KR100664466B1 - Microparticle for delivering oral vaccines and the method of manufacturing thereof - Google Patents
Microparticle for delivering oral vaccines and the method of manufacturing thereof Download PDFInfo
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- KR100664466B1 KR100664466B1 KR1020040028800A KR20040028800A KR100664466B1 KR 100664466 B1 KR100664466 B1 KR 100664466B1 KR 1020040028800 A KR1020040028800 A KR 1020040028800A KR 20040028800 A KR20040028800 A KR 20040028800A KR 100664466 B1 KR100664466 B1 KR 100664466B1
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- microparticles
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- palmitoyl
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/02—Bacterial antigens
- A61K39/025—Enterobacteriales, e.g. Enterobacter
- A61K39/0275—Salmonella
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
본 발명은 세포외 다당류를 이용한 백신 운반용 미세입자에 관한 것으로, 보다 상세하게는 GFP 발현 Salmonella typhimurium 균체를 항원모델로 하고 이들을 함유하는 세포외 다당류를 사용하여 제조한 경구용 백신 운반용 미세입자 및 그 제조방법에 관한 것이다.The present invention relates to microparticles for vaccine delivery using extracellular polysaccharide, and more specifically, oral vaccine delivery microparticles prepared using GFP expressing Salmonella typhimurium cells as an antigen model and using extracellular polysaccharides containing them, and their preparation. It is about a method.
약물운반 시스템, 미세입자, 리포좀, 다당류, β-1,3-글루칸, 커들란-설페이트, O-팔미토일 커들란-설페이트, O-팔미토일 스클레로글루칸-설페이트Drug delivery systems, microparticles, liposomes, polysaccharides, β-1,3-glucan, curdlan-sulfate, O-palmitoyl curdlan-sulfate, O-palmitoyl scleroglucan-sulfate
Description
도 1은 OPP 및 OPCS가 코팅된 리포좀의 TEM 이미지 사진이다[(a) 코팅안된 리포좀, (b) OPP가 코팅된 리포좀, (c) OPCS가 코팅된 리포좀).1 is a TEM image of OPP and OPCS-coated liposomes ((a) uncoated liposomes, (b) OPP-coated liposomes, (c) OPCS-coated liposomes).
도 2는 OPCS가 코팅된 리포좀의 TEM 이미지 사진이다[(a) 지질:OPCS = 9:1, (b) 지질:OPCS = 6:4].Figure 2 is a TEM image of OPCS coated liposomes ((a) lipid: OPCS = 9: 1, (b) lipid: OPCS = 6: 4].
도 3은 OPCS가 코팅된 리포좀의 크기 분포를 보여주는 그래프이다[각각의 지질대 OPCS 비율은 다음과 같다: (a) 10:0, (b) 9:1, (c) 8:2, (d) 7:3 및 (e) 6:4].3 is a graph showing the size distribution of OPCS coated liposomes [each lipid to OPCS ratio is as follows: (a) 10: 0, (b) 9: 1, (c) 8: 2, (d 7: 3 and (e) 6: 4].
도 4는 OPCS 농도별로 OPCS가 코팅된 리포좀의 제타 전위를 보여주는 그래프이다.Figure 4 is a graph showing the zeta potential of the OPCS-coated liposomes by OPCS concentration.
도 5는 SGF(Stimulated Gastric Fluid)에서 OPCS가 코팅된 리포좀의 평균 크기 변화를 보여주는 그래프이다.5 is a graph showing the average size change of the OPCS-coated liposomes in SGF (Stimulated Gastric Fluid).
도 6은 OPCS가 코팅된 리포좀으로부터 5-carboxyflourescien의 방출 정도를 보여주는 그래프이다.6 is a graph showing the degree of release of 5-carboxyflourescien from OPCS coated liposomes.
도 7은 S. typhimurium 염색체의 단면도이다.7 is a cross-sectional view of the S. typhimurium chromosome.
도 8은 GFP+ S. typhimurium 의 녹색 형광 염색을 실시하여 배양한 사진이다.Figure 8 is a photo cultured by performing a green fluorescent staining of GFP + S. typhimurium .
도 9는 GFP+ S. typhimurium 가 로딩된 미세입자의 다중초점 현미경 사진을 보여준다9 shows multifocal micrographs of microparticles loaded with GFP + S. typhimurium
도 10은 GFP+ S. typhimurium 의 크기 분포를 보여주는 그래프이다[(A) ST만 로딩된 리포좀, (B) OPCS가 코팅된 리포좀, (C) OPP가 코팅된 리포좀, (D) 풀루란 아세테이트로 제조한 리포좀, (E) 코팅안된 ST].10 is a graph showing the size distribution of GFP + S. typhimurium [(A) liposome loaded with ST only, (B) liposome coated with OPCS, (C) liposome coated with OPP, (D) pullulan acetate Prepared liposomes, (E) uncoated ST].
도 11은 균체입자를 담지한 미세입자제제를 경구투여한 후 Peyer's patch와 장간막 림프절에 도달한 GFP+ 세균들을 관찰한 결과를 보여주는 그래프이다.FIG. 11 is a graph showing the results of observing GFP + bacteria reaching Peyer's patch and mesenteric lymph nodes after oral administration of microparticles carrying cell particles.
도 12는 미세입자를 경구투여한 후 혈청내 항체가를 측정한 결과를 보여주는 그래프이다.12 is a graph showing the results of measuring antibody titers in serum after oral administration of microparticles.
도 13은 미세입자를 경구투여한 후 대변내 sIgA를 측정한 결과를 보여주는 그래프이다.Figure 13 is a graph showing the result of measuring the sIgA in feces after oral administration of the microparticles.
도 14는 미세입자로 면역한 후 병독성 S. typhimurium을 경구투여한 후 Peyer's patch와 비장에서 살아있는 세균 수를 측정한 결과를 보여주는 그래프이다.14 is a graph showing the results of measuring the number of living bacteria in Peyer's patch and spleen after oral administration of virulent S. typhimurium after immunization with microparticles.
