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¿ø¹®º¸±â Microfluidics Application Note


INTRODUCTION

Liposome Àº Çϳª ¶Ç´Â ±× ÀÌ»óÀÇ bi-layered membrane À¸·Î ±¸¼ºµÈ spherical lipid vesicle ÀÌ´Ù. ÀÌ·¯ÇÑ liposome ÀÇ Á¦Á¶¿¡ °¡Àå ¸¹ÀÌ »ç¿ëµÇ´Â °ÍÀº Phospholipids ÀÌ´Ù. ¿Ö³ÄÇϸé Lipid bi-layers ·Î µÑ·¯½ÎÀÎ aqueous cores ±¸Á¶Àû Ư¼º»ó liposome Àº ÇÙ»êÀ» Æ÷ÇÔÇÑ hydrophilic actives ¸¦ ĸ½¶È­ÇÏ°í Àü´ÞÇÒ ¼ö Àֱ⠶§¹®ÀÌ´Ù. ±×·¡¼­ ´Ù¸¥ lipid-based delivery systems ¿Í ºñ±³ ½Ã °¡Àå ¿ì¼öÇÏ´Ù



ÀÌ·¯ÇÑ Liposome À» ¼Ò·®À¸·Î Á¦Á¶ÇÏ´Â ¹æ¹ýÀº ¸¹Áö¸¸ »ý»ê ±Ô¸ð·Î Á¦Á¶ÇÏ´Â ¹æ¹ýÀº ¼Õ¿¡ ²ÅÀ» ¸¸Å­ Àû´Ù. ¿Ö³ÄÇϸé lipids, ½Ã°£, ¿¹»ê, ÇÊ¿äÇÑ lipids ÀÇ Ç°Áú, ĸ½¶È­ ÇÒ active ingredients µî ¸¹Àº ¿ä¼Ò¸¦ °í·ÁÇÏ¿© Á¦Á¶ÇؾßÇϱ⠶§¹®ÀÌ´Ù.


CHOICE OF PREPARATION TECHNOLOGIES

Liposome Á¦ÀÛ¹ýÀº ´ÙÀ½ µÎ °¡Áö°¡ ÀÖ´Ù.

(1) lipid hydration ¹æ¹ý¿¡ ±â¹ÝÇÑ top-down ¹ý

(2) solvent ¿Í non-solvent streams À» È¥ÇÕÇÑ ÈÄ precipitation À» »ç¿ëÇÏ¿© ¿øÇÏ´Â Å©±â¿Í lamellarity ¸¦ °¡Áø liposomeÀ» Á¦Á¶ÇÏ´Â bottom-up ¹ý

Lipid hydration method (Top-down)

ÀüÅëÀûÀÎ top-down ¹æ¹ýÀ̶ó Çϸé (1) thin film ¶Ç´Â (2) lipid ¿Í lipophilic active ingredients ÀÇ È¥ÇÕ¹°À» Á¦Á¶ÇÑ ÈÄ ÀÌ °á°ú¹°À» ¼ö¿ë¾×¿¡ ¼öÈ­ÇÑ´Ù.

±×·¯³ª Phospholipids Àº ¼öÈ­ »óÅ¿¡¼­ ´Ù½Ã bi-layered ±¸Á¶·Î Á¶¸³µÇ¸é¼­ ºÒ±ÕÁúÇÑ large unilamellar vesicles (LUVs) ¹× multilayered vesicles (MLVs) ¸¦ Çü¼ºÇϱ⠶§¹®¿¡ ¿øÇÏ´Â Å©±â¿Í lamellarity ¸¦ ¾ò±â À§Çؼ­´Â ´Ù¾çÇÑ ±â°èÀû ±â¼úÀ» ÅëÇØ Å©±â¸¦ ÁÙÀÌ°í conditioning ÀÛ¾÷ÀÌ ÇÊ¿äÇÏ´Ù.

Solvent injection method (Bottom-up)

Top-down ¹æ¹ý°ú ¹Ý´ë·Î bottom-up ¹æ¹ýÀº ħÀüÀ» ÀÌ¿ëÇÏ¿© liposome vesicles ¸¦ Á÷Á¢ »ý¼ºÇÑ´Ù.

