Patent Application: US-87069310-A

Abstract:
disclosed is a device for preparation of liposomes , comprises a reaction tank and an infusion unit . the reaction tank comprises a collector mounted in a predetermined position of the reaction tank ; two inlet ports are included : the first inlet port for infusing an aqueous solution ; and the second inlet port for infusing an organic solution . the infusion unit can introduce a bioactive agent containing - aqueous solution into the reaction tank . the infusion unit comprises a filter connected to one end of the infusion unit and being adjacent to the collector . the method using the device comprises the steps of infusing an aqueous solution and an organic solution into the reaction tank of the device and thus forming an interface between the filter and the collector ; infusing a bioactive agent containing - aqueous solution and being filtered by the filter , the bioactive agent is encapsulated to form a water - in - oil emulsion ; the water - in - oil emulsion is passing through the aqueous solution and thus to form a water - in - oil - in - water double emulsion . finally the removal of the organic phase of water - in - oil - in - water double emulsion enables the harvest a plurality of liposomes . it has advantages such that simple - used and automation production . thus nano size or sub - micro size liposomes can be prepared with high encapsulation efficiency without sonicators or delicate microfluidic systems .

Description:
liposomes are holy spheres composed of lipid bilayers , which are structurally similar to cell membranes , may contain certain amounts of aqueous solution . liposomes can be used for gene delivery and therapy , targeted drug delivery and control release , and immunoassay . double emulsion method is a technique that tiny bubbles are dispersed from two or more immiscible liquids . firstly , high concentration lipids are dissolved in an organic solvent . a small amount of aqueous solution is added to the organic solvent and thus be stably dispersed therein to form an emulsion . then the emulsion is added into water to form a water - in - oil - in - water double emulsion ( w / o / w double emulsion ). finally , the organic solvent is evaporated by a rotary evaporator or by aging for a while to obtain bilayer liposemes ( fig1 e ). the w / o / w double emulsion is an emulsion , which can be considered as emulsions within emulsions . it composed of an internal aqueous part , and a hydrophobic component surrounded with an aqueous continuous phase . with reference to fig2 , a sectional view of the device according to the first embodiment of the present invention . as shown in fig2 , the device 100 comprises a reaction tank 1 and an infusion unit 2 . in this embodiment , reaction tank 1 is made of glass . however , reaction tank is , including but not limited to , glass device or any proper size container made of chemical / physical compatible material . the reaction tank 1 comprises a collector 11 , a first inlet port 12 and a second inlet port 13 . the collector 11 is mounted at the bottom of the reaction tank 1 to collect the water - in - oil - in - water double emulsion l ( w / o / w double emulsion ). the first inlet port 12 and second inlet port 13 are adapted to infuse an aqueous solution s 1 and an organic solution s 2 into the collector 11 to form an interface . so that the organic solution s 2 can be used to form liposomes , and the aqueous solution s 1 can be provided to be an external part surrounding thereby . after that , liposomes can be harvested after removing the organic phase of the w / o / w double emulsion . the infusion unit 2 comprises a filter 21 . in this embodiment , filter 21 is , but not limited to a glass sieve , or other filter devices made of chemical / physical compatible material . for example , it can be a syringe filters . nevertheless , the pore sizes of the filter devices relate to the formation of liposome size , so the ideal filter devices should be selected depend on actual requirements . the filter 21 is connected to one end of the infusion unit 2 , and is adjacent to the collector 11 . the other end of the infusion unit 2 is used to introduce a bioactive agent containing - aqueous solution s 3 into the reaction tank 1 . according to this embodiment , the bioactive agent is fluorescence dye . furthermore , the bioactive agent can be a drug , protein , aptamer or contrast agent . the manufacture process of liposomes using the device 100 of the present invention is described as follow . at first , the aqueous solution s 1 and the organic solution s 2 are infused into the reaction tank 1 . due to insolubility property between the organic and aqueous phase , two solutions contact each other and thus form an interface between the filter unit 21 and the collector 11 . once being infused into the reaction tank 1 , the bioactive agent containing - aqueous solution s 3 is filtered by the filter unit 21 firstly , and is then sequentially passed through