Amphiphilic methacrylate monomers having cholesteryl or dialkyl groups as hydrophobic moiety were synthesized and the vesicle formation were carried out by sonication and by polymerization. Morphological and physical properties of these vesicles were investigated by electron microscopy, spectroscopic methods, differential scanning calorimetry, liquid scintillation counting, and transport of substrates through polymeric bilayer membranes.
Cholesteryl amphiphilic methacrylate monomer, cholesteryl-oxycarbonyl-methyl 2-(methacryloyloxy)ethyl dimethyl ammonium chloride, coded as CHODAMA was synthesized from cholesteryl chloroacetate by reacting with 2-(dimethylamino)ethyl methacrylate. CHODAMA was soluble in water giving a viscous solution. Upon polymerization of CHODAMA (0.5% in water) by free radical initiator or by UV irradiation the viscosity of the solution dropped as the Polymerization proceeded, indicating that the vesicles were being formed during the polymerization process. Electron micrographs and entrapment of [$^{14}C$]sucrose confirmed that the polymer of CHODAMA thus prepared formed well defined closed microvesicles (diameters ranging between 200 and 500 Å). Poly(CHODAMA) vesicles were stable for at least several months and were highly stable thermally, exhibiting no notable change upon heating up to 80℃.
A double alkyl chain amphiphilic methacrylate monomer, α,$α^1$-(dihexadecyloxy)glyceryl-oxycarbonyl-methyl 2-(methacryloyloxy)ethyl dimethyl ammonium chloride, coded as HELDAMA was synthesized from α, $α^1$-(dihexadecyloxy)glycerol according to the similar procedure to that employed for CHODAMA. Didodecyl amphiphilic methacrylate monomer, coded as DOLDAMA was synthesized in the similar way from α, $α^1$-(didodecyloxy)glycerol. DOLDAMA was readily dispersed in water at room temperature, whereas HELDAMA which was more hydrophobic than DOLDAMA could be dispersed in water only at temperatures above its phase transition temperature (41℃).
Dialkyl amphiphilic vesicles were prepared by sonication of DOLDAMA or HELDAMA above their phase transition temperatures. Polymeric vesicles were formed by polymerization either by irradiation with a UV lamp or heating the monomer solution with an initiator. The sizes of these dialkyl amphiphilic vesicles were determined by electron microscope as having diameters ranging from 300 to 1500 Å. The phase transition temperature of HELDAMA vesicles rose from 42.5 ℃ to 56.5 ℃ after polymerization, whereas the phase transition of poly(CHODAMA) vesicles was not observed below 100 ℃. Poly(HELDAMA) which were separated from free [$^{14}C$]sucrose and Brilliant Green dye on a Sephadex G-50-80 column entrapped 1.07% of [$^{14}C$]sucrose and 2.9% of Brilliant Green dye.
The size of copolymeric vesicles prepared from CHODAMA and HELDAMA could be increased by increasing the content of HELDAMA in the vesicle-forming copolymers. The phase transitions of these copolymeric vesicles were lowered in proportion to the increase in the portion of CHODAMA. These results clearly demonstrate that copolymeric vesicles according to the feed mole ratio of CHODAMA and HELDAMA were formed uniformly.
Poly(HELDAMA) vesicles were ruptured in 35% ethanol or by addition of surfactant Triton X-100 (0.5% aqueous solution). However, even the addition of ethanol or surfactant could not induce the rupture of the poly(CHODAMA) vesicles, again confirming the high stability of these vesicles.
The stability of poly(CHODAMA) vesicles by polymerizing at different salt concentration was examined. As the salt concentration was increased, the vesicles became larger, untill precipitated at the 0.5% level of KCl. It was notable that when formed once, poly(CHODAMA) vesicles did not precipitate upon the addition of KCl even in the saturated solution, whereas HELDAMA vesicles precipitated by the addition of some salt.
When the poly(CHODAMA) vesicles prepared by the polymerization in the 0.4% uranyl acetate solution were dialyzed and aged at room temperature for one day after dilution by acetic acid solution, electron micrographs of these vesicles showed the presence of right-handed helical tubular structures which were supposed to be resulted from successive fusions of vesicles.
The leakage rate of [$^{14}C$]sucrose by poly(CHODAMA) vesicles was considerably slower than that by poly(HELDAMA) vesicles. Poly(HELDAMA) vesicles were ruptured thermally above 40 ℃, whereas the rupture of copolymeric vesicles containing CHODAMA more than 25 % of total monomer was not detected even at 80 ℃.
