Abstract:
A microfluidic device to produce polymersomes having three coaxial passageways of increasing size with fluid flowing in one direction. The first and smallest passageway contains the content of the polymersome, the middle passageway contains a block copolymer, and the largest and outer passageway contains an aqueous medium or water. The device can produce polymersomes with control of size and membrane thickness. The device will allow quantitative loading of the polymersomes in high quantities. The device is robust and easily assembled and has the ability to independently control the three streams involved in making the polymersomes.

Description:
CROSS REFERENCE TO RELATED APPLICATIONS 
       [0001]    This application claims the benefit of U.S. Provisional Application No. 61/373,341, filed Aug. 13, 2010, the entire contents of which is herein incorporated by reference in its entirety. 
     
    
     BACKGROUND 
       [0002]    1. Technical Field 
         [0003]    The present disclosure generally relates to a microfluidic device, and more particularly to a microfluidic device suitable for encapsulation of pharmaceutical, flavors, fragrances and the like by forming polymersomes and liposomes as encapsulation and delivery agents, viscosity modifiers and/or thickening agents. 
         [0004]    2. Background Art 
         [0005]    In general, polymersomes represent a class of vesicles, tiny hollow spheres that enclose a solution. Polymersomes are typically made using amphiphilic synthetic block copolymers to form the vesicle membrane, and generally range from about 50 nm to about 5 um in radius or more. Polymersomes generally contain an aqueous medium in their core and are useful for encapsulating and protecting sensitive molecules, such as, for example, drugs, enzymes, other proteins and peptides, and DNA and RNA fragments. In general, the polymersome membrane provides a physical barrier that isolates the encapsulated material from external materials, such as those found in biological systems. 
         [0006]    Polymersomes are similar to liposomes, which are formed from naturally occurring lipids. While having many of the properties of natural liposomes, polymersomes typically exhibit increased stability and reduced permeability. Furthermore, the use of synthetic polymers allows manipulation of the characteristics of the membrane and thus control permeability, release rates, stability and other properties of the polymersome. 
         [0007]    Encapsulated actives such as fragrances, flavors, pharmaceutical materials, etc., may be used in a variety of cosmetic, pharmaceutical and food related areas. Such applications include, but are not limited to, fragrance, drug and flavor encapsulation using well-defined particle size encapsulation agents. Polymersomes containing active enzymes that provide a way to selectively transport substrates for conversion have been described as nanoreactors and have been used to create controlled release drug delivery systems while being substantially invisible to the immune system. 
         [0008]    There are various methods to protect active compounds from environmental and processing conditions, avoid loss of volatiles and release actives at desired times. Active encapsulation generally requires using a carrier material for protection, delivery and controlled release. Liposomes and polymersomes can be used as active delivery agents. Successful encapsulation processes require high encapsulation efficiency, protection of actives from unfavorable process and storage conditions and favorable release mechanisms. 
         [0009]    Liposomes are used in various cosmetics applications such as cosmetic stick formulations and anhydrous spray formulations. Liposome encapsulated actives are generally spray dried with other hydrocolloids and dispersed in various formulations. Successful encapsulation in the fragrance industry, for example, for hair care products, depends on factors such as availability, cost and compatibility of ingredients and deposition of the actives onto hair. The active should survive washing, rinsing and even drying of hair. Even though liposomes have been used extensively for delivery and deposition of actives onto the hair, they may have limitations in terms of shelf life and after administration. However, polymersomes or nanoparticles of lipids/polymers may overcome some limitations experienced with liposomes. In general, polymersomes are tougher and stronger than liposomes. The ability to modify the surface of liposomes and polymersomes with anchoring molecules may enhance the survival of actives during various stages of use. 
         [0010]    Encapsulation of flavors has enjoyed numerous available techniques. Spray drying and extrusion are some common encapsulation techniques. These processes generally require high temperature exposure, and heat and/or oxygen sensitive flavors are adversely affected from these methods. Other techniques are freeze drying and hot melting. Liposomes are also used as flavor encapsulation agents. 
         [0011]    Encapsulation of physiologically active compounds can enhance bioavailability and therapeutic index over extended time scales. Lipid and polymer based drug delivery systems utilize the ability to form micro-spheres. These micron size particles can be used for a variety of purposes ranging from direct injection to aerosols for inhalation. Unilamellar and multilamellar liposomes have also been used as lipid-based drug delivery systems. 
