivery systems, temperature et al., 2009).the The naturally balanced et al., 2005).(Salas Typically, saturated fatty acids have high having amorphous solid existing core room temperature that 2017). It is considered alipid type of at solid lipid nanoparticles temperature (Salas et al., 2009). The naturally balanced accommodates hydrophobic compounds (Abdelaziz et al., vegetable butter. Understanding the melting nanostructured lipid carriers compounds (NLCs) are (Abdelaziz characterized for amount of fatty acidsunsaturated define thepresent meltinglow point of a melting point while melting accommodates hydrophobic et al., (SLN) by blend aoftype lipidsoftosolid resultlipid in non-crystalline amount of fatty acidsconstituents define the ofmelting point of a 2017). prepared It is considered nanoparticles temperature and the the the butter allows having amorphous solid lipid core at room temperature that vegetable butter. Understanding melting temperature (Salas et al., 2009). The naturally balanced 2017). It islipid considered aoftype ofto solid nanoparticles amorphous coreblend (Muller et al., 2002). vegetable butter. Understanding the melting (SLN) prepared by lipids resultlipid in non-crystalline the elaboration of the exact melting point of the lipid to accommodates hydrophobic compounds (Abdelaziz et al., temperature and the the butter amount of fatty acidsconstituents define the of pointallows of a (SLN) prepared by of lipids to result in non-crystalline temperature andofthe constituents ofmelting theetof butter allows amorphous lipid coreblend (Muller et al., 2002). be used to construct the NLCs (Rehman al., 2017). 2017). It is considered a type of solid lipid nanoparticles the elaboration the exact melting point the lipid to The NLCs are after et theal., homogenization of the lipid vegetable butter. Understanding melting amorphous lipidobtained core (Muller 2002). theused elaboration of thethe exact melting pointetthe ofal., the2017). lipid to (SLN) prepared by blend ofhigh lipids to result in non-crystalline be to construct NLCs (Rehman and NLCs aqueous phase atafter followed by temperature and the constituents of the butter allows The are obtained the temperatures homogenization of the lipid Several works use synthetic lipids (Brezaniova et al., be used to construct the NLCs (Rehman et al., 2017). amorphous lipid core (Muller al., 2002). The NLCs are theallow homogenization of theoflipid decreasing the obtained temperature toet recrystallization the the elaboration of the exact melting point of the lipid to and aqueous phase atafter high temperatures followed by 2016; Pandiya et al., 2017; Stella et al., 2018), natural Several works use synthetic lipids (Brezaniova et al., and core. aqueous phase at of high temperatures by lipid The production a less ordered solidfollowed lipid core is be used to construct the NLCs (Rehman et al., 2017). Several works use synthetic lipids (Brezaniova et al., decreasing the temperature to allow recrystallization of the lipids (Mandawgade and Patravale, 2008) or even The NLCs are obtained aftertotheallow homogenization of theoflipid 2016; Pandiya et al., 2017; Stella et al., 2018), natural decreasing the temperature recrystallization essential for loading drugsof into the ordered core. The drug is usually 2016; et al., lipids 2017; Stella et al., 2018), lipid core. The production a less solid lipid corethe is mixture of synthetic et 2008) al., 2017) with and aqueous phase at high temperatures followed by lipids Pandiya (Mandawgade and (Zoubari Patravale, ornatural even Several works use synthetic lipids (Brezaniova eteven al., lipid core. The production of a less ordered solid lipid core located infor between the chains of the fatty acids, thus it is lipids (Mandawgade and Patravale, 2008) or essential loading drugs into the core. The drug is usually known melting points to produce SLNs. However the decreasing the temperature to allow recrystallization of the mixturePandiya of synthetic lipids (Zoubari et al., 2017) with 2016; et al., 2017; Stella et al., 2018), natural essential for loading drugs into the core. The drug is usually important to have fatty acids with different chain length (Dal of synthetic (Zoubari