Liposome À» Çü¼ºÇÏ´Â ¼ººÐÀº À¯±â¿ë¸Å¿¡ ¿ëÇصǴµ¥ À̶§ »ç¿ëµÇ´Â À¯±â¿ë¸Å´Â water miscible Ư¼ºÀ» ¶í´Ù. ÀÌ ¿ëÇؾ×À» ¼ö¿ë¾× ¹öÆÛ ½Ã½ºÅÛ (aqueous buffer system) ¿¡ õõÈ÷ ÁÖÀÔÇÑ´Ù. ÁÖÀÔÇÒ ¶§ small unilamellar vesicles (SUV) ¿Í LUV °¡ Çü¼ºµÈ´Ù. ÀÌ´Â ÁÖÀÔÀ» ÅëÇØ À¯±â¿ë¸Å°¡ ¼ö¿ë¾×À¸·Î º¯°æµÇ¸é¼­ ¿ëÇصµ°¡ º¯Çϱ⠶§¹®ÀÌ´Ù.


ALTERNATIVE APPROACHES USING MICROFLUIDIZER TECHNOLOGY

Microfluidizer processor ¸¦ »ç¿ëÇϸé À§¿¡¼­ ³ª¿­ÇÑ lipid filmÀ» ¸¸µå´Â °úÁ¤À» °ÅÄ¡Áö ¾Ê°íµµ liposomes À» »ý»êÇÒ ¼ö ÀÖ´Ù. ¾Æ·¡¿¡¼­ ÀÌ¿¡ °ü·ÃÇÑ ³»¿ëÀ» ¼Ò°³ÇÏ°íÀÚ ÇÑ´Ù.

Lipid solution method

1. ¸ðµç lipid, lipophilic ingredients ´Â ¿¡Åº¿Ã°ú °°Àº water miscible solvent ¿¡ ¿ëÇØ ÈÄ aqueous phase ¿¡ Ãß°¡ÇÑ´Ù. ÀÌ °á°ú¹°Àº low shear mixer ·Î Àüó¸® ÇÑ ÈÄ Microfludizer processor ·Î ó¸®ÇÑ´Ù. ÀÌ´Â À§¿¡¼­ ¼Ò°³ÇÑ solvent injection method (bottom-up) ¹æ¹ý°ú À¯»çÇϳª ´Ù¸¥ Á¡ÀÌ ÀÖ´Ù. Àüó¸® °úÁ¤¿¡¼­ ÀÌ¹Ì MLV ÀÌ »ý¼ºµÇ°í Microfluldizer ¸¦ ÅëÇÑ ÈÄó¸® °úÁ¤¿¡¼­ SUV °¡ »ý¼ºµÇ±â ¶§¹®¿¡ vesicle formation mechanism ÀÌ ´Ù¸£´Ù´Â °ÍÀÌ´Ù. µû¶ó¼­ ÈÄó¸® ÈÄ solvent °¡ Á¦°ÅµÇ¾î¾ß ÇÑ´Ù.
 
2. Double emulsion method ¸¦ ÀÌ¿ëÇÑ hydrophilic active encapsulation À» ÇÒ ¼ö ÀÖ´Ù. Hydrophilic drugs ¸¦ Æ÷ÇÔÇÏ°í ÀÖ´Â Aqueous phase (W1) À» lipid solution (O) ¿Í ¼¯¾î¼­ water-in-oil (W1/O) ¿¡¸ÖÁ¯À» ¸¸µç´Ù. (À̶§ O ´Â water immiscible solvent ¿¡ ¸ðµç lipid ingredients °¡ ¿ëÇصǾî ÀÖ´Â »óÅÂÀÌ´Ù. ) W1/O ¿¡¸ÖÁ¯Àº ´Ù½Ã second aqueous (W2) ¿Í ¼¯¾î¼­ W1/O/W2 ¿¡¸ÖÁ¯À» ¸¸µç´Ù. ±× ÈÄ Microfluidizer processor ·Î ó¸®ÇÏ°í solvent ¸¦ Á¦°ÅÇϸé final solution ÀÌ Æ÷ÇÔµÈ liposome vesicles À» ¾òÀ» ¼ö ÀÖ´Ù.

ÀÌ ¹æ¹ýÀº encapsulation efficiency °¡ ¸Å¿ì ³ô´Ù. ÃÖ±Ù ¹®Çå*¿¡ µû¸£¸é plasmid DNA (inside two different cationic liposome formulations) ÀÇ °æ¿ì 80% ÀÇ encapsulation ¼º°øÀ» º¸°íÇß´Ù.
* Lajunen, T., et al. 'Topical drug delivery to retinal pigment epithelium with microfluidizer produced small liposomes.' European Journal of Pharmaceutical Sciences 62 (2014): 23-32.

