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Formulation of Poorly Water-Soluble Compound Loaded Solid Lipid Nanoparticles in a Microchannel System Fabricated by Mechanical Microcutting Method: Puerarin as a Model Drug Linhong Xu,†,‡ Xu Tan,† Junxian Yun,*,§ Shaochuan Shen,§ Songhong Zhang,§ Changming Tu,§ Wei Zhao,§ Bing Tian,*,∥ Gensheng Yang,⊥ and Kejian Yao§ †

Faculty of Mechanical & Electronic Information, China University of Geosciences (Wuhan), Wuhan 430074, China State Key Laboratory of Fluid Power Transmission and Control, Zhejiang University, Hangzhou 310027, China § State Key Laboratory Breeding Base of Green Chemistry Synthesis Technology, College of Chemical Engineering and Materials Science, and ⊥College of Pharmaceutical Science, Zhejiang University of Technology, Hangzhou 310032, China ∥ Key Laboratory for Nuclear-Agricultural Sciences of Chinese Ministry of Agriculture and Zhejiang Province, Institute of Nuclear-Agricultural Sciences, Zhejiang University, Hangzhou 310029, China ‡

ABSTRACT: Delivery of poorly water-soluble compounds by use of nanosized lipid carriers has attracted much attention in pharmaceutical and therapeutic areas in recent years. However, it is difficult to formulate poorly water-soluble compound loaded lipid nanoparticles with narrow size distributions and proper drug load properties. In the present work, we introduce a precise mechanical manufacture method, the microcutting approach, to fabricate a microchannel system on a stainless steel slab, which was then assembled and employed to prepare poorly water-soluble drug loaded solid lipid nanoparticles (SLNs) by liquid flowfocusing and gas displacing techniques. A poorly water-soluble drug, puerarin, was used as the model drug, and the production of puerarin-loaded SLNs was achieved under various conditions. Particle size distribution of the obtained drug-loaded SLNs was measured by dynamic light scattering (DLS), and the particle morphology was observed by transmission electron microscopy (TEM). The state of the drug-loaded SLNs was analyzed by differential scanning calorimetry (DSC), and the drug load was determined by high-performance liquid chromatography (HPLC). The results showed that microchannels fabricated by the present mechanical microcutting method were excellent in surface quality and precise in channel sizes and shapes, which are easily scaled-up. The puerarin-loaded SLNs prepared by the present microchannel system have a narrow size distribution and the mean particle size varied with the velocities of fluids and the lipid concentration. The drug load capacity of puerarin was influenced by preparation parameters, like the flow velocities of liquids and the concentrations of lipid and puerarin. Therefore, by employing suitable preparation parameters, one can produce poorly water-soluble drug loaded SLNs with expected sizes and drug load capacities by use of microchannels fabricated by the microcutting method, which could be an effective and alternative approach for scale-up production of those new nanosized delivery systems containing poorly water-soluble drugs for potential pharmaceutical and therapeutic applications.

1. INTRODUCTION Poorly water-soluble drugs and compounds play a major role in various therapeutic areas and are thus of significant importance in the pharmaceutical industry. A large percentage (more than ∼30−40%) of drugs in the established pharmaceutical pipelines or drug candidates generated by chemical synthesis, highthroughput screening, and combinatorial chemistry approaches are known to be poorly water-soluble.1−3 However, due to the aqueous insolubility it is always a challenge task to deliver poorly water-soluble drug substances effectively in clinical administration. Numerous formulation approaches, such as molecular complexes, polymeric micellar systems, nanosuspensions, and lipid formulations as well as chemically modified prodrugs, have been proposed to deliver poorly water-soluble drugs in the last few decades, as reviewed in refs 3−5. Among them, the colloidal delivery system of solid lipid nanoparticles (SLNs) has attracted much attention as one of the new nanosuspensions, owing to excellent properties such as high biocompatibility, nontoxicity, long-term stability, good physiological options, and controlled release.6−11 This interesting class © 2012 American Chemical Society

of carriers could permit various administration routes, that is, peroral, parenteral, dermal, ocular, and pulmonary approaches, and thus potential delivery applications not only for watersoluble biopharmaceutical substances12,13 but also for poorly water-soluble drugs.3,5,11,14 SLNs can be prepared by several methods,7−9,14−16 such as solvent injection approach,17 membrane contactor method,18,19 supercritical fluid technology,20 microemulsion,21−23 dropletphase aerosol synthesis,24 as well as homogenization with grinding, high pressure, or high shear. However, it is difficult to produce SLNs with small mean size and a narrow size distribution, especially on the industrial scale. Recently, the liquid flow-focusing method in concentric, T-shaped or crossshaped microchannels has been suggested as a novel approach to prepare SLNs with a narrow size distribution.25−27 By Received: Revised: Accepted: Published: 11373