본 발명은 세포외 다당류를 이용한 백신 운반용 미세입자에 관한 것으로, 보다 상세하게는 GFP 발현 Salmonella typhimurium 균체를 항원모델로 하고 이들을 함유하는 세포외 다당류를 사용하여 제조한 경구용 백신 운반용 미세입자 및 그 제조방법에 관한 것이다.The present invention relates to microparticles for vaccine delivery using extracellular polysaccharide, and more specifically, oral vaccine delivery microparticles prepared using GFP expressing Salmonella typhimurium cells as an antigen model and using extracellular polysaccharides containing them, and their preparation. It is about a method.
인체의 점막 표면은 병원균의 주요 도입 부위가 된다. 점막의 면역성은 첫 번째 면역학적 방어기작을 제공한다. 점막 표면에 항체를 유도하기 위하여, 점막 표면에 직접 항원을 투여하여야 한다. 이 경우 경구용 백신 접종은 여러 가지 잇점을 가진다. 항원을 표출시키고 대부분의 통상적인 비경구용 백신 접종이 대부분의 감염성 병원균에 대하여 우세한 부위가 되는 점막 표면에 항상 도달하지 못하는 IgG 우성 항체 반응을 유도하는 동안 유도되는 IgA 우성 항체 반응을 일으키는 생리적 루트이다. 또한 경구용 백신 접종은 점막 면역성을 유도하는 대부분의 통상적인 방법을 제공한다.The mucosal surface of the human body becomes the main entry site for pathogens. Mucosal immunity provides the first immunological defense mechanism. To induce antibodies on mucosal surfaces, antigens must be administered directly to mucosal surfaces. In this case, oral vaccination has several advantages. It is a physiological route that expresses antigens and induces an IgA dominant antibody response that induces an IgG dominant antibody response that does not always reach the mucosal surface, which is the predominant site for most infectious pathogens. Oral vaccination also provides most common methods of inducing mucosal immunity.
항원의 경구용 백신 접종은 여러 가지 면역성 검사를 극복해야만 한다. 일반적으로 백신의 경구용 투여는 많은 양의 항원을 필요로 한다. 많은 소화 효소들이 투여된 백신을 감소시킨다. 항원의 경구용 투여는 경구 내성(oral tolerance)으로 알려진 항원 특이성 말초 면역 관용(peripheral immune tolerance)을 유도한다. 이러한 단점을 해결하기 위하여, 항원을 포집시킨 캡슐 및 이를 안전하게 운반하는 방법이 연구되고 있다.Oral vaccination of antigens must overcome several immunological tests. Oral administration of vaccines generally requires large amounts of antigen. Many digestive enzymes reduce the vaccine administered. Oral administration of the antigen leads to antigen specific peripheral immune tolerance known as oral tolerance. In order to solve this disadvantage, a capsule for collecting antigen and a method of safely transporting it have been studied.
경구용 백신의 미세입자 운반 시스템은 전신 운반(systemic delivery)을 통해 많은 수의 현저한 이점을 제공하고 있어 상당한 주목을 받고 있다. 고분자 운반 시스템은 많은 방법을 통해 점막에 투여되는 백신의 효능을 증강시키도록 제작할 수 있다: 예를 들면, 저하를 통해 항원을 방어할 수 있고, 점막 조직 한 부분의 흡수력을 높임으로써 응집할 수 있으며, 체내의 잔류 시간을 연장하거나 또는 항원의 흡수 부위를 표적으로 할 수도 있다(소화관내의 Peyer's patches).Microparticle delivery systems of oral vaccines have received considerable attention as they provide a number of significant benefits through systemic delivery. Polymer delivery systems can be engineered to enhance the efficacy of vaccines administered to the mucosa in many ways: for example, to defend against antigen through degradation, to aggregate by increasing the absorption of a portion of mucosal tissue, It may also extend the retention time in the body or target the site of absorption of the antigen (Pyer's patches in the digestive tract).
이에 본 발명자들은 항산성, 점막접착성 및 면역 증진성 고분자를 탐색한 결과, 항산성과 면역, 항암 활성이 있는 β-1,3-글루칸의 유도체를 발견하게 되었고, 이를 이용한 면역 증진성 유도체 합성 기술과 리포좀 코팅 기술을 통해 경구용 백신 운반용 미세입자를 제조하고 본 발명을 완성하게 되었다.Therefore, the present inventors have found a derivative of β-1,3-glucan having anti-acid, mucoadhesive and immuno-promoting polymers, and have anti-acid, immunity and anti-cancer activity. And through liposome coating technology to prepare oral vaccine transport microparticles to complete the present invention.
따라서, 본 발명의 목적은 GFP 발현 Salmonella typhimurium 균체를 항원모델로 하고 이들을 함유하는 세포외 다당류를 코팅하여 제조한 경구용 백신 운반용 미세입자 및 그 제조방법을 제공하는 것이다.
Accordingly, it is an object of the present invention to provide an oral vaccine delivery microparticles prepared by coating an extracellular polysaccharide containing Salmonella typhimurium cells expressing GFP as an antigenic model and a method for producing the same.
본 발명은 GFP 발현 Salmonella typhimurium 균체를 항원모델로 하고 이들을 함유하는 세포외 다당류를 코팅하여 제조한 경구용 백신 운반용 미세입자 및 그 제조방법에 관한 것이며, 바람직하게는 상기 미세입자를 리포좀 형태로 제조한다.The present invention relates to oral vaccine transport microparticles prepared by coating GFP-expressing Salmonella typhimurium cells as an antigen model and coating extracellular polysaccharides containing them, and preferably, the microparticles are prepared in the form of liposomes. .