Solvent free method

Áö±ÝºÎÅÍ ¼Ò°³ÇÒ solvent free method ´Â ¸î¸î lipsome formulation ¿¡ Àû¿ëµÇ´Â ½Å±â¼úÀÌ´Ù. Lipid films À̳ª solution À» ÅëÇÑ liposome forming ÀÌ ¾Æ´Ï±â ¶§¹®¿¡ À¯±â¿ë¸Å°¡ ¾ø´Â °á°ú¹°À» ¸¸µé ¼ö ÀÖ´Ù.

Microfluidizer ·Î ó¸®ÇÏ¿© ´Ù¿î»çÀÌ¡µÈ lipophilic actives (with aqueous buffer solutions) ¿Í dry lipid ingrdients ¸¦ ¹Ù·Î È¥ÇÕÇÏ¿© liposomes À» ¸¸µç´Ù. ÇØ´ç ¹æ¹ýÀº ÀÌ¹Ì »ó¾÷È­µÈ ¸®Æ÷Á» ¾à¹° Á¦Ç°À» ¸ð¹æÇÏ´Â Á¦ÇüÀ» »ý¼ºÇÒ ¶§ ÀûÇÕÇÏ´Ù. ¶ÇÇÑ ÀÓ»óÀûÀ¸·Î ÀûÀýÇÑ ¹üÀ§ ³»¿¡¼­ vesicle size ¸¦ ¸¸µé ¼ö ÀÖ´Ù.


WHY MICROFLUIDICS?

High quality liposomes - Fixed Interaction Chamber¢â ¹× Microfluidizer processor ÀÇ ±ÕÀÏÇÑ ¾Ð·ÂÀ» À¯ÁöÇÏ´Â ÆßÇÁ ½Ã½ºÅÛ¿¡ ÀÇÇØ ¾ÈÁ¤ÀûÀÌ°í ±ÕÀÏÇÑ shear °¡ »ý¼ºµÇ°í µû¶ó¼­ ±ÕÀÏÇÑ Å©±âÀÇ SUV Á¦ÀÛÀÌ °¡´ÉÇÏ´Ù. Microfluidizer ±â¼úÀº liposome Å©±â¸¦ 40 - 50 nm Á¤µµÀÇ ÀÛÀº »çÀÌÁî·Î ÁÙÀÏ ¼ö ÀÖ´Ù.

High Concentration - Microfluidics ´Â Á¦Ç°À» ³óÃàÇϱâ À§ÇÑ Ãß°¡ÀûÀÎ downstream process °úÁ¤ÀÌ ÇÊ¿ä ¾øÀÌ therapeutic level ¿¡ ºÎÇյǴ °í³óµµÀÇ liposome solution ÀÇ »ý»êÀÌ °¡´ÉÇÏ´Ù. Lipid ³óµµ ¹üÀ§´Â 5mM ¿¡¼­ 25mM ÀÌ´Ù. Microfluidizer ±â¼úÀº ready¡©to-be-administered liposome ¾à¹°ÀÇ lipid ³óµµ ¹üÁÖ ¶Ç´Â ±× ÀÌ»óÀÇ ³óµµ¸¦ °¡Áø liposome À» »ý»êÇÒ ¼ö ÀÖÀ¸¸ç, ÀÌ´Â ÀϹÝÀûÀ¸·Î solvent injection ¹æ¹ý¿¡¼­ ´Þ¼ºÇÒ ¼ö ÀÖ´Â °Íº¸´Ù ÃÖ´ë 2¹è ÀÌ»ó ³ô´Ù.

Very fast size reduction - ƯÈ÷ extrusion ¶Ç´Â microfluidic chip ±â¼ú°ú °°Àº slow process ¿Í ºñ±³Çϸé liposome vesicle ÀÇ Å©±â¸¦ ÁÙÀ̴ ó¸®½Ã°£ÀÌ ÈξÀ ª´Ù. ½ÇÇè½Ç ±Ô¸ðÀÇ Microfluidizer processor ·Î 1~3¸®Å͸¦ »ý¼ºÇϴµ¥ ¾à 10ºÐÀÌ ¼Ò¿äµÈ´Ù. »ý»ê¿ë ¸ðµ¨Àº ½Ã°£´ç ¼ö¹é ¸®Å͸¦ »ý»êÇÑ´Ù.