March 5, 2012 August 6, 2012 August 16, 2012 August 16, 2012 dx.doi.org/10.1021/ie300592u | Ind. Eng. Chem. Res. 2012, 51, 11373−11380

Industrial & Engineering Chemistry Research

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utilizing gas slug flow in the microchannels, the production of SLNs by this method could be performed continuously.28,29 Compared with other preparation techniques, this method is simple and has no need of overcritical operations, and the particle sizes can also be controlled by changing process parameters such as flow velocities and concentrations of solutes. Therefore, it has potential applications in preparation of SLNs for delivery of poorly water-soluble drugs. However, from the application view it is necessary to develop novel precise fabrication techniques of microchannels, which are convenient and easily scaled-up. Various methods for the fabrication of microchannels on metal plates have been suggested,30 such as mechanical dicing,31 electrochemical machining,32 electrical discharge machining,33,26−29 photolithography etching,34 vibration cutting,35 and micromachining using laser beam, electron beam, or ion beams, as well as combinations of those techniques36 and other mechanical approaches.37−40 The energy beam based techniques have some limitations, such as the need for special facilities and the thermally induced microcracks or defects on the workpiece, while the tool-based methods always suffer the lack of ultrasmall and special micromachining tools. However, with advances in machining tools, novel microcutting tools are being developed and some are commercially available, which could promote the tool-based micromachining as a competitive approach for the fabrication of microchannels.41 Puerarin (8-glucosyldaidzein) is an isoflavone C-glycoside isolated from the plant root of Pueraria lobata Ohwi,42−45 a traditional Chinese herbal medicine for several centuries. This major bioactive component has comprehensive pharmacological actions in the treatment of cardiovascular and cerebrovascular diseases, diabetes, and other diseases like hyperlipidemia or hypercholesterolemia, platelet aggregation, and inflammation.43−47 However, the bioavailability of puerarin in the peroral route by traditional capsules or tablets is very low due to its aqueous insolubility, and thus there is a lack of oral formulations currently.23 The injection formulation of puerarin is available widely nowadays, but adverse drug reactions such as allergic responses, hemolytic anemia, allergic shock, fever, and kidney or liver damage were sometimes observed in clinical parenteral administration.48,49 There is thus an urgent need to develop novel formulations of puerarin or different delivery routes in order to enhance the bioavailability and decrease the risk of serious adverse effects in the injection route. Li et al.21 prepared puerarin loaded monocaprate nanoparticles by double emulsion method. The obtained nanoparticles were amorphous, which may be benefited to the incorporation of puerarin. Chen et al.50 prepared puerarin loaded by poly(Llactide) nanoparticles by supercritical CO2 process. In this work, we prepare puerarin-loaded SLNs by using the liquid flow-focusing and gas displacing method in a microchannel with T-shaped and cross junctions.28 Different from previously reported work,25−27 the present rectangular microchannel system was fabricated on stainless steel slab by a precise manufacturing method, the mechanical microcutting method, using a micromilling cutter with two cutting edges and helix grooves, and is easy to scale up. The fabrication of microchannels was presented and the drug loading capacities, particle size distribution, and morphology as well as the crystallinity of the obtained SLNs at various preparation conditions were characterized experimentally.

2. MATERIALS AND METHODS 2.1. Materials. Puerarin (99%) were purchased from Nanjing Zelang Medical Technology Co. Ltd. (Nanjing, China). Softisan 100 (a triglyceride mixture of natural, saturated, even-numbered, and unbranched fatty acids with a chain length from C10 to C18) was from Sasol Chemical Co. Ltd. (Nanjing, China). Poloxamer 188 was from BASF Co. Ltd. (Shanghai, China). Other chemicals used were of analytical grade from local sources. The 304 stainless steel was obtained from a local market. 2.2. Fabrication of Rectangular Microchannels on Stainless Steel Slab. Stainless steel was used for manufacture of the basic microchannel slab with length of 280 mm, width of 80 mm, and thickness of 13 mm. The top surface of the slab was milled and ground carefully to improve the surface roughness. A solid carbide end micromilling cutter of about 400 μm diameter with two cutting edges and helix grooves was employed to fabricate microchannels on the stainless steel slab, and the fabrication process was achieved in a high-speed computerized numerical control engraving and milling machine (JTCK-500, Kejie Automation Co., Ltd., Jiangmen, China). The main microchannel has width 480 μm, depth 295 μm, and length 215 mm, with cross branch channels of 30 mm length each located 60 mm from the inlet of the main channel, followed by a T-shaped branch channel of the same size 60 mm downstream thereafter, as shown schematically in Figure 1. The