여러 가지 미생물 다당류는 생물공학의 생성물로 널리 수용되고 있으며, 그 외의 다당류는 다양한 개발 단계에 있다. Aureobasidium pullulans는 α-D-글루칸(pullulan)을 합성한다. 이러한 플루란(pullulan)은 높은 친수성을 가지며, 내유성(oil-resistant), 수용성 필름을 형성하는데 사용한다. Agrobacterium 및 Rhizobium 종을 포함하는 여러 가지 세균들은 각각 여러 가지 세포외 다당류를 제조한다. 그 중 하나가 중성 겔을 생성하는 비교적 저분자량인 1,3-β-D-글루칸인 커들란(curdlan)이다.Many microbial polysaccharides are widely accepted as products of biotechnology, and other polysaccharides are in various stages of development. Aureobasidium pullulans synthesizes α-D-glucan. These pullulans have high hydrophilicity and are used to form oil-resistant, water-soluble films. Several bacteria, including Agrobacterium and Rhizobium species, each produce several extracellular polysaccharides. One of them is curdlan, a relatively low
본 발명에서는 상기 커들란을 리포좀에 코팅하기 위하여 1,3-β-D-글루칸을 소수성화하고, 이러한 소수성 1,3-β-D-글루칸의 유도체들을 사용하여 리포좀을 코팅하여 경구용 백신 운반용 미세입자를 제조한다.In the present invention, hydrophobization of 1,3-β-D-glucan to coat the curdlan on liposomes, and coating liposomes using derivatives of hydrophobic 1,3-β-D-glucan for oral vaccine delivery Prepare microparticles.
본 발명에 의한 1,3-β-D-글루칸의 유도체로는 커들란-설페이트(Curdlan-sulfate), O-팔미토일 커들란-설페이트(O-palmitoyl curdlan-sulfate), O-팔미토일 스클레로글루칸-설페이트(O-palmitoyl scleroglucan-sulfate), O-팔미토일 풀루란(O-palmitoyl pullulan, OPP) 등을 들 수 있다.Derivatives of 1,3-β-D- glucan according to the invention is larger them up-sulfated (Curdlan-sulfate), O-palmitoyl grow them up-sulfated (O -palmitoyl-curdlan sulfate), O-palmitoyl's Klee a glucan -, and the like palmitoyl pullulan (O -palmitoyl pullulan, OPP) - sulfate (O -palmitoyl scleroglucan-sulfate), O.
이하, 실시예를 통하여 본 발명을 보다 더 구체적으로 설명하지만, 본 발명이 이들 실시예로만 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these Examples.
[실시예 1] β-1,3-글루칸의 유도체인 커들란-설페이트(Curdlan-sulfate) 합성Example 1 Curdlan-sulfate Synthesis of β-1,3-Glucan
항산성과 항암활성을 가진 β-1,3-글루칸인 커들란(curdlan)(Wako, Osaka, Japan 에서 구입)(Takashi Yoshida, et al., Synthesis of curdlan sulfates having inhibitory effects in vitro against AIDS viruses HIV-1 and HIV-2, Carbohydrate Research, (1995) 276: 425-436)의 면역 활성을 증진시키기 위하여 설페이트가 결합된 유도체를 합성하였다. Curdlan (purchased from Wako, Osaka, Japan), β-1,3-glucan with anti-acid and anticancer activity (Takashi Yoshida, et al ., Synthesis of curdlan sulfates having inhibitory effects in vitro against AIDS viruses HIV- 1 and HIV-2, Carbohydrate Research, (1995) 276: 425-436) was synthesized sulfate bound derivatives to enhance the immune activity.
커들란 0.5g을 피리딘 40ml에 분산시킨 후 SO3-피리딘 복합체 6.0g을 첨가하여 85℃에서 2시간 동안 교반한 다음 얼음 욕조로 옮겨 냉각하였다. 상기 혼합용액에 10% NaOH 용액 50ml를 교반하면서 첨가한 후 상분리가 일어날 때까지 아세톤을 첨가하고 3차례 세척단계를 거친 다음 투석과정을 거쳐 합성을 완료하였다.0.5 g of Curdlan was dispersed in 40 ml of pyridine, 6.0 g of SO 3 -pyridine complex was added thereto, stirred at 85 ° C. for 2 hours, and then transferred to an ice bath for cooling. 50 ml of 10% NaOH solution was added to the mixed solution with stirring. Acetone was added until phase separation occurred, followed by three washing steps, followed by dialysis to complete the synthesis.
FT-IR 스펙트럼을 통해 커들란-설페이트의 합성 여부를 확인한 결과, 1259 cm-1 근처에서 S=O 결합흡수 피크를 보였고, 619 cm-1 근처에서 C-O-S 결합 흡수 피크를 나타내어 그 합성이 확인되었다.The FT-IR spectra confirmed the synthesis of curdlan-sulfate, and showed S = O binding absorption peaks near 1259 cm −1 and COS binding absorption peaks near 619 cm −1 to confirm the synthesis.
[실시예 2] O-팔미토일 커들란-설페이트(O-palmitoyl curdlan-sulfate, OPCS) 합성[Example 2] O-palmitoyl grow them up-sulfated (O -palmitoyl-curdlan sulfate, OPCS) synthesis
Sunamoto(Macromolecules 25, 5665-5670, 1992) 그룹에서 제시한 방법을 일부 수정하여 합성하였다. 먼저, 0.5g 커들란-설페이트를 11ml 디메틸포름아미드(dimethylformamide)에 용해한 후 1ml 피리딘과 0.05g 팔미토일 클로라이드(palmitoyl chloride)가 포함된 0.24ml 디메틸포름아미드 용액을 첨가하 였다. 혼합액을 60℃에서 8시간 동안 교반하면서 반응시킨 다음 1시간 동안 상온에서 냉각한 후 에탄올과 디에틸에테르를 이용하여 침출시키고 세척하여 진공 건조하였다.The method proposed by Sunamoto (Macromolecules 25 , 5665-5670, 1992) group was synthesized with some modifications. First, 0.5 g curdlan-sulfate was dissolved in 11 ml dimethylformamide, and then 0.24 ml dimethylformamide solution containing 1 ml pyridine and 0.05 g palmitoyl chloride was added. The mixture was reacted with stirring at 60 ° C. for 8 hours, cooled at room temperature for 1 hour, then leached with ethanol and diethyl ether, washed and dried in vacuo.