et al.,However 2017) with located in The between the chains of the fattysolid acids, thus it is is mixture of lipid core. production of a less ordered lipid core known (Mandawgade melting pointslipids to produce SLNs. the lipids Patravale, 2008) or even located the acids chains of different the fattychain acids, thus it is Pizzol etinal., 2014; Muller et al., 2002). known melting points and to produce SLNs. However the important tobetween have fatty with length (Dal essential for loading drugs into the core. The drug is usually mixture of mixture important to 2014; have fatty acids with different chain length (Dal mixture of synthetic lipids (Zoubari et al., 2017) with of Pizzol etinal., Muller et al., 2002). located between the chains of the fatty acids, thus it is known melting points to produce SLNs. However the Pizzol et al., 2014; Muller et al., 2002). Copyright IFAC important©to2018 have fatty acids with different chain length (Dal 1 mixture of Pizzol et al., 2014; Muller et al., 2002). Copyright 2018 IFAC 1 Hosting by Elsevier Ltd. All rights reserved. 2405-8963 © 2018, IFAC (International Federation of Automatic Control) Copyright 2018 responsibility IFAC 1 Control. Peer review©under of International Federation of Automatic 10.1016/j.ifacol.2018.11.598 Copyright © 2018 IFAC 1
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vegetable lipids in order to reach a new melting temperature is a little explored area.
NLCs were prepared through the high shear homogenization method based on Zoubari et al. (2017). Briefly, the butter associations (1% w/v) were melted in water bath at 10°C above respective melting points. Sudan Red III (0.1% w/v) was used as hydrophobic active and was added to the lipid phase. The aqueous phase was obtained by dissolving Pluronic F127 (0.7% w/v) in distilled water and heated with the lipid phase. The last phase was poured over the first one and high shear homogenization was carried out at 17,000 rpm for 15 minutes by using a FSH-2 High Speed Homogenizer. Finally, the nanoparticles were maintained at room temperature for solidification of the lipid core.
Thus, in this work, the lipid phase was set to melt in defined temperature combining distinct vegetable butters based on its melting point and constituents. Three formulation of NLCs were prepared with lipids to melt at the defined temperatures of 29°C, 37°C and 42°C, generating the drug release triggered by precise temperature. 2. MATERIAL AND METHODS 2.1 Materials The vegetable butters extracted from Theobroma cacao (Lot 08DS07), Passiflora incarnata (Lot PFB018/03) and Butyrospermum parkii (Lot OLV012546) were produced by Ebpm Comercial Ltda and purchased from Armazém Peter Paiva Exclusividades Artesanais Ltda (Brazil); the lipid extract from Olea europaea was purchased from Sovena Group (Portugal). Pluronic F127 was obtained from SigmaAldrich. Sudan Red III and Bromothymol Blue were generously donated by the Laboratorio de Farmacognosia (UFJF, Brazil). Amicon Ultra 0.5mL (Centrifugal Filters) 10K were purchased from Millipore (Germany). Distilled water was prepared freshly whenever required. All other chemicals and solvents were of analytical grade.
2.6 Characterization of NLCs For the determination of the hydrodynamic diameter of the particles a Laser Diffraction Particle Size Analyzer LS 13320 (Beckman Coulter, USA) was used. The PdI (Polydispersity Index) was calculated according to the following formula (1): (1)
PdI:
(𝑑𝑑²) (𝑑𝑑′ )²
Where d is the average value of squares of measured diameters, and d’ is the arithmetic average of measured diameters.
2.2 Gas chromatography An aliquot of each vegetable lipid was subjected to analysis by high-resolution gas chromatography (HR-GC) (HP7820A, Agilent, USA) equipped with flame ionization detector. A SUPELCOWAX-10 15mm x 0.2mm x 0.2µm column was used, with a temperature gradient of (A) 120°C, (0 min), 10°C/min to 1240°C (B) 60°C, (0 min), 10°C/min to 240°C; injector (split of 1/50) at 250°C and detector at 260 °C. The carrier gas used was hydrogen (4 mL/min) and the injection volume was of 1 µL. Identification of peaks was made by comparison with standard methylated fatty acids Supelco37 Fame mix (Supelco cat no 47885-U).