Figure 1. Schematic diagram of the sizes of the microchannels.

slab with microchannels was covered by a polypropylene plate with thickness about 2 mm, followed by another milled and ground stainless steel slab with the same sizes. The manufacture and assemblage of the microchannel system were achieved in Wuhan Redywoods Bioengineering Co. Ltd. (Wuhan, China). 2.3. Preparation of Puerarin-Loaded SLNs. The preparation of puerarin-loaded SLNs was carried out in an experiment setup the same as reported previously.28 Typically, the drug−lipid solutions with different concentrations were prepared by dissolving puerarin and Softisan 100 in ethanol, and the aqueous solution phase was obtained by dissolving poloxamer 188 in water (0.5%, w/w). In each run, the drug− lipid solution of a given concentration was pumped into the main microchannel, the aqueous phase was pumped simultaneously through the two branch channels of the cross-junction to induce flow focusing, and the gas phase of N2 was injected through the branch of the T-junction to generate the gas− liquid slug flow in the channel. The flow streams were observed by use of a microscope connected with a charge-coupled device (CCD) camera (model TS1000ME, Fastec Imaging Inc.), as reported previously.26,28,29 The nanoparticles were formed within the main channel and the particle suspension was 11374

dx.doi.org/10.1021/ie300592u | Ind. Eng. Chem. Res. 2012, 51, 11373−11380


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collected at the outlet by a stirred and sealed vessel for further analysis. 2.4. Characterization of Puerarin-Loaded SLNs. Size distributions of the puerarin-loaded SLNs obtained under various conditions were tested by dynamic light scattering (DLS) conducted at 90° scattered light and the temperature was 25 °C (90 plus, Brookhaven Instruments Corp., New York), as in our previous work.26−28 Particle morphology was examined by depositing drops of the puerarin-loaded SLN suspension onto a copper grid covered with a thin carbon film, which was dried in air and then observed by transmission electron microscopy (TEM) (JEM-1230, JEOL Ltd., Japan). Samples of the puerarin-loaded SLN suspensions were centrifuged at 17000g for 20 min, and the nanoparticles were separated and dried by freezing for differential scanning calorimetry (DSC) measurements. Thermal responses of the freeze-dried samples were tested by DSC (Netzsch STA 409 PC/PG, Netzsch-Gerätebau GmbH, Germany) at a heating rate of 5 K/min from room temperature to 100 °C under a continuous argon flow. The puerarin loading capacity of the SLNs was measured by HPLC in a Waters Alliance liquid chromatograph system (2695) equipped with a photodiode array detector (2998) and a SunFire C18 column (5 μm, 4.6 mm × 250 mm) with acetonitrile as mobile phase. Several milligrams of the freezedried nanoparticle samples were dissolved in methanol and diluted 5-fold in acetonitrile for analysis. The peak signals were calibrated by use of pure puerarin and methanol solutions with known concentrations, and the established standard curve was used for quantitative determination of the amount of puerarin in each sample. The drug loading capacity was then calculated by the mass ratio of puerarin and SLNs. Flow rate of 1 mL/min, injection volume of 20 μL, and column temperature of 35 °C were used in the HPLC analysis, with detection at UV 249 nm.

In the present work, we performed the fabrication of the rectangular microchannel with cross and T-shaped junctions on an ordinary high-speed computerized numerical control engraving and milling machine, widely used in many manufacturing factories. The cutting speed was kept at 15 000 rpm, the feed rate was maintained at 600 mm/min, and the cutting depth in each step was 0.005 mm during machining. We also fabricated rectangular microchannels at a cutting speed of 20 000 rpm, feed rate of 300 mm/min, and cutting depth of 0.04 mm. We observed that the tool life and surface quality could be improved by increasing the spindle speed. However, the surface quality is not very excellent for the microfluid flowing requirements. Then, the polishing process was considered after fabrication, and a fiber oil-stone was employed to polish the microchannel surfaces (including the channel bottom and both side faces). We found that the final channel surface roughness was improved and satisfactory to be used in the preparation of SLNs. The present fabrication method is low-cost and the obtained microchannel is easy to scale up. Figure 2 shows the microchannels fabricated by the mechanical

Figure 2. (a) Stainless steel slab with rectangle microchannels and (b) assembled microchannel unit.