FT-IR 스펙트럼을 통해 O-팔미토일 커들란-설페이트의 합성 여부를 확인한 결과, 커들란-설페이트에서 약하게 나타난 2917 cm-1 근처에서 CH2CH3의 보다 강한 결합흡수 피크를 보여 그 합성이 확인되었다.The FT-IR spectra confirmed the synthesis of O -palmitoyl curdlan-sulfate, showing a stronger binding absorption peak of CH 2 CH 3 near 2917 cm -1 , which was weak in curdlan-sulfate. It became.
[실시예 3] O-팔미토일 스클레로글루칸-설페이트(O-palmitoyl scleroglucan-sulfate) 합성[Example 3] O-palmitate in weekends's Klee glucan-sulfated (O -palmitoyl scleroglucan-sulfate) synthesis
Sunamoto(Macromolecules 25, 5665-5670, 1992) 그룹에서 제시한 방법을 일부 수정하여 합성하였다. 먼저, 0.5g 스클레로글루칸-설페이트를 11ml 디메틸포름아미드에 용해한 용액에 1ml 피리딘과 0.05g 팔미토일 클로라이드가 포함된 0.24ml 디메틸포름아미드 용액을 첨가하였다. 상기 혼합액을 60℃에서 8시간 동안 교반하면서 반응시킨 다음 1시간 동안 상온에서 냉각한 후 에탄올과 디에틸에테르를 이용하여 침출시키고 세척하여 진공 건조하였다.The method proposed by Sunamoto (Macromolecules 25 , 5665-5670, 1992) group was synthesized with some modifications. First, a 0.24 ml dimethylformamide solution containing 1 ml pyridine and 0.05 g palmitoyl chloride was added to a solution of 0.5 g scleroglucan-sulfate dissolved in 11 ml dimethylformamide. The mixture was reacted with stirring at 60 ° C. for 8 hours, cooled at room temperature for 1 hour, then leached with ethanol and diethyl ether, washed and dried in vacuo.
FT-IR 스펙트럼을 통해 O-팔미토일 스클레로글루칸-설페이트의 합성 여부를 확인한 결과, 스클레로글루칸-설페이트에서 약하게 나타난 2922cm-1 근처에서 CH2CH 3의 보다 강한 결합흡수 피크를 보여 그 합성이 확인되었다.The FT-IR spectra confirmed the synthesis of O -palmitoyl scleroglucan-sulfate, showing a stronger binding absorption peak of CH 2 CH 3 near 2922 cm -1 , which is weak in scleroglucan-sulfate. Synthesis was confirmed.
[실시예 4] O-팔미토일 플루란(O-palmitoyl pullulan, OPP) 합성[Example 4] O - palmitoyl pullulan (O -palmitoyl pullulan, OPP) synthesis
플루란 0.5g을 피리딘 40ml에 분산시킨 후 SO3-피리딘 복합체 6.0g을 첨가하여 85℃에서 2시간 동안 교반한 다음 얼음 욕조로 옮겨 냉각하였다. 상기 혼합용액에 10% NaOH 용액 50ml을 교반하면서 첨가한 후 상분리가 일어날 때까지 아세톤을 첨가하고 3차례 세척단계를 거친 다음 투석과정을 거쳐 합성을 완료하였다. 0.5 g of pullulan was dispersed in 40 ml of pyridine, and 6.0 g of SO 3 -pyridine complex was added thereto, stirred at 85 ° C. for 2 hours, and then transferred to an ice bath for cooling. 50 ml of 10% NaOH solution was added to the mixed solution with stirring. Acetone was added until phase separation occurred, followed by three washing steps, followed by dialysis to complete the synthesis.
FT-IR 스펙트럼을 통해 O-팔미토일 플루란의 합성 여부를 확인한 결과, 2927 cm-1 근처에서 CH2CH3 결합흡수 피크를 보였고, 1153 cm-1 근처에서 C-O 결합 흡수 피크를 나타내어 그 합성이 확인되었다.FT-IR spectra confirmed the synthesis of O -palmitoyl flurane, and showed a CH 2 CH 3 binding absorption peak near 2927 cm -1 and a CO binding absorption peak near 1153 cm -1 Confirmed.
[실시예 5] O-팔미토일 플루란(O-palmitoyl pullulan, OPP)이 코팅된 리포좀 제조Example 5 Preparation of Liposomes Coated with O -palmitoyl Pullulan ( O- palmitoyl pullulan, OPP)
계란의 포스파티딜콜린(phosphatidylcholine, PC)과 포스파티딜에탄올아민(phosphatidylethanolamine, PE) 및 콜레스테롤(cholesterol, CH)을 7:1:2의 몰비로 클로로포름/메탄올 혼합용액 5ml에 용해시킨 후 회전식 농축증발기(rotary evaporator)를 이용하여 용매를 증발시켜서 얇은 층(thin film)을 제조한 다음 PBS 완충액을 가수하여 베시클(vesicles)을 제조하였다. 제조한 리포좀 용액에 상기 실시예 4에서 제조한 OPP를 리포좀 인지질과 7:3 (lipid:OPP molar)이 되도록 2 ml의 PBS 완충액에 용해하여 리포좀 현탁액에 첨가한 후 1시간 동안 교반하여 OPP가 코팅된 리포좀을 제조하였다.Phosphatidylcholine (PC), phosphatidylethanolamine (PE), and cholesterol (cholesterol, CH) in eggs are dissolved in 5 ml of a chloroform / methanol mixture at a molar ratio of 7: 1: 2, followed by a rotary evaporator. By evaporating the solvent to prepare a thin film (thin film) to prepare a vesicle (vesicles) by hydrolyzing the PBS buffer. In the prepared liposome solution, the OPP prepared in Example 4 was dissolved in 2 ml of PBS buffer so as to be liposome phospholipid and 7: 3 (lipid: OPP molar), added to the liposome suspension, and stirred for 1 hour to coat the OPP. Prepared liposomes.