         [0012]    Currently, polymersomes are made nearly exclusively by a process of film rehydration. The method involves simply the spontaneous budding off of polymersomes from a polymer surface. It is a slow process and with very low levels of material encapsulation. This process provides little control of polymersome size or membrane thickness and loading is inefficient. It is believed that polymersomes are currently not used commercially due to these problems. 
         [0013]    One known device is used to make polymersomes by microfluidics using opposing fluid flows. The device is tedious to produce with a high failure rate. It is fragile, and has not been shown to allow control of size or membrane thickness of the polymersomes produced. 
         [0014]    In view of the various known beneficial uses of polymersomes as useful carriers for targeted medication and the lack of suitable ways to prepare them, it is desirable to provide an improved apparatus to prepare polymersomes and its method of its use. These and other inefficiencies and opportunities for improvement are addressed and/or overcome by the systems and methods of the present disclosure. 
       SUMMARY 
       [0015]    Generally speaking, in accordance with exemplary embodiments of the present disclosure, a substantially rigid block with three concentric holes of increasing size into which are placed three tubes or capillaries is provided. The innermost capillary has an opening of from between about 100 to about 500 nanometers to about 10 to 100&#39;s of microns produced by a capillary puller and includes the active ingredient in an aqueous medium. The second, or middle, capillary has a somewhat larger opening and surrounds a portion of the inner capillary and provides a polymer for forming the polymersome. A third larger capillary surrounds the other two providing an aqueous medium. The scaffold of the device is a small polymer block chosen for its chemical compatibility and machinability. In exemplary embodiments of the present disclosure, the capillaries are all held in position by insertion into the block. 
         [0016]    Fluid is introduced to each tube in the same direction. A Teflon tube is directly connected to the inner capillary that introduces the active ingredient in an aqueous medium and is directly connected to the glass outlet tube of the device. Fluid including the polymer is introduced in the middle tube, and water or an aqueous medium is introduced into the outer capillary by way of channels cut into the device block perpendicular to the capillaries and at the end of each drill hole. Fluid flow rates are controlled to provide laminar flow. 
         [0017]    The present disclosure provides for a device for the preparation of polymersomes. 
         [0018]    The present disclosure also provides for a device for preparation of polymersomes and for controlling the size of the polymersomes prepared. 
         [0019]    The present disclosure also provides for a device to prepare polymersomes that allows loading of material inside the polymersomes with near 100% efficiency. 
         [0020]    The present disclosure also provides for a device to prepare polymersomes that allows control of the composition of the bilayer that defines the polymersome. 
         [0021]    The present disclosure also provides for a device to prepare polymersomes having multiple bilayers resulting in a thicker “skin” and a more robust polymersome. 
         [0022]    The present disclosure also provides for a device that allows for much faster production of polymersomes. 
         [0023]    The present disclosure also provides for a method for producing polymersomes. 
         [0024]    Exemplary embodiments of the present disclosure accordingly includes a product possessing the features, properties, and the relation of components and the several steps and the relation of one or more of each steps with re-respect to each of the others which will be exemplified in the product hereinafter described, and the scope of the present disclosure will be indicated in the claims. 
         [0025]    Additional advantageous features, functions and applications of the disclosed systems and methods of the present disclosure will be apparent from the description which follows, particularly when read in conjunction with the appended figures. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0026]    To assist those of ordinary skill in the art in making and using the disclosed systems and methods, reference is made to the appended figures, wherein: 
           [0027]      FIG. 1  is a schematic view in section of a unitary flow direction concentric capillary microfluidic device constructed and arranged in accordance with an exemplary embodiment of the present disclosure; 
           [0028]      FIG. 2  is a schematic view in section of the concentric capillary microfluidic device of  FIG. 1  showing the fluid inlets; 
           [0029]      FIG. 3  is a schematic view in section of the concentric capillary microfluidic device of  FIG. 2  taken along line  3 - 3 ; 
           [0030]      FIG. 4  is a light microscopy image of polymer stabilized vesicular structures filled with fluorescein added to internal deionized water flow made in the microfluidic device of  FIGS. 1-3 ; 
           [0031]      FIG. 5  is a fluorescent microscopy image of the polymer stabilized vesicular structures of  FIG. 4 ; 
           [0032]      FIG. 6  is an electron microscopy image of a polymersome prepared in a device of  FIGS. 1-3 ; 
           [0033]      FIG. 7  is a transmission electron microscopy image of a OsO 4  stained/fixed polymersome captured on a copper TEM grid prepared in a device of  FIGS. 1-3 ; 
           [0034]      FIG. 8  is a side elevational view of a concentric capillary microfluidic device constructed and arranged in accordance with an embodiment of the present disclosure; 
           [0035]      FIG. 9  is a schematic view showing the concentric holes and channels drilled into a rigid block to prepare the microfluidic device in accordance with an exemplary embodiment of the present disclosure; 
           [0036]      FIG. 10  is a schematic view showing the concentric capillaries placed in the holes drilled into the block of  FIG. 9  to prepare the microfluidic device in accordance with the present disclosure; and 
           [0037]      FIG. 11  is a light microscopy image showing the formation of polymersomes according to an exemplary embodiment of the present disclosure. 