Scalability - Preclinical ¿¡¼­ ÀÓ»ó ¶Ç´Â »ý»ê ±Ô¸ð·Î º¯°æÇÒ ¶§ scalability ¸¦ °í·ÁÇØ¾ß ÇÑ´Ù. ÀüÅëÀûÀÎ top-down ¹æ½Ä°ú bottom-up ¹æ½ÄÀº scalable ÀÌ ¾î·Æ´Ù. Microfluidizer ±â¼úÀº º»ÁúÀûÀ¸·Î ÃÖ´ë ¼öõ ¸®ÅÍÀÇ ¾çÀ¸·Î scalable °¡´ÉÇϱ⠶§¹®¿¡ ¸ðµç ±Ô¸ðÀÇ Àåºñ¿¡¼­ therapeutic concentrations ¸¦ »ý¼ºÀÌ °¡´ÉÇϸç ÀÌ´Â ¼ö½Ê ³â µ¿¾È ÀÔÁõµÇ¾ú´Ù.

Solvent independent - Microfluidizer ·Î liposome vesicle ÀÇ Å©±â¸¦ ÁÙÀÌ´Â °øÁ¤¿¡ ÀϹÝÀûÀ¸·Î ¿ë¸Å°¡ »ç¿ëµÇÁö ¾Ê±â ¶§¹®¿¡ Ãß°¡ÀûÀÎ ¿ë¸Å Á¦°Å °øÁ¤ÀÌ ÇÊ¿äÇÏÁö ¾Ê´Ù. ±×·¯³ª formulation °øÁ¤¿¡ ¿ë¸Å°¡ ÇÊ¿äÇÑ °æ¿ì Microfluidizer processor ÀÇ ³»ºÎ ºÎÇ°µéÀº ´ëºÎºÐÀÇ ¿ë¸Å¿Í ȣȯµÇ¹Ç·Î ¿À¿°À» ¿ì·ÁÇÒ ÇÊ¿ä´Â ¾ø´Ù.

cGMP ready systems - CIP ¹× SIP ±â´ÉÀ» °®Ãá cGMP Áö¿ø ½Ã½ºÅÛÀ» Á¦°øÇÑ´Ù. Microfluidizer processor ´Â cGMP ¿¡ °¡Àå ÀûÇÕÇÏ°Ô ¼³°èµÇ¾úÀ¸¸ç CIP/SIP ÀÛ¾÷ÀÌ °¡´ÉÇÏ´Ù.

Flexibility - ÀÛµ¿ ¸Å°³º¯¼ö¸¦ º¯°æÇÏ¿© µ¿ÀÏÇÑ Microfluidizer processor ·Î ´Ù¾çÇÑ Å©±â¿Í formulation ÀÇ liposome À» ¸¸µé ¼ö ÀÖÀ» »Ó¸¸ ¾Æ´Ï¶ó liposome ÀÇ Å©±â¸¦ ¿¹ÃøÇÒ ¼ö ÀÖ´Ù.

Sterile Filtration - Pall Life Sciences ÀÇ ¿¬±¸´Â Microfluidizer processor ¸¦ »ç¿ëÇÏ¿© »ý»êµÈ liposome ÀÌ 220nm ÇÊÅÍ·Î ¼º°øÀûÀ¸·Î ¿©°úµÉ ¼ö ÀÖÀ½À» Áõ¸íÇÏ¿´´Ù.
* http://www.pall.com/en/biotech/webinars/sterile-filtration-complex-fluid.html


CHOOSING THE RIGHT METHODOLOGY




CASE STUDY 1 - COMPARING TO THE BOTTOM-UP METHOD

¾Æ·¡ µÎ °¡Áö ¹æ½ÄÀ¸·Î Á¦Á¶ÇÑ liposome À» ºñ±³ÇÏ¿´´Ù.
(1) Microfluidizer processor
(2) Microfluidic chip À» ÀÌ¿ëÇÑ bottom-up ¹æ½Ä

Results

Table 2 ¿¡¼­¿Í °°ÀÌ µÎ ¹æ¹ý ¸ðµÎ ¸Å¿ì ±ÕÀÏÇÑ ºÐÆ÷ (Pdls °¡ 0.1¿¡ °¡±î¿ò) ¸¦ °¡Áø ÀÛÀº Å©±âÀÇ ( <100nm ) liposome À» Á¦Á¶ÇÏ¿´´Ù. ÇÏÁö¸¸ Fig.1 À» º¸¸é Microfluidizer ó¸® °á°ú°¡ ÈξÀ ´õ ÀÛÀº vesicle À» Á¦Á¶Çß°í, ÀüüÀûÀÎ »çÀÌÁî ºÐÆ÷µµ¿¡¼­µµ ÈξÀ ´õ ÀÛÀº °á°ú¸¦ º¸¿©ÁØ´Ù.