O-팔미토일 플루란이 코팅된 리포좀은 투과형 전자현미경(Transmittance Electronic Microscopy, TEM)을 통해 형태를 관찰하였고, 동적광산란법(Dynamic Light Scattering, DLS)을 이용하여 입자의 크기를 측정하였으며, 그 결과를 도 1(b)에 나타내었다.The liposomes coated with O -palmitoyl flurane were observed by transmission electron microscopy (TEM), and the particle size was measured by dynamic light scattering (DLS). Is shown in Figure 1 (b).
① 입자의 형태: TEM을 통해 관찰한 결과 입자의 크기는 100-200 nm이었으며, 모양은 구형을 이루고 있었다. 또한, 첨가된 OPP로 인해 리포좀 표면에 얇은 막이 형성되었음을 관찰할 수 있었다.① Particle morphology: The size of particles was 100-200 nm and the shape was spherical. In addition, it was observed that a thin film was formed on the surface of the liposome due to the added OPP.
[실시예 6] O-팔미토일 커들란 설페이트(O-palmitoyl curdlan sulfate, OPCS)가 코팅된 리포좀 제조[Example 6] O - palmitoyl grow them up the coated liposomes prepared sulfate (O-palmitoyl curdlan sulfate, OPCS )
계란의 포스파티딜콜린(phosphatidylcholine, PC)과 포스파티딜에탄올아민(phosphatidylethanolamine, PE) 및 콜레스테롤(cholesterol, CH)을 일정 7:1:2의 몰비로 클로로포름/메탄올 혼합용액 5ml에 용해시킨 후 회전식 농축증발기(rotary evaporator)를 이용하여 용매를 증발시켜서 얇은 층(thin film)을 제조한 다음 PBS 완충액을 가수하여 베시클(vesicles)을 제조하였다. 제조한 리포좀 용액에 상기 실시예 2에서 제조한 OPCS를 리포좀 인지질과 7:3(lipid:OPCS molar)이 되도록 2 ml의 PBS 완충액에 용해하여 리포좀 현탁액에 첨가한 다음 1시간 동안 교반하여 OPCS가 코팅된 리포좀을 제조하였다. Phosphatidylcholine (PC), phosphatidylethanolamine (PE), and cholesterol (cholesterol, CH) in eggs were dissolved in 5 ml of a chloroform / methanol mixture at a molar ratio of 7: 1: 2, followed by a rotary evaporator. The thin film was prepared by evaporation of the solvent, followed by hydrolysis of the PBS buffer to prepare vesicles. In the prepared liposome solution, the OPCS prepared in Example 2 was dissolved in 2 ml of PBS buffer so as to be liposome phospholipid and 7: 3 (lipid: OPCS molar), added to the liposome suspension, and stirred for 1 hour to coat the OPCS. Prepared liposomes.
제조한 O-팔미토일 커들란 설페이트가 코팅된 리포좀은 투과형 전자현미경을 통해 형태를 관찰하였고, 동적광산란법(Dynamic Light Scattering, DLS)을 이용하여 입자의 크기를 측정하였으며, 그 결과를 도 1(c)에 나타내었다.The prepared O -palmitoyl curdlan sulfate-coated liposomes were observed through a transmission electron microscope, and the particle size was measured using a dynamic light scattering method (DLS). c).
① 입자의 형태: TEM을 통해 관찰한 결과 입자의 크기는 100-200 nm이었으며, 모양은 구형을 이루고 있었다. 또한, 첨가된 OPCS로 인해 리포좀 표면에 얇은 막이 형성되었음을 관찰할 수 있었다.① Particle morphology: The size of particles was 100-200 nm and the shape was spherical. In addition, it was observed that a thin film was formed on the surface of the liposome due to the added OPCS.
[시험예 1] OPCS가 코팅된 리포좀의 특성 및 안정성 확인[Test Example 1] Check the properties and stability of the OPCS-coated liposomes
투과형 전자현미경을 통해 상기 실시예 6의 방법으로 제조한 OPCS가 코팅된 리포좀의 형태를 관찰하였고, 동적광산란법을 이용하여 입자의 크기를 측정하였으며, 제타 전위(zeta potential) 측정을 통해 OPCS의 도입 여부를 판단하였고, 인공위액에서의 안정성을 조사하였으며, 그 결과는 아래와 같다:The type of the OPCS-coated liposome prepared by the method of Example 6 was observed through a transmission electron microscope, the particle size was measured by dynamic light scattering, and the introduction of the OPCS through the measurement of zeta potential. The results were evaluated and their stability in gastric juice was investigated. The results are as follows:
① 입자의 형태: 투과형 전자현미경을 통해 관찰한 결과 입자의 크기는 100-200nm 이었으며, 모양은 구형을 이루고 있었다. ① Particle shape: The size of particles was 100-200nm, and the shape was spherical when observed through transmission electron microscope.
도 2는 지질과 OPCS의 혼합비율을 달리하여 OPCS의 함량에 따른 리포좀 표면의 변화를 관찰한 TEM 사진이며, 첨가된 OPCS의 양이 증가할수록 리포좀 표면의 코팅 부분이 확실하게 관찰되었다.2 is a TEM photograph of the change of the liposome surface according to the content of the OPCS by varying the mixing ratio of lipid and OPCS, the coating portion of the surface of the liposome was reliably observed as the amount of the added OPCS increases.