       
    
    
     DETAILED DESCRIPTION 
       [0038]    In the description which follows, like parts are marked throughout the specification and drawings with the same reference numerals, respectively. Drawing figures are not necessarily to scale and in certain views, parts may have been exaggerated for purposes of clarity. 
         [0039]    A unitary flow direction microfluidic device  11  constructed and arranged in accordance with an exemplary embodiment of the present disclosure is shown in elevation in  FIG. 8 . In general, device  11  is formed of a substantially rigid block or housing  12  having an input end  13  and an output end  14  that has been machined to include three concentric tubes. These tubes are shown in the schematic views of  FIGS. 1-3 . Here, an innermost tube  16  is referred to as tube # 1 , a middle tube  17  is referred to as tube # 2 , and an outer tube  18  is referred to as tube # 3 . In exemplary embodiments, each tube  16 ,  17  and  18  is separately fed by a syringe pump or the like. 
         [0040]    In the construction illustrated in  FIGS. 1-3 , smallest tube  16  (tube # 1 ) contains an aqueous medium containing any material that will be loaded into a polymersome. Middle tube  17  (tube # 2 ) contains a block copolymer dissolved in a non-selective solvent. Largest tube  18  (tube # 3 ) contains an aqueous solution which may be pure water or may be a solution formulated to control the osmotic pressure mismatch between the inside and the outside of the polymersome. Fluid is introduced into the inlet end of each tube by a fitting  16   a ,  17   a  and  18   a  as shown in  FIG. 2 . Fittings  16   a ,  17   a  and  18   a  may be luer locks for connecting Teflon tubes to tube  16 ,  17  and  18 . 
         [0041]    In exemplary embodiments, tubes  16 ,  17  and  18  are kept concentric and kept from touching each other by block  12  in which they are held. Device  11  is generally formed by drilling three holes. First, smallest hole  21  is drilled through the entire length of block  12 . A middle size hole  22  is drilled roughly two thirds of the way through block  12 . Finally, a hole  23 , the largest hole, is drilled to roughly one third the length of block  12 . Holes  21 ,  22  and  23  are concentric and their size closely matches the outside diameters of tubes  16 ,  17  and  18 , respectively and mentioned previously. 
         [0042]    In general, block  12  is ultra-high molecular weight polyethylene, chosen due to its high degree of chemical compatibility and machinability, although the present disclosure is not limited thereto. Other types of blocks (e.g., polymer blocks) are suitable, for example, the materials of construction of which may include a wide variety of materials. For example, these include non-ferrous metals, silica-based materials, carbonaceous materials, polymeric materials, such as nylon, polyacetals, polyvinylchloride, polyethylene, polypropylene and fluorine containing polymers, such as polytetrafluoroethylene (PTFE), perfluoroalkoxy (PFA), or other suitable plastic material. The polymeric materials may be filled or unfilled, so long as it is chemically compatible. 
         [0043]    In order to construct device  11 , smallest tube  16  (tube # 1 ) is typically inserted substantially all the way through block  12  by way of first hole  21 . Middle tube  17  (tube # 2 ) is inserted over smallest tube  16  and is pushed into block  12  just short of the end of second drilled hole  22 . Largest tube  18  (tube # 3 ) is then inserted to just short of the end of the third and largest diameter drilled hole  23 .  FIG. 3  shows this schematically as, does  FIG. 9 . 