the organic solution s 2 and the aqueous solution s 1 . finally , w / o / w double emulsion l is formed and enters into the collector 11 to be harvested . with reference to fig3 , this is a flow chart of the device according to the second embodiment of the present invention . this embodiment is based on double emulsion method and uses the device 100 mentioned above ( as shown in fig2 ). accordingly , the manufacture process of liposomes is simple and programmable , and particularly has high encapsulation efficiency . step 101 : providing an aqueous solution s 1 , an organic solution s 2 , and a bioactive agent containing - aqueous solution s 3 . the details of the process are described as follow : ( 1 ) the organic solution s 2 according to this embodiment is consisting of an organic solvent and at least one phospholipid . the organic solvent used here is chloroform , and the phospholipid includes dipalmitoylphosphatidylcholine ( dppc ), dipalmitoylphosphatidylglycerol ( dppg ) or other kinds of phospholipids are also workable . dppg can be used to lower the aggregation level and increase the stability of liposomes ), or other phospholipids . dppc and dppg ( w / w ration = 10 : 1 ) are added and dissolve in a proper volume of chloroform , so as to obtain the dppc / dppg solution . the solution is loaded in the syringe and to be the organic solution s 2 . on the other hand , the organic solution s 2 is added with 0 . 1 mm nile red ( a hydrophobic fluorescence dye , uv / vis absorbance maximum : 543 nm , emission wavelength maximum : 610 nm ). it is facilitated to observe the distribution of water and oil by fluorescence or laser scanning confocal microscope . the substance contained in the organic solution s 2 is , including but not limited to fluorescence , drug or contrast agent . ( 2 ) 5 ml of 5 ( 6 )- carboxyfluorescein ( a hydrophilic fluorescence dye , uv / vis absorbance maximum : 492 nm , emission wavelength maximum : 517 nm ) solution with a determined concentration , is prepared by ddh 2 o and loaded in a plastic syringe ( 3 ml ). it is to be the bioactive agent containing - aqueous solution s 3 of the present embodiment . ( 3 ) a plastic syringe ( 3 ml ) is provided and loaded with ddh 2 o which is the aqueous solution s 1 of the present embodiment . ( 4 ) while the preparation is done , the filter unit 21 ( glass sieve ) and the infusion unit 2 are installed in the reaction tank 1 . through a pvc tube , the syringe is connected with an infusion pump ( figures not shown ), so as to infuse the solutions with a determined flow speed into the reaction tank 1 , respectively . in this embodiment , the flow speeds are set as follow : bioactive agent containing - aqueous solution : 0 . 30 ml / h ( relative mid - speed ), organic solution : 0 . 15 ml / h ( relative low - speed ), aqueous solution : 0 . 50 ml / h ( relative high - speed ). step 102 : infusing the aqueous solution s 1 and organic solution s 2 into the reaction tank 1 of the device 1 and thus forming an interface between the filter 21 and the collector 11 ( as shown in fig2 ). step 103 : infusing the bioactive agent containing - aqueous solution s 3 and being filtered by the filter 21 , the bioactive agent droplet is surrounded by the organic solution s 2 to form a water - in - oil emulsion ( as shown in fig1 c ). step 104 : the water - in - oil emulsion is passing through the aqueous solution 51 and form a water - in - oil - in - water double emulsion ( as shown in fig1 d ). step 105 : removing the organic phase of water - in - oil - in - water double emulsion in step 104 so as to harvest a plurality of liposomes . for example , in this embodiment , a rotary evaporator is used to remove the chloroform to obtain the liposomes . step 106 : the unencapsulated part of the bioactive agent containing - aqueous solution s 3 in step 105 is removed . for example , dialysis method is used to remove fluorescence dye ( 5 ( 6 )- carboxyfluorescein ) of the solution . for the results can be easily detected , a glass sieve having pore size of 5 - 10 μm can used in foregoing steps to obtain larger size liposomes . thus , after chloroform is removed , the results can be observed by fluorescence microscopes . fig4 ( a )- 4 ( c ) depict the fluorescence microscopy photos of ( a ) under phase contrast mode , ( b ) nile red fluorescence , and ( c ) carboxyfluorescein fluorescence . as shown in photos , sphere particles are observed and their diameters are mostly less than 10 μm . as results shown in fig4 ( a )- 4 ( c ) that nile red and carboxyfluorescein are simultaneously observed in the liposeomes . to further detect the distribution of fluorescence dye in solution , a laser scanning confocal microscope can be used to observe the liposomes . fig5 ( a )- 5 ( c ) depict the fluorescence microscopy photos of ( a ) nile red fluorescence , ( b ) carboxyfluorescein fluorescence , ( c ) overlapping image of ( a ) and ( b ). as compared with fig5 ( a ) and 5 ( b ) , hydrophobic fluorescence dye , nile red , is distributed around the surface of spheres , and thus exhibits the distribution of phospholipid molecules of the spheres . on the other hand , hydrophilic fluorescence dye , carboxyfluorescein , is distributed inside the spheres . as shown in fig5 ( c ) , an overlapped image of fig5 ( a ) and 5 ( b ) , distribution of bioactive agents and phospholipids of a sphere is exhibited . to sum up , the spherical vehicles as manufactured in the present embodiment are the liposomes formed by the w / o / w double emulsion . in contrast with large size liposomes produced in embodiment 2 , a syringe filter having membrane pore size of 0 . 