② 입자의 크기: 도 3에서는 지질과 OPCS의 혼합비율을 달리하여 제조한 OPCS가 코팅된 리포좀의 입자 크기를 DLS를 이용하여 측정하였으며, OPCS로 코팅하지 않은 리포좀의 입자크기는 평균 120nm인 반면에, OPCS를 첨가한 리포좀의 입자크기는 OPCS의 첨가량에 따라 증가하고 있음을 보여준다. 그러나 지질과 OPCS의 비 율이 7:3을 넘을 경우 입자의 크기의 변화는 적었다.② Particle size: In FIG. 3, the particle size of the OPCS-coated liposomes prepared by varying the mixing ratio of lipids and OPCS was measured by using DLS, whereas the particle size of the liposomes not coated with OPCS was 120 nm on average. The particle size of the liposome added with OPCS increases with increasing amount of OPCS. However, when the ratio of lipid to OPCS exceeds 7: 3, the particle size change was small.
③ 제타 전위 측정: 도 4에서는 지질과 OPCS의 혼합비율을 달리하여 제조한 OPCS가 코팅된 리포좀의 제타 전위 값을 보여주고 있으며, OPCS의 첨가량이 증가함에 따라 제타 전위 값은 감소하였다. 이는 OPCS의 전하가 음전하를 가지고 있어 리포좀에 도입되어 제타 전위 값을 낮추어 주기 때문이다. 따라서, 이 결과는 투과형 전자현미경으로 관찰한 결과를 뒷받침해 주는 것으로서 OPCS가 리포좀을 코팅하였음을 확인할 수 있었다.③ Zeta potential measurement: Figure 4 shows the zeta potential value of the OPCS-coated liposome prepared by varying the mixing ratio of lipid and OPCS, the zeta potential value was decreased with the addition amount of OPCS. This is because the charge of the OPCS has a negative charge and is introduced into the liposome to lower the zeta potential value. Therefore, these results support the results observed with a transmission electron microscope, confirming that the OPCS coated liposomes.
④ SGF(Stimulated Gastric Fluid)에서의 안정성 측정: OPCS가 코팅된 리포좀을 OPCS의 첨가량을 달리하여 제조한 후 SGF에 분산시킨 다음 5시간 후에 입자의 크기 변화를 측정함으로써 안정성을 평가하였으며, 그 결과를 도 5에 나타내었다. 그 결과 OPCS의 첨가량이 증가함에 따라 입자의 크기 변화가 적었다. 이는 OPCS에 의해 항산성이 리포좀에 부가되었고, 그 정도가 OPCS에 첨가량에 따라 향상되었음을 의미한다.④ Stability measurement in SGF (Stimulated Gastric Fluid): The OPCS-coated liposomes were prepared by varying the amount of OPCS added, dispersed in SGF, and then evaluated for stability by measuring the change in particle size after 5 hours. 5 is shown. As a result, the change in particle size was small as the amount of OPCS added increased. This means that the acidity was added to the liposomes by OPCS, and the extent was improved according to the amount added to the OPCS.
⑤ 투과성(Permeability) 측정: 리포좀에 5-carboxyflourescien(CF)를 로딩(loading)한 다음 OPCS(lipid:OPCS = 7:3)를 첨가하여 반응시킴으로써 CF를 로딩한 OPCS 코팅된 리포좀을 제조하고 PBS 완충액에서 CF의 방출 경향을 측정하였으며, 그 결과를 도 6에 나타내었다. 그 결과, OPCS를 첨가하지 않은 리포좀으로부터 CF는 5시간 후에 90% 이상 방출된 반면 OPCS가 코팅된 리포좀은 5시간 후에 40% 적은 양의 CF를 방출하였다. 이는 OPCS의 첨가로 인해 리포좀의 안정성을 보다 증가시켰음을 의미한다.⑤ Permeability measurement: A CF loaded OPCS coated liposome was prepared by loading 5-carboxyflourescien (CF) into liposomes and then reacting with the addition of OPCS (lipid: OPCS = 7: 3) and preparing PCS buffer The release trend of CF was measured at, and the results are shown in FIG. 6. As a result, CF was released more than 90% after 5 hours from the liposome without the OPCS while the OPCS-coated liposome released 40% less CF after 5 hours. This means that the addition of OPCS further increased the stability of the liposomes.
[시험예 2] 합성 미세입자/리포좀의 장관내 표적전달Test Example 2 Intestinal Target Delivery of Synthetic Microparticles / Liposomes
1) 녹색형광 단백질(Green Fluorescence Protein, GFP) 양성 S. typhimurium (ST)의 제조1) Preparation of Green Fluorescence Protein (GFP) positive S. typhimurium (ST)
먼저, 클론한 CAT(Chloramphenicol Acetyl Transferase; CAT) 유전자를 pEGFP(clonetech, USA)의 EGFP 유전자 하류에 위치하는 멀티플 클로닝 부위내의 EcoRI 및 Spe1에 삽입하여 pEGFP-CAT 구조물을 제조하였다. CAT 유전자는 pKD3를 템플레이트로 하여 서열번호 1(5'- GGA ATT CGT GTA GGC TGG AGC TGC TTC -3') 및 서열번호 2(5'- GAC TAG TCC TAC CTG TGA CGG AAG ATC -3')의 프라이머를 사용하여 PCR 반응을 거쳐 증폭하여 얻었다. 이때, PCR 반응은 94℃에서 5분간 초기변성한 후, 94℃에서 30초간 변성, 58℃에서 30초간 어닐링, 72℃에서 1분간 확장하는 과정을 30 사이클로 반복한 다음, 마지막으로 72℃에서 7분간 반응시켰다.First, the cloned CAT (Chloramphenicol Acetyl Transferase; CAT) gene was inserted into EcoRI and Spe1 in a multiple cloning site located downstream of the EGFP gene of pEGFP (clonetech, USA) to prepare a pEGFP-CAT construct. The CAT gene is composed of pKD3 as a template for SEQ ID NO: 1 (5'- GGA ATT CGT GTA GGC TGG AGC TGC TTC-3 ') and SEQ ID NO: 2 (5'- GAC TAG TCC TAC CTG TGA CGG AAG ATC -3'). The primers were amplified by PCR reaction. At this time, the PCR reaction was initially denatured at 94 ° C. for 5 minutes, denatured at 94 ° C. for 30 seconds, annealed at 58 ° C. for 30 seconds, and extended for 1 minute at 72 ° C. in 30 cycles, and finally, at 7 ° C. at 7 ° C. The reaction was carried out for a minute.