2.7 Encapsulation efficiency The encapsulation efficiency (EE) was measured based on Kushwaha et al. (2013) and Zoubari et al. (2017) using bromothymol blue (0.1% w/v). Briefly, the formulation was added in a 10K MWCO Amicon Ultra Centrifugal Filters and centrifuged for 20 minutes at 15,000 rpm. The filtrate was then analyzed in Nanodrop 2000 (Thermo Fisher Scientific Inc., USA) at a wavelength of 433 nm (Shimada and Hasegawa 2017). Absorbance was fitted to the calibration curve to find the amount of dye that was incorporated. The percentage of encapsulation efficiency (%EE) was calculated according to the formula 2:
2.3 Analysis of melting point To determine the melting point of the lipid, an aliquot of each butter was analyzed in the Digital Apparatus of Melting Point MQAPF-301 (Microquímica Ind. And Com. LTDA, Brazil), in triplicate.
(2) % EE =
𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇 𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑 𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐− 𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝐹 𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑 𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇 𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑 𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐
x 100
2.8 Release assay
2.4 Preparation of defined melting temperature lipid blend
To determine the release efficiency (RE) of the drug from NLCs, aqueous solution containing ethanol (10% v/v) was preheated at the aimed melting temperature. Then, NLCs were added to the solution (1:6 v/v) and the tube was incubated at the aimed melting temperature for 10, 30 and 60 minutes. The solution was placed in 30K MWCO Amicon Ultra centrifugal filters and centrifuged for 10 minutes at 8000 rpm. The filtrate volume was measured, analyzed in Nanodrop 2000 at 433 nm (Shimada and Hasegawa 2017) and fitted to the bromothymol blue calibration curve. The amount of released active was adjusted to percentage of mass in the flow through. As control, the
The lipid associations were chosen based on the melting point of each vegetable butter. The aimed melting temperatures to be produced were 29, 37 and 42°C. Predetermined amount of each fat was weighted, based on the melting point and proportion of constituents. Thereafter, the butters were pooled, heated 10 degrees above aimed melting temperature and homogenized for 2 minutes to form the blend of lipids. The analysis of the melting point of the butter mixture was made as described in Section 2.3. 2.5 Preparation of Nanostructured Lipid Carriers (NLC)
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3
Table 2. Melting point of vegetable butters
nanoparticle formulated to melt at 37°C was incubated at 35°C, and the release was evaluated. 2.9 Statistical analysis All assays were performed in triplicate. The results presented were expressed as mean values ± SD (standard deviation). Data were analyzed by one-way ANOVA using the GraphPad Prism software. P-values less than 0.05 were considered statistically significant.
Vegetable butter
Melting temperature (°C)
1
35.6
2
42.5
3
39.9
4
10
3. RESULTS AND DISCUSSION 3.1 Characterization of vegetable lipids In order to characterize the chemical composition of the vegetable butters, HR-GC analysis was carried out. Table 1 shows the fatty acids identified by HR-GC in each butter (1T. cacao; 2- P. incarnate; 3- B. parkii; 4- O. europaea). Interestingly, the major constituent present in all of the vegetable butter was oleic acid (C18:1) (over 30%) followed by stearic acid (C18:0); except for O. europaea that had the second major constituent as palmitic acid (C16:0). The third major constituent was palmitic acid for T. cacao and P. incarnata butter, while it was linoleic acid (C18:2) for B. parkii and O. europaea.
The melting point of the butters can be influenced by number of unsaturation in carbon chain of the fatty acids, which causes it to decrease its melting point. This is due to the fact that double bonds make the system more unstable when compared to a saturated chain. In addition, the length of the fatty acid chain may also influence the melting point of the butter. In this way, the greater number of carbons in the chain causes the melting point to increase. Since a large chain requires more energy to be broken, a higher temperature to break the bonds and melt the butter is necessary (Rustan and Drevon, 2005). This can be observed in butter number 4 that has a lower melting point since it contains a greater number of unsaturations and also fatty acids with shorter carbon chain length when compared to other butters.