The lyophilized powder of poly(CHODAMA) vesicles entrapping [$^{14}C$]sucrose was not only regenerable to polymeric vesicles by repeated suspension in water but also these regenerated vesicles were found to retain 90% of [$^{14}C$]sucrose entrapped into the original vesicles. Upon standing for 4 months after the regeneration, amount of [$^{14}C$]sucrose entrapped into these vesicles were dropped to 43% level.
콜레스테롤 양친매성 메타크릴 모노머(CHODAMA)는 콜레스테롤클로로 아세테이트와 2-(디메틸아미노)에틸 메타크릴레이트 (DMAEMA)의 4가 암모니움염 반응으로 부터 합성하였다. CHODAMA수용액에 중합개시제를 첨가하거나 자외선을 조사하면 중합이 진행되어 초음파 진동처리 없이도 크기가 200~500Å 정도인 구형의 고분자 미세액포가 형성되는 것을 전자현미경 사진과 $[^{14}C]^{su}$ CROSE의 포획에 의해 확인하였다. 이 Poly(CHODAMA) 미세액포는 여러달 동안 안정한 상태로 존재할 뿐만 아니라 열이나 염에 대한 안정성도 매우 좋았다.
이중알킬 양친매성 메타크릴 모노머(HELDAMA, DOLDAMA)는 α, $α^1$ -디알킬옥시글리세롤과 클로로아세트산과의 탈수반응에 의한 에스테르를 합성한 후 DMAEMA와 4가 암모니움염 반응을 시켜서 합성하였다. 디알킬 양친매성 미세액포는 DOLDAMA나 HELDAMA의 상변이 온도 이상에서 초음파 진동을 가하여 만들었다. 이 미세액포에 중합개시제를 가하고 가열하거나 자외선을 조사하여 이중충막을 중합시키면 구형의 300~1,500 Å의 크기를 갖는 안정한 고분자 미세액포가 형성되었다.
100℃ 이하에서는 상변이 온도가 관찰되지 않는 Poly (CHODAMA) 미세액포와는 달리 42.5℃의 상변이 온도를 갖고 있던 HELDAMA 미세액포가 중합후에는 56.5℃로 상변이 온도가 상승 하였다. CHODAMA와 HELDAMA의 공중합에 의해 형성된 미세액포에 있어서, CHODAMA의 몰비가 증가할수록 점차 크기는 감소하고 상변이 온도는 내려가는 현상으로 볼때 CHODAMA와 HELDAMA의 몰비에 따라 균일하게 공중합된 미세액포가 형성된다는것을 명확히 알 수 있다.
이중층막으로 밀폐된 구형의 poly(HELDAMA) 미세액포는 액포의 내부 수용액상에 비이온 물질인 $[^{14}C]$ sucrose가 미세액포 형성전에 첨가된 총량의 1.07%, 양이온인 Brilliant Green 염료가 2.9%정도 포획되었다.
Poly(HELDAMA) 미세액포는 유기용매인 에탄올의 농도가 3.5%, 또는 계면활성제인 Triton X-100의 농도가 0.17%일때 미세액포의 내부에 포획되었던 염료가 상온에서도 갑자기 누출되기 시작하였다. 그러나, poly(CHODAMA) 미세액포는 에탄올이나 계면활성제가 첨가되어도 누출현상을 일으키지 않을 정도로 매우 안정함을 확인할 수 있었다.
우라닐 아세테이트 용액(0.4%)에서 중합시킨 poly(CHODAMA) 미세액포를 투석하고 초산을 약간 첨가한 후 실온에서 하루동안 방치시킨 미세액폭의 전자현미경 사진은 미세액포의 연속적인 융합에 의한 결과로 추정되는 우선성 튜브구조를 보여주었다.
이중층막의 소수성부분이 콜레스테롤인 Poly(CHODAMA) 미세액포는 소수성부분이 알킬 사슬인 Poly(HELDAMA) 미세액포 보다 포획된 $[^{14}C]$ sucrose의 누출속도가 훨씬 느렸다. 또한 poly(HELDAMA) 미세액포는 40℃ 이상의 온도에서 포획된 양이온 염료를 30분 이내에 대부분 누출시키지만 CHODAMA 모노머를 25% 이상 포함하는 공중합체인 미세액포는 80℃ 에서도 비이온 물질인 sucrose의 누출이 전혀 일어나지 않았다.
$[^{14}C]$ sucrose를 포획하고 있는 poly(CHODAMA)를 미세액포를 냉동건조 시킨 후 물에 재분산시킨 시료를 젤컬럼분리한결과 미세액포의 구조가 파괴되지 않았을 뿐만 아니라 초기 포획된 $[^{14}C]$ sucrose양의 90%를 유지하고 있을 정도로 매우 안정하였으며, 4개월 동안 상온에서 방치한 시료의 경우도 $[^{14}C]$ sucrose양의 43%를 포획하고 있었다.