Linear DNA transformation method(Detsanko and Wanner, 2000)를 이용하여 S. typhimurium 염색체내에 융합 구조물(fusion construct)을 삽입하고 전기이동(electrophoration)에 의하여 플라스미드(XXX)를 함유한 S. typhimurium를 형질전환시켰다. lacZP::EGFP::CAT 구조물(SHJ2033)을 갖고 있는 균주를 최종적으로 서열번호 3(5'- GGC CGA TTC ATT AAT GCA GC -3') 및 서열번호 4(5'- GCA TGT GTC AGA GGT TTT CGC C -3')의 프라이머를 사용하여 PCR 반응을 거쳐 증폭하였고, DNA sequencing 에 의하여 확정하였으며, 상기 S. typhimurium 염색체의 단면도를 도 7에 도시하였다. 이때, PCR 반응은 94℃에서 5분간 초기변성한 후, 94℃에서 30 초간 변성, 58℃에서 30초간 어닐링, 72℃에서 1분간 확장하는 과정을 30 사이클로 반복한 다음, 마지막으로 72℃에서 7분간 반응시켰다.A fusion construct was inserted into the S. typhimurium chromosome using the linear DNA transformation method (Detsanko and Wanner, 2000) and transformed S. typhimurium containing plasmid (XXX) by electrophoresis. The strain carrying the lacZP :: EGFP :: CAT construct (SHJ2033) was finally isolated from SEQ ID NO: 3 (5'- GGC CGA TTC ATT AAT GCA GC -3 ') and SEQ ID NO: 4 (5'- GCA TGT GTC AGA GGT TTT Amplification was carried out by PCR using a primer of CGC C-3 ′), confirmed by DNA sequencing, and a cross-sectional view of the S. typhimurium chromosome is shown in FIG. 7. In this case, the PCR reaction was initially denatured at 94 ° C. for 5 minutes, then denatured at 94 ° C. for 30 seconds, annealed at 58 ° C. for 30 seconds, and extended for 1 minute at 72 ° C. in 30 cycles. The reaction was carried out for a minute.
2) 항원이 포함된 미세입자의 점막성 기관(mucosal organs)으로의 전달시험2) Delivery test of microparticles containing antigen to mucosal organs
효과적인 항원의 운반을 위해서는 위의 강한 산을 통과하여 소장에 위치한 Peyer's patch와 장간막 림프절에 도달하여야 한다. 이를 확인하기 위하여 GFP+ ST를 고정한 후(2% 파라포름알데히드(paraformaldehyde))(도 8 참조) 이를 리포좀에 포집시킨 다음 이 리포좀을 각각 실시예 4의 O-팔미토일플루란(OPP)(실시예 5의 방법으로 제조), 실시예 2의 O-팔미토일커들란 설페이트(OPCS)(실시예 6의 방법으로 제조) 및 플루란 아세테이트(pullulan acetate, PA)로 제조한 리포좀을 다중초점 현미경(confocal microscope)으로 관찰하였으며, 그 결과를 도 9에 나타내었다.To carry an effective antigen, it must pass through the strong acid in the stomach to reach the Peyer's patch in the small intestine and the mesenteric lymph nodes. In order to confirm this, after fixing GFP + ST (2% paraformaldehyde) (see FIG. 8), it was collected in liposomes, and then the liposomes were prepared by O -palmitoylflurane (OPP) of Example 4, respectively. Prepared by the method of Example 5), O -palmitoylkerlan sulfate (OPCS) of Example 2 (prepared by the method of Example 6) and liposomes prepared by pullulan acetate (PA) were subjected to multifocal microscopy ( observation with a confocal microscope, and the results are shown in FIG. 9.
상기 플루란 아세테이트를 이용하여 미세입자를 제조하는 과정은 다음과 같다: 플루란 아세테이트 30mg을 5ml의 디클로로메탄(dichloromethane, DCM)에 용해하고, 여기에 GFP+ ST를 일정량 첨가한 다음 PVA 10%(w/v) 수용액에 천천히 부은 후 교반하면서 DCM을 충분히 증발시켜 미세입자를 제조하였다.The procedure for preparing the microparticles using the furan acetate is as follows: 30 mg of the furan acetate is dissolved in 5 ml of dichloromethane (DCM), and a predetermined amount of GFP + ST is added thereto, followed by
그 결과, 상기 미세입자들은 대체로 1-3 ㎛의 직경을 보였고, 도 10에서 보여지는 바와 같이, 플루란 아세테이트(pullulan acetate)의 경우 입자 크기가 가장 크게 나타났다.As a result, the microparticles generally had a diameter of 1-3 μm, and as shown in FIG. 10, the largest particle size was shown in the case of pullulan acetate.