Table 1. Analysis of fatty acids in each butter by HR-GC
Compound
1
2
3
4
Percentage (%) C8:0 - Caprilic acid
0.0
0.7
0.0
0.0
C10:0 - Capric acid
0.0
0.9
0.0
0.0
C12:0 - Lauric acid
0.0
12.2
0.0
0.0
C14:0 - Myristic acid
0.1
4.9
0.2
0.0
C16:0 - Palmitic acid
26.5
13.6
4.0
10.6
C16:1 - Palmitoleic acid
0.6
0.3
0.1
1.1
C18:0 - Stearic acid
32.8
15.8
40.0
3.7
C18:1 - Oleic acid
33.3
38.7
45.1
76.8
C18:2 - Linoleic acid
4.1
10.6
6.7
5.7
C18:3 - Linolenic acid
0.4
0.2
0.2
0.7
C20:0 - Arachidic acid
1.0
0.3
1.2
0.3
Others
1.2
1.7
2.4
1.0
Next, the samples of the lipid associations were subjected to melting temperature analysis. Table 3 summarizes the results and indicate the proportion of mass used to obtain the exact melting point.
Table 3. Melting temperature obtained by lipid associations and their mass proportions Lipid association
Melting point obtained (°C)
Proportion of Mass (%)
1+4
29
75.00 + 25.00
1+3
37
98.03 + 1.96
1+2
42
92.60 + 7.40
These fat samples constitute the solid lipid core of the nanoparticle. As mentioned by Siafaka et al. (2016), intelligent nanocarriers can transport the drug into the target tissue and, once in the presence of a stimulus such as temperature, release the encapsulated active.
In addition to the percentage of constituents, the melting point of each lipid was determined as shown in Table 2.
3.2 Characterization of Solid Lipid Nanoparticle
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The lipids used in this paper have similar composition of saturated and unsaturated fatty acids, but distinct on proportions of each one. This fact allows easy homogenization by giving them individual characteristics, such as their melting temperature. In this work, the combination of vegetable butters at right proportions was used to form specific melting points. Pluronic block copolymer was used to stabilize the lipid core. Hypothetically, the hydrophobic poly (propylene oxide) block connected to the solid lipidic core, and the hydrophilic PEG block surround the core providing size control to the nanoparticles (Kabanov; Batrakova; Alakhov, 2002; Betrakova and Kabanov, 2008). The Figures 1A, 1B and 1C, shows the frequency curve of the prepared NLCs. The average diameter of nanoparticles were 124 nm ± 39 nm, 146 nm ± 51 nm and 127 nm ± 54 nm, for NCLs made of lipids with melting points on 29°C, 37°C and 42°C, respectively. The calculated PdI was 0,197, 0,192 and 0,184 for NLCs with melting point at 29°C, 37°C and 42°C, respectively. The results indicated that the proportion of lipids did not significantly change the diameter distribution of the nanoparticles, using the same method of preparation.
Figure 1. Particle size distribution of NLCs prepared with lipid with melting point of (A) 29°C, (B) 37°C and (C) 42°C.
The size of a lipid particle depends on many variants. By mixing natural fatty acids, Rehman et al. (2017) found similar results of size and PdI for their thermosensitive SLNs. In addition, Dal Pizzol et al. (2014) tested not only different lipids, but also different surfactants to study the influence of the components in the formulation. Yet, the amount of active to be loaded is also important and may interfere in the formation of the nanoparticle. 3.3 Encapsulation efficiency and release assay Encapsulation efficiency was found to be 82.07%, 88.63%, and 91.63% for the 29°C, 37°C and 42°C, respectively. This is in accordance to other authors as seen by Kushwaha et al. (2013) (70% EE), Zoubari et al. (2017) and Stella et al. (2018) (above 80% EE), Venkateswarlu e Manjunath (2004) and Padiya et al. (2017) (above 90% EE). In fact, the non-organized lipid core formed from fatty acids with different carbon chain length allows the accommodation of drugs in the NLCs (Muller et al, 2002). The release assay was carried out to test the release after submitting the formulations to the defined temperature. As observed in Figure 2, there is increased release of the active after 10 minutes of incubation in defined temperature, followed to a continued reduction in the content of drug in the nanoparticles. This indicate that the NCLs prepared having defined melting point are indeed releasing the drug in the specific temperatures.