         [0044]    In exemplary embodiments of the present disclosure, the dimension of smallest hole  21  is typically about 1 mm in diameter but can be smaller or larger and range from about 0.1 mm to about 2 mm, but preferable is between about 0.5 and about 1.5 mm. A size is selected to facilitate insertion of tube  16  that may have an internal diameter of about 10 to about 500 μm that is tapered to about 5 to about 50 μm at the outlet. Middle hole  22  is larger than smallest hole  21  and drilled to about 2 mm in diameter, but may range from about 1.5 to about 3.0 mm to accommodate middle tube  17  having an inner diameter from about 40 to about 1000 μm. Outer hole  23  is about 5 mm in diameter, but may range from about 3 to about 6 mm for receiving outer tube  18  having an inner diameter of from about 1000 to about 4000 μm for transporting the outer water or fluid phase and receiving the polymersomes at outlet side  14  of block  12 . 
         [0045]    With reference now to  FIG. 8 , fluid is introduced directly into each tube  16 ,  17  and  18 . For smallest tube  16 , a Teflon tube or the like from a pump, preferably a small pump, such as a syringe pump, an HPLC pump, or a small infusion pump is connected (e.g., directly connected) by a leur lock  16   a  to tube  16  that is generally glass at input side  13  of block  12 . For middle tube  17  and largest tube  18 , fluid is introduced by way of a pair of channels  27  and  28 , respectively, cut into block  12  substantially perpendicular to tubes  16 ,  17  and  18  at the end of each drill hole  22  and  23 . Block  12  is tapped and leur locks  17   a  and  18   a  are screwed into block  12  to attach Teflon tubes. Fluid then enters tubes  16 ,  17  and  18  through the ends of each tube inserted into block  12 . Flow rates are controlled to insure laminar flow of all fluids. 
         [0046]    The formation of polymersomes occurs by surrounding drops formed from smallest tube  16  with the polymer solution of middle tube  17 . The resulting droplets surrounded by polymer solution is carried along by the flow from largest tube  18 . During this time, the solvent surrounding the droplet from smallest tube  16  diffuses into the bulk aqueous solution, leaving the block copolymers behind. As these copolymers consist of a hydrophobic and a hydrophilic block or layer, the polymer self-assembles into a bilayer. That is, the hydrophilic block or layer faces the aqueous phase both inside and outside of the polymersome, while the hydrophobic blocks are buried in the core of the polymer layer. The result is a thin skin of outer polymer defining a sphere or sphere-like shape or layer. Conceptually this is much like a balloon. The inner solution is separated from the outer solution by the block copolymer bilayer. 
         [0047]    The following examples are set forth for purposes of illustration only, and not intended to be presented in a limiting sense. In each case a block  12  including three concentric tubes as described above in connection with  FIG. 8  is used. Inner tube or capillary  16  is designated Q 1 , middle tube  17  is designated Q 2  and outer tube  18  is designated Q 3 . The diameters of the tubes or capillaries were Q 1 =1 mm with the tip being drawn down to approximately 50 microns, Q 2 =2 mm, and Q 3 =5 mm. 
       Example 1 
       [0048]    The flow rates to the capillaries are Q 1 =0.2 ml/min, Q 2 =0.4 ml/min, and Q 3 =3.3 ml/min. The inner fluid (Q 1 ) was water, the middle fluid (Q 2 ) was a solution of chloroform and dioxane in a 17:3 v/v ratio containing 3 mg/ml of PA60-30 (a poly(butadiene-b-acrylic acid) block copolymer of 30 K molecular weight containing 30 wt % of the hydrophilic poly(acrylic acid) block). The outer flow Q 3  contained water. 
         [0049]    These parameters resulted in drop sizes of approximately 100 microns. 
       Example 2 
       [0050]    The fluids are the same as in Example 1, with Q 1 =water, Q 2 =block copolymer solution of chloroform and dioxane, and Q 3 =water. Tubes or capillaries are the same size as in Example 1 but the flow rate for Q 2  is increased. The flow rates were Q 1 =0.2 ml/min, Q 2 =0.5 ml/min and Q 3 =3.3 ml/min. 
         [0051]    The resulting drops produced are approximately 80 microns in size. 