3. RESULTS AND DISCUSSION 3.1. Fabrication of Microchannels by the Mechanical Microcutting Method. Mechanical microcutting is different from the usual milling machining because the cutting tools used in the microcutting process are very much smaller than those used in the usual milling procedures. Those micromilling cutters have small diameters of about several hundreds of micrometers and break down very easily. Therefore, they could have a bad influence on the tool life and the machined surface quality. In general, austenitic stainless steel is a good candidate material for the basic structure for microchannels, owing to its inert and noncorrosive properties for those fluids used for the preparation of SLNs. The microchannel system fabricated on austenitic stainless steel slab is convenient to assemble and easy to scale up, which is obviously attractive to produce puerarinloaded SLNs continuously from the industrial view. However, austenitic stainless steel is considered as a typical difficult-to-cut material due to its high tendency to strain hardening, high ductility, and adhesiveness as well as low conductivity, which could make the cutting edge often exposed to severe conditions during metal cutting. Therefore, in order to achieve the precise fabrication of microchannels with regular shapes on the austenitic stainless steel slab, it is of significant importance to choose the milling machine and suitable machining parameters like cutting speed, feed rate, and cutting depth. Moreover, careful experience is also necessary for the operators.

microcutting method and the assembled microchannel unit, which was then applied in the preparation of puerarin-loaded SLNs in this work. 3.2. Morphology of Puerarin-Loaded SLNs. In this work, the microchannel system with two junctions was employed in order to achieve both liquid flow-focusing and gas-slug displacing processes in the main channel simultaneously, which has been demonstrated to be effective for the continuous production of SLNs, as reported in our previous work.28 The liquid flow-focusing process occurs at the cross junction, where the flow stream of lipid solution is focused to occupy the channel center region and surrounded by the aqueous phase streams and flows stably along the main microchannel. The diffusive and convective mass transfer of water in the aqueous stream and water-miscible organic solvent in the lipid solution can induce local lipid−puerarin supersaturation and thus the formation of puerarin-loaded SLNs. The complex suspension of fluids containing SLNs passes freely through the microchannel without deposition or blockage by the gas-displacing process, which is achieved at the downstream T-shaped junction by introducing inert gas slugs through the branch channel. The transfers of both organic solvent and water are enhanced by the complex mixing induced by gas−liquid slug flow, resulting in a more homogeneous solute concentration field and SLNs with narrower size distribution.28 11375



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Preparation of puerarin-loaded SLNs was achieved at various velocities of the aqueous phase and the drug−lipid solution phase and different lipid and puerarin concentrations, as summarized in Table 1. Values of the Reynolds numbers of the Table 1. Summary of Experimental Parametersa lipid concn (mg/mL)

puerarin concn (mg/mL)

UA (m/s)

UDL (m/s)

UG (m/s)

ReAb

ReDLc

ReG

5 5 5 5 5 5 5 5 5 5 5 5 15d 15d 15d 15d

0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 2.0 4.0 6.0 8.0 0.5 0.5 0.5 0.5

0.05 0.10 0.15 0.20 0.25 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.10 0.15 0.20

0.010 0.010 0.010 0.010 0.010 0.015 0.020 0.025 0.010 0.010 0.010 0.010 0.010 0.010 0.010 0.010

0.0117 0.0117 0.0117 0.0117 0.0117 0.0117 0.0117 0.0117 0.0117 0.0117 0.0117 0.0117 0.0117 0.0117 0.0117 0.0117

19.6 39.2 58.8 78.4 98.0 19.6 19.6 19.6 19.6 19.6 19.6 19.6 19.6 39.2 58.8 78.4

2.6 2.6 2.6 2.6 2.6 4.0 5.3 6.6 2.6 2.6 2.6 2.5 2.7 2.7 2.7 2.7

0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3

a

A, aqueous phase (0.5% poloxamer 188); DL, drug−lipid solution; G, gas (N2). Operation temperature was 27 °C. bDensities and viscosities of the aqueous solution were calculated from literature.52 cDensities and viscosities of the drug−lipid solutions were measured experimentally. dTemperature was about 31 °C.