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Brezaniova, I., Hruby, M., Kralova, J., Kral, V., Cernochova, Z., Cernoch, P., Slouf, M., Kredatusova, J., Stepanek, P. (2016). Temoporfin-loaded 1tetradecanol-based thermoresponsive solid lipid nanoparticles for photodynamic therapy. Journal of Controlled Release, 241, 34-44.
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Chilkoti, A., Dreher, M.R., Meyer, D. E., Raucher, D. (2002). Targeted drug delivery by thermally responsive polymers. Advanced Drug Delivery Reviews, 54, 613– 630.
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Dal Pizzol. C., Filippin-Monteiro, F. B., Restrepo, J. A. S., Pittella, F., Silva, A. H., Souza, P. A., Campos, A. M., Creczynski-Pasa, T. B. (2014). Influence of Surfactant and Lipid Type on the Physicochemical Properties and Biocompatibility of Solid Lipid Nanoparticles. International Journal of Environmental Research and Public Health, 11, 8581-8596.
Figure 2. Percentage of drug content in the NCLs, after individual incubation at 29°C (open circle), 37°C (triangle), 42°C (diamond), control 35°C (open square).
Approximately 15% of the drug was released from the control nanoparticles incubated at 35°C. In fact, the direct addition of the nanoparticles to the water phase containing ethanol allows some of the active to migrate to water phase. This was possible mainly because of the characteristic of bromothymol that is soluble in water but also in oil. In addition, the incubation at precise temperature started melting the lipid core and allowed the bromothymol to partition from the melted lipid to the water phase, increasing its concentration in the flow through.
El-Housiny, S., Eldeen, M. A. S., El-Attar, Y. A., Salem, H. A., Attia, D. Bendas, E. R., El-Nabarawi, M. A. (2018). Fluconazole-loaded solid lipid nanoparticles topical gel for treatment of pityriasis versicolor: formulation and clinical study. Drug delivery, 25:1, 7890. Garcês, A., Amaral, M. H., Sousa Lobo J. M., Silva, A. C. (2018). Formulations based on solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC) for cutaneous use: A review. European Journal of Pharmaceutical Sciences, 112, 159-167.
4. CONCLUSIONS In this work we prepared NLCs using blend of lipids from vegetable origin to adjust the melting temperature of the lipid phase. The vegetable butters were characterized by gas chromatography and the melting point was determined. Based on the proportion of the constituents, it was possible to set precise melting points for the blend of lipids with which the nanoparticles were prepared. The particles size and PdI of NLCs were not affected by the lipid constitution. Drug incorporation and release assay confirmed that the NLCs could accommodate the drug in the core and were responsive to the defined temperature. The method to set precise temperature for triggered release from NLCs presented here might be applicable to a broad range of temperatures.
Hatakeyama, H. (2017). Recent advances in Endogenous and Exogenous Stimuli-Responsive Nanocarriers for Drug Delivery and Therapeutics. Chemical and Pharmaceutical Bulletin, 65:7, 612-617. Hou, D., Xie, C., Huang, K., Zhu, C. (2003). The production and characteristics of solid lipid nanoparticles (SLNs). Biomaterials, 24, 1781-1785. Kabanov, A. V., Batrakova, E. V., Alakhov, V. Y. (2002). Pluronic ® block copolymers as novel polymer therapeutics for drug and gene delivery. Journal of Controlled Release, 82, 182-212. Kushwaha, A. K., Vuddanda, P. R., Karunanidhi, P., Singh, S. K., Singh, S. (2013). Development and evaluation of solid lipid nanoparticles of raloxifene hydrochloride for enhanced bioavailability. BioMed Research International, 1-9.
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