45 μm is used in this embodiment to produce smaller size liposomes . as using the syringe filter according to this embodiment , the flow speed of infusion pump is set to 0 . 20 ml / h . except that , other steps are similar to embodiment 2 , so the details are not repeated here . the results are displayed in fig6 ( a )- 6 ( d ) . the figures depict the particle size distribution of the double emulsion after natural evaporation , separately after ( a ) 14 hours , ( b ) 22 hours , ( c ) 36 hours , and ( d ) 46 hours . in order to produce liposomes from the double emulsion in step 104 , the double emulsion is placed for a while to evaporate the chloroform . a particle size analyzer is used to analyze the continuous change of the double emulsion size as chloroform evaporates . as shown in fig6 ( a )- 6 ( d ) , the size of the double emulsion according to this embodiment is decreased due to evaporation of chloroform ( 14 , 22 , 38 and 46 hours ). after 46 hours , the size of the double emulsion is invariable and so as to form liposomes whose diameter is ¼ of the membrane pore size . to further shorten the preparation time , rotary evaporator is used to accelerate evaporation of chloroform . under the condition as follow , 100 mbar , 50 ° c ., evaporate for 30 min , liposomes are obtained with similar size as treated as nature evaporation for 46 hours . the size of liposome produced by double emulsion method is determined by the size of water droplets ( bioactive agent containing - aqueous solution ). in this embodiment , two syringe filters having membrane pore size of 0 . 22 μm and 0 . 45 μm are used respectively , so as to examine liposome size influenced by different membrane pore size of filter , and thus analyzed by particle size analyzer . as shown in fig7 ( a )- 7 ( b ) , the figures depict the particle size distribution of the liposomes prepared by syringe filter having membrane pore size of ( a ) 0 . 22 μm and ( b ) 0 . 45 μm . as analyzed by particle size analyzer , two liposome sizes are shown in fig7 ( a )- 7 ( b ) , respectively . the results identify that the liposome size generated by the device of the present invention is directly related with the membrane pore size of filter . and its diameter is approximately ¼ of the membrane pore size . by a fluorescence spectrophotometer , the encapsulation efficiency of liposomes that encapsulate fluorescence dye can be obtained . the definition of encapsulation efficiency is defined as follow : as calculated results , according to the calibration curve obtained from carboxyfluorescein standard ( coefficient of determination , r 2 = 0 . 994 ), the encapsulation efficiency of carboxyfluorescein of liposomes generated by the device of the present invention is shown in table . 1 . the encapsulation efficiency percentages of the liposomes filtered by filters having membrane pore size of 0 . 22 μm and 0 . 45 μm are 24 % and 28 %, respectively . the encapsulation efficiency of agents encapsulated by liposomes is significantly related with the method of producing liposomes . the composition of the liposomes may also be influential the encapsulation efficiency . as compared with the documents [ 1 - 5 ] and the method according to the present invention , the encapsulation efficiencies are exhibited in table . 2 . as shown in table . 2 , it is found that the encapsulation efficiencies of liposomes generated by thin - film hydration methods or reverse phase evaporation methods are commonly much lower than 30 %. however , the encapsulation efficiency of liposomes according to the present invention is much higher . the encapsulation efficiency of liposomes generated by the device of the present invention is approximately 0 . 3 , which is higher than the results in documents 1 and 3 . it is proved that the device and method of the present invention can generate liposomes with high encapsulation efficiency . although the present invention has been described with reference to the preferred embodiments thereof , it is apparent to those skilled in the art that a variety of modifications and changes may be made without departing from the scope of the present invention which is intended to be defined by the appended claims . volodkin , d . ; mohwald , h . ; voegel , j . c . ; ball , v . j . control . release 2007 , 117 , 111 - 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