aqueous phase flow (ReA = ρAUADb/μA, where ρA and μA are the density and viscosity and UA is the superficial velocity of the aqueous solution), the drug−lipid solution phase (ReDL = ρDLUDLDb/μDL, where ρDL and μDL are the density and viscosity and UDL is the superficial velocity of the drug−lipid solution phase), and gas (ReG = ρGUGDb/μG, where ρG and μG are the gas density and viscosity, and UG is the gas superficial velocity) were also calculated and are listed in Table 1. For the drug− lipid solution with lipid concentration of 15 mg/mL, the operation temperature was maintained at about 31 °C to ensure the complete dissolution of lipid in the drug−lipid solution since the solubility of Softisan 100 in ethanol is low,51 which was then employed to produce the nanoparticles. For the drug−lipid solution with lipid concentration of 5 mg/mL, the temperature was about 27 °C. The liquid flow-focusing and gas displacing performance during the formation of puerarin-loaded SLNs in the present microchannel system were similar to those observed in the microchannel system fabricated in stainless steel plates by wire electrical discharge machining in previous work.28,29 Figure 3 shows examples of the images of flow focusing of puerarin− lipid solution and aqueous phase at the cross junction, liquid streams between cross and T-shaped junctions, formation of Taylor bubbles at the T-shaped junction, and gas−liquid slug flow downstream of the T-shaped junction. As can be seen, at the cross junction the liquid flow-focusing occurred as the puerarin−lipid solution met the flow streams of the aqueous solution phase from the branch channels. The flow stream of the puerarin−lipid solution was suppressed and occupied the center area of the main channel, while the aqueous solution took the region outside the center of the channel. Transfer of miscible solvents (water and ethanol) between the flow streams

Figure 3. Images of (a) flow focusing at the cross junction, (b) liquid streams between cross and T-shaped junctions, (c−e) formation of Taylor bubbles at the T-shaped junction, and (f) gas−liquid slug flow downstream of the T-shaped junction. ReA = 19.6, ReG = 2.6, and ReDL = 0.5. The aqueous phase was 0.5% poloxamer in water, the drug−lipid solution phase was 0.5 mg/mL puerarin and 5 mg/mL Softan 100, and the gas phase was N2.

induced the increase of local puerarin and lipid concentrations and thus resulted in the rapid formation of puerarin-loaded SLNs. The introduction of Taylor bubbles could not only enhance the mass transfer of this process but also prevent the possible blockage of particles within the microchannel. The generation of Taylor bubbles and the flow performance of gas− liquid slug flow were similar as those observed in the case without loading drug.28,29 The morphology of the puerarin-loaded SLNs was investigated by TEM. Actually, the morphology of one solid lipid nanoparticle could be different from another even under the same conditions, due to the incorporation of drug particulates. Here, as typical examples, Figure 4 shows TEM images of puerarin-loaded SLNs obtained at UA = 0.05 m/s, UDL = 0.01 m/s, and UG = 0.012 m/s. The lipid concentration was 5 mg/mL and the puerarin concentration was 8 mg/mL. It is seen that these SLNs are slightly more nonspherical in shape than those without puerarin prepared under similar conditions as reported previously.26,28 In fact, the present lipid Softisan 100 is a triglyceride mixture of fatty acids with chain lengths of C10−C18. The molecular structures of these contents are much different from that of puerarin. Therefore, in the particle 11376



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Figure 5. Particle size distributions of puerarin-loaded SLNs prepared at various Reynolds numbers of aqueous phase flow (ReG = 2.7, ReDL = 0.3). (■) ReA=19.6, (●)ReA=39.2, (□) ReA=58.8, and (○) ReA=78.4. The aqueous phase was 0.5% poloxamer in water, the drug−lipid solution phase was 0.5 mg/mL puerarin and 15 mg/mL Softan 100 in ethanol, and the gas phase was N2.

solution was maintained at 0.085 m/s and the gas velocity was kept at 0.012 m/s in these preparations. It is seen that the sizes of these puerarin-loaded SLNs are in nanosize range from about 50 to 600 nm depending on the preparation conditions. The size distribution of the SLNs at a given condition is relatively narrow. Figure 6 shows the changes of mean size and