CASE STUDY 2 - COMPARE TO HIGH PRESSURE HOMOGENIZER (HPH)

¾Æ·¡ µÎ °¡Áö ±â±â¸¦ ÀÌ¿ëÇÏ¿© µ¿ÀÏÇÑ ½Ã·á, µ¿ÀÏÇÑ ¾Ð·Â, µ¿ÀÏÇÑ pass ¼ö¸¦ ó¸®ÇÑ ¸®Æ÷Á»À» ºñ±³ÇÏ¿´´Ù.
(1) Microfluidizer processor
(2) ±âÁ¸ÀÇ °í¾Ð ±ÕÁúÈ­±â±â (HIGH PRESSURE MONOGENIZER, HPH)

Benefits of fixed geometry & constant pressure

Microfluidizer ±â¼úÀº fixed geometry high shear zone °ú ±ÕÀÏÇÑ ¾Ð·ÂÀÇ ÆßÇÁ¸¦ »ç¿ëÇÑ´Ù. Fig. 2 ¿¡¼­ º¼ ¼ö ÀÖµíÀÌ Microfluidizer processor ´Â ÀÌ¿Í °°Àº ƯÀ¯ÀÇ Á¶ÇÕÀ» ÅëÇØ °¢ ó¸® °øÁ¤ °úÁ¤¿¡¼­ ÀÏÁ¤ÇÑ ¾Ð·ÂÀ» ´Þ¼ºÇÒ ¼ö ÀÖ´Ù. ÇÏÁö¸¸ HPH´Â ¸ñÇ¥ ¾Ð·Â¿¡¼­ ÃÖ´ë 50% ³·Àº °¡º¯ ¾Ð·ÂÀ¸·Î »ùÇÃÀ» ó¸®ÇÑ´Ù. ó¸®ÇÏ°íÀÚ ÇÏ´Â »ùÇà ¿ë·®ÀÌ 1ml ¿¡¼­ ¼öõ ¸®ÅÍ·Î ´Ù¾çÇÒÁö¶óµµ Microfluidizer ÀÇ ÀÏÁ¤ÇÑ ¾Ð·ÂÀ» À¯ÁöÇÏ´Â ±â¼ú¿¡ ÀÇÇØ ¸ðµç »ùÇÿ¡ Àü´ÞµÇ´Â ¾Ð·ÂÀÌ ÀÏÁ¤ÇÏ°Ô À¯ÁöµÈ´Ù.



µ¿ÀÏÇÑ Á¶°ÇÀÇ Ã³¸® °á°ú´Â Fig 3 °ú Table 3°ú °°´Ù.

Fig 3 ÀÌ º¸¿©ÁÖµíÀÌ Microfluidizer processor ¿¡ ÀÇÇØ »ý¼ºµÈ liposome ÀÔÀÚ Å©±â ºÐÆ÷µµ´Â single peak, Áï ¾ÆÁÖ ±ÕÀÏÇÑ ÀÔµµ ºÐÆ÷¸¦ º¸¿©ÁÖ´Â ¹Ý¸é HPH ¿¡ ÀÇÇØ »ý¼ºµÈ °á°ú´Â ³ÐÀº ¹üÀ§¿¡ °ÉÃÄ three peaks, Áï ´Ù¾çÇÑ Å©±â ÀÔµµ ºÐÆ÷¸¦ º¸¿©ÁØ´Ù.

Æò±Õ liposome ÀÔÀÚ Å©±â¸¦ ³ªÅ¸³½ Table 3ÀÇ °á°ú´Â Microfluidizer ±â¼úÀÌ liposome Å©±â¸¦ ÁÙÀÌ´Â µ¥ ÈξÀ ´õ È¿À²ÀûÀ̶ó´Â °ÍÀ» Áõ¸íÇÑ´Ù. ¶ÇÇÑ Microfluidizer °øÁ¤Àº ´Ü 2 pass ¿¡ 100nm ¹Ì¸¸ÀÇ liposome À» »ý¼ºÇÒ ¼ö ÀÖ¾ú´Ù. ¹Ý¸é¿¡ HPH´Â 3 pass ó¸® ÈÄ¿¡¾ß 183nm ÀÌ µÇ¾ú´Ù. ÀÌ´Â Microfluidizer processor ¿¡¼­ 1 pass ó¸®ÇÑ °á°úÀÎ 113nm º¸´Ùµµ ÈξÀ Å« °á°ú¿´´Ù.














 
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