         [0052]    Examples of polymersomes made utilizing exemplary device  11  are shown in  FIGS. 4-7  and  11 .  FIGS. 4 and 5 , respectively are light and fluorescent microscopy images of polymer stabilized vesicular structures filled with fluorescein added to internal deionized water flow made by microfluidic device  11 .  FIG. 6  is a microscopy image of a solution that includes polymersomes in accordance with an embodiment of the present disclosure. The scale bars in  FIGS. 4-6  are 10 μm.  FIG. 7  is a transmission electron microscopy image of a OsO 4  stained/fixed polymersome captured on a copper TEM grid. 
         [0053]      FIG. 11  is a light microscopy image showing the formation of polymersomes according to another exemplary embodiment of the present disclosure. The polymer used was Pluronics 127, a commercially available amphiphilic block copolymer. The organic solvent used to dissolve the polymer was a mixture of toluene and chloroform, which is removed by dialysis. The relative flow rates of the tubes/capillaries were 0.35 ml/minute for the inner flow (Q 1 ), 1.00 ml/minute for the middle flow (Q 2 ), and 5.00 ml/minute for the outer flow (Q 3 ). The initial droplet formation was in the jetting regime. As shown in  FIG. 11 , the polymersomes are about a micron in diameter. It is noted that the size distribution largely comes from the issue of focusing (i.e., the polymersomes shown are not all in the same plane, so some polymersomes are focused on the center while others are focused near the top or bottom, so they appear smaller). 
         [0054]    A wide variety of active ingredients can be included in the inner phases as set forth in WO/2009/148598, the entire contents of which is incorporated herein by reference in its entirety. Examples of block copolymers including a hydrophobic block and a hydrophilic block suitable for use in preparing polymersomes in accordance with the present disclosure are described in WO/2009/148598 and U.S. Pat. No. 7,151,077, the entire contents of which U.S. patent is also incorporated herein by reference in its entirety. 
         [0055]    A microfluidic device prepared in accordance with the present disclosure is capable of producing monodisperse polymersomes in a highly controlled manner. The device is highly robust, portable, and contains very few components that are all very easy and convenient to change. The scaffold of the device is a small block chosen due to its high degree of chemical compatibility and machinability. The polymer block is designed in such a way that three capillaries are held in place while being individually supplied with fluid from a pump (e.g., syringe pump). This allows for independent control of each fluid stream and control of polymersome size, membrane thickness, and quantitative encapsulation. Each of the channels has a luer lock or the like attached to it, to which a tube is attached that connects each to the syringe and syringe pump that supplies the fluid. The use of this system greatly increases versatility, where parts can be quickly and easily interchanged, and fluid flow rates can be easily tailored independent of each other. 
         [0056]    The advantages of preparing polymersomes by this technique over traditional film rehydration techniques include:
       1. The ability to control the size of the polymersomes.   2. Loading of material inside the polymersomes with near 100% efficiency.   3. Ability to control the composition of the bilayer that defines the polymersome.   4. Allows for much faster production of polymersomes.   5. Allows for increasing the number of bilayers defining each polymersome, thus enabling the tailoring of the robustness of the polymersomes.       
 
         [0062]    While the present disclosure has been described with reference to certain preferred embodiments, numerous changes, alterations and modifications to the described embodiments are possible without departing from the spirit and scope of the present disclosure as defined in the appended claims, and equivalents thereof. 
         [0063]    It will thus be seen that the objects set forth above, among those made apparent from the preceding description, are efficiently attained and, since certain changes may be made in the above method product without departing from the spirit and scope of the present disclosure, it is intended that all matter contained in the above description and shown in the accompanying drawings shall be interpreted as illustrative and not in a limiting sense. 
         [0064]    Although the systems and methods of the present disclosure have been described with reference to exemplary embodiments thereof, the present disclosure is not limited to such exemplary embodiments and/or implementations. Rather, the systems and methods of the present disclosure are susceptible to many implementations and applications, as will be readily apparent to persons skilled in the art from the disclosure hereof. The present disclosure expressly encompasses such modifications, enhancements and/or variations of the disclosed embodiments. Since many changes could be made in the above construction and many widely different embodiments of this disclosure could be made without departing from the scope thereof, it is intended that all matter contained in the drawings and specification shall be interpreted as illustrative and not in a limiting sense. Additional modifications, changes, and substitutions are intended in the foregoing disclosure. Accordingly, it is appropriate that the appended claims be construed broadly and in a manner consistent with the scope of the disclosure.