Figure 4. TEM photographs of puerarin-loaded SLNs prepared by the microchannel system (ReA = 19.6, ReG = 2.7, ReDL = 0.3). The aqueous phase was 0.5% poloxamer in water, the drug−lipid solution phase was 8 mg/mL puerarin and 5 mg/mL Softan 100, and the gas phase was N2.

formation process the incorporation of puerarin could cause a more amorphous structure in the formation of SLNs, which consequently induce the variations of particle morphology compared with those without puerarin loaded. 3.3. Size Distributions of Puerarin-Loaded SLNs. In general, the sizes of SLNs prepared by the liquid flow-focusing approach in microchannels were strongly influenced by the velocities of the aqueous phase and the lipid phase.26,28 In this work, we also observed that the mean size and the size distributions of puerarin-loaded SLNs were affected by the characteristics of flow streams of the aqueous phase and the drug−lipid solution phase. As an example, Figure 5 shows the variation of particle size distribution of puerarin-loaded SLNs with ReA at a given ReDL of 2.7 and a certain gas ReG of 0.3. The velocity of the drug

Figure 6. Variation of (a) mean diameter and (b) polydispersity index of puerarin-loaded SLNs with Reynolds number of the aqueous phase. The drug−lipid solution phase was (●) 0.5 mg/mL puerarin and 15 mg/mL Softan 100 in ethanol (ReDL = 2.7, ReG = 0.3) or (○) 0.5 mg/ mL puerarin and 5 mg/mL Softan 100 in ethanol (ReDL = 2.6, ReG = 0.3). The aqueous phase was 0.5% poloxamer in water, and the gas phase was N2.

polydispersity index with ReA at fixed ReDL of 2.6 and ReG of 2.7. As can be seen, the polydispersity index values are below about 0.2 and close to those of SLNs without puerarin content.26,28 The mean particle sizes decreased with increasimg ReA or velocity of the aqueous phase for drug−lipid solutions with different concentrations of lipid. This trend is also similar to those observed for SLNs without puerarin.26,28 The reason is 11377



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that the increase of ReA could enhance solvent mass transfer between the aqueous phase and the drug−lipid solution streams, which accelerate the formation of numerous small local-concentrated zones for particle precipitation. However, the size distributions of the present puerarin-loaded SLNs are slightly broader and the mean sizes are larger than those SLNs without puerarin, prepared by the same method under similar conditions.26,28 It is also seen that the distribution of particle size tends to be slightly broader with decreasing ReA. The reason is that tiny puerarin particles occurred concurrently during the formation of puerarin-loaded SLNs. These concomitant puerarin particles always have nonspherical shape and are nanosized, much smaller than puerarin-loaded SLNs, which consequently contributes to the broader changes of the particle size distributions. Figure 7 shows the variation of mean particle size distributions and polydispersity index of puerarin-loaded

Figure 8. DSC curves of (A) drug-free lipid and (B, C) puerarinloaded SLNs prepared from (B) 8 mg/mL puerarin and 5 mg/mL Softan 100 in ethanol (ReA = 19.6, ReG = 2.5, ReDL = 0.3) or (C) 2 mg/ mL puerarin and 5 mg/mL Softan 100 in ethanol (ReA = 19.6, ReG = 2.6, ReDL = 0.3) respectively. The aqueous phase was 0.5% poloxamer in water and the gas phase was N2.

lipid was observed, indicating the decreased melting point of the lipid particles. This was caused by the formation of heterogeneous defects within the matrix of SLNs. Figure 9 displays the drug loading capacities within the SLNs prepared from drug−lipid solutions containing different

Figure 7. Variations of (○) mean diameter and (■) polydispersity index of puerarin-loaded SLNs with Reynolds number of the drug− lipid solution phase (ReA = 19.6, ReG = 2.6). The drug−lipid solution phase was 0.5 mg/mL puerarin and 5 mg/mL Softan 100 in ethanol, the aqueous phase was 0.5% poloxamer in water, and the gas phase was N2. Figure 9. Effects of puerarin concentration in the drug−lipid solution phase on the drug loading capacity (ReA = 19.6, ReDL = 2.5−2.6, ReG = 0.3). The aqueous phase was 0.5% poloxamer in water and the gas phase was N2.