상기 미세입자를 구강으로 투여하고 24시간이 경과한 후 현광 현미경 또는 다중초점 현미경을 사용하여 Peyer's patch와 장간막 림프절에서 GFP+ 세균을 관찰하였으며, 그 결과를 도 11에 나타내었다. Peyer's patch 또는 장간막 림프절로 운반되는 GFP+ ST를 포함한 미세입자 중 OPCS가 코팅된 미세입자의 운반이 유의하게 증가하였다. 이로 인해, OPCS를 사용하여 미세입자를 만드는 경우 항원이 강 산성 환경의 위를 안정적으로 통과하여 점막성 기관에 효과적으로 운반할 수 있음을 알 수 있었다. After 24 hours after oral administration of the microparticles, GFP + bacteria were observed in Peyer's patch and mesenteric lymph nodes using a fluorescence microscope or a multifocal microscope. The results are shown in FIG. 11. The transport of OPCS-coated microparticles was significantly increased in microparticles including GFP + ST delivered to Peyer's patch or mesenteric lymph nodes. For this reason, it was found that when OPCS is used to make microparticles, antigens can stably pass through the highly acidic environment and effectively transport to mucosal organs.
[시험예 2] 균체담지 미세입자 투여후 면역반응 조사Test Example 2 Investigation of Immune Response after Administration of Cell Support Microparticles
1) 면역원-미세입자/리포좀을 이용한 면역 1) Immunization using immunogen-microparticles / liposomes
각각의 면역원-미세입자/리포좀을 0.25ml의 용적을 사용하였다. 항원의 조성 및 투여 경로에 따라 마우스를 각각 실험군으로 구별하고 각 군은 10 - 12 마리로 하였다. 경구투여는 가는 비닐튜브나 경구실험 전용 곡선 니들(curved needle)을 사용하였다. 최종투여 7일 후에 마우스로부터 비장과 혈액을 채취하여 실험에 사용하였다. 실험동물로는 생후 6 - 8 주된 정상 BALB/c 암컷 마우스를 사용하였다. 실험중 모든 마우스들은 크기가 18cm × 28cm인 cage당 6마리 이하를 넣어 항온 항습 및 무균 상태로 유지되는 동물사육시설에서 사육하고, 수돗물과 시판되는 실험동물 사료를 공급하였다.Each immunogen-microparticle / liposome was used at a volume of 0.25 ml. Mice were divided into experimental groups according to the composition of the antigen and the route of administration, and each group was 10-12 mice. For oral administration, thin vinyl tubes or curved needles for oral experiments were used. Seven days after the final administration, spleen and blood were collected from mice and used for the experiment. As experimental animals, normal BALB / c
2) 항체 검사 2) antibody test
면역혈청과 비장세포 배양액, 그리고 타액 내에서 항원-특이 항체 IgG, IgM, IgA와 모든 sIgA를 검출하였다. 즉, 항원과 anti-mouse IgA를 카보네이트 완충액(carbonate buffer)에 각각 5㎕/ml의 농도로 용해한 후 96-well 배양판에 50㎕씩 분주하여 실온에서 1주야 두어 부착시키고 일정배율로 희석한 시료를 4℃에서 1주야 반응시켰다. 그런 다음 항원에 부착된 항체를 퍼옥시다아제(peroxidase)가 부착된 항-마우스 IgA(1:1000 희석액)를 반응시킨 후, 효소색소기질인 OPD(orthophenylenediamine)과 반응시키고 5N 황산액으로 정지시켜 최종적으로 생산된 색소를 490nm 파장하의 ELISA 판독기(Molecular Devices Inc.)를 사용하여 판독하였다.Antigen-specific antibodies IgG, IgM, IgA and all sIgA were detected in immune serum, splenocyte culture and saliva. In other words, the antigen and anti-mouse IgA were dissolved in carbonate buffer at a concentration of 5 μl / ml, and then 50 μl of the mixture was placed in a 96-well culture plate. Was reacted overnight at 4 ° C. Then, the antibody attached to the antigen is reacted with anti-mouse IgA (1: 1000 dilution) attached with peroxidase, and then reacted with the enzyme pigment substrate OPD (orthophenylenediamine) and stopped with 5N sulfate solution. The resulting pigment was read using an ELISA reader (Molecular Devices Inc.) at 490 nm wavelength.
< 결과 ><Result>
a) 혈청내 항체생산능력: 구강 면역에 대한 항체 반응을 측정하기 위하여 다앙한 종류의 미세입자를 2주에 한번씩 2회 1011의 미세입자를 구강으로 투여한 다음 최종 투여 1주일후 혈청을 얻어서 ELISA 검사를 이용하여 혈청내 항체가를 측정하였으며, 그 결과를 도 12에 나타내었다. ST 항원에 대한 IgG와 IgA 항체는 모든 면역 동물에서 측정되었다. OPCS가 코팅된 ST를 포함한 리포좀은 IgG와 IgA 역가가 ST를 포장한 리포좀이나 OPP가 코팅된 ST 포함 리포좀이나 ST 단독 투여에 비하여 유의하게 증가되었다.a) Serum antibody production capacity: To measure antibody response to oral immunity, various types of microparticles were administered twice a week for 10 11 microparticles orally and then serum was obtained one week after the final administration. The antibody titer in serum was measured using an ELISA test, and the results are shown in FIG. 12. IgG and IgA antibodies against ST antigens were measured in all immune animals. Liposomes containing OPCS-coated STs were significantly increased in IgG and IgA titers compared to liposomes packed with STs or those containing OPP-coated STs or ST alone.
b) 분비형 항체 (sIgA) 생산능력: 점액성 면역의 가장 큰 특징은 분비되는 IgA이다. 분비되는 IgA는 상피세포의 감염을 예방 할 수 있고 상피 세포를 통과한 항원에 대해서도 제거하는 기능을 가지고 있다. 구강 면역에 대한 점액성 면역반응을 측정하기 위하여 상기와 같은 방법으로 각 미세입자를 구강으로 투여한 다음 최종 투여 1주일 후 대변을 얻어 대변내 sIgA를 ELISA 검사를 이용하여 측정하였으며, 그 결과를 도 13에 나타내었다. 역시 OPCS가 코팅된 ST를 포함한 리포좀은 다른 미세입자의 투여군들에 비하여 유의하게 증가되었다.b) Secretory antibody (sIgA) production capacity