SLNs with ReDL at fixed ReA of 19.6 and ReG of 0.3. Velocities of the aqueous phase and the gas were maintained at 0.05 and 0.012 m/s, respectively. As can be seen, the polydispersity index values are about from 0.005 to 0.161, indicating the size distributions are narrow. The mean particle sizes increased with increasing ReDL. This was caused by the increase of the total amounts of solvents needing to be transferred from the drug− lipid solution into the aqueous phase with increasing ReDL at a given time within the main microchannel. From these results, we can also see that it is possible to produce puerarin-loaded SLNs with expected different sizes by changing only the flow conditions. 3.4. DSC Thermograms and Drug Loading Capacity of SLNs. Figure 8 shows DSC thermograms of the drug-free lipid and the puerarin-loaded SLN prepared from drug−lipid solutions containing different amounts of puerarin. Preparation velocities of the puerarin-loaded SLN were UA = 0.05 m/s, UDL = 0.01 m/s, and UG = 0.012 m/s. There are no peaks for puerarin from room temperature to 100 °C as demonstrated by Li et al.,21 and therefore we did not measure the DSC thermograms of puerarin. As can be seen from Figure 8, there is a single peak near the melting point of the lipid (33.5−35.5 °C for Softan 100) for the drug-free lipid sample, while another new peak was observed at about 51 °C, indicating the incorporation of puerarin into the matrix of the SLNs. The early occurrence of the first peak near the melting point of the

amounts of puerarin. As can be seen, the drug loading capacity increased with increasing puerarin concentration of the drug− lipid solutions. In this work, the maximum capacity of about 7.4% (w/w) was achieved for the drug−lipid solution phase containing 5 mg/mL Softan 100 and 8 mg/mL puerarin at ReDL of 2.5, ReA of 19.6, and ReG of 0.3. In the HPLC measurements, the sample was first dissolved in methanol and then diluted with acetonitrile, and the final content of methanol was about 13% (w/w). The effects of methanol peaks on the final measurements of the drug loading capacities were calibrated, and the averaged deviation was about 20.9%. It should be noted that accurate determination of the puerarin loading capacities was difficult because both particles of pure puerarin and lipid particles containing puerarin existed in the suspension of SLNs, and these particles were difficult to separate. The samples used in the present measurements were separated by centrifugation, and only partial puerarin-loaded SLNs were recovered successfully. Therefore, the drug encapsulation efficiency of puerarin was not explored, and further investigation is needed in future work. Moreover, in actual cases the puerarin loading capacity of each nanoparticle could be different, which could 11378



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also influence the overall release performance of drug at the prospective location with desired amount. Therefore, the mutual effects of particle size distribution and drug loading capacity in clinical administration need to be explored in the future too.

4. CONCLUSIONS By using the mechanical microcutting method with the micromill tool for milling, the ordinary machining center for manufacturing, and the fiber oil-stone for polishing, it is easy to fabricate microchannels with precise sizes and regular shapes on the stainless steel slab. The present rectangular microchannel with cross and T-shaped junctions was suitable and effective to be employed in the preparation of SLNs loaded with the poorly water-soluble drug puerarin under various conditions by using the liquid flow-focusing and gas displacing technique. The prepared puerarin SLNs have amorphous morphology and are slightly more nonspherical in shape than those SLNs without puerarin. The size distributions of these SLNs are narrow and the mean particle sizes decreased with increasing ReA but increased with increasing ReDL. Therefore, the particle sizes of puerarin-loaded SLNs can be controlled by changing the flow conditions in the microchannels. The drug loading capacity of puerarin increased with increasing the puerarin concentration of the drug−lipid solutions. A maximum capacity of 7.4% was observed in the present work, and further improvement of the capacity is needed in the future.

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AUTHOR INFORMATION

Corresponding Author

*Telephone: +86-571-88320951. Fax: +86-571-460 88033331. E-mail: [email protected] (J.X. Yun). Telephone: +86-461 571-86971215. Fax: +86-571-86971703. E-mail: tianbing@zju. 462 edu.cn (B.Tian). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge partial financial support by the National Natural Science Foundation of China (20606031, 21036005), the International Science & Technology Cooperation Program of China (1017), the Visiting Scholar Foundation of the State Key Laboratory of Fluid Power Transmission and Control in Zhejiang University (GZKF-201010), the Hubei Provincial Natural Science Foundation of China (2010CDB04101) and the Zhejiang Provincial Natural Science Foundation of China (Y4080326).

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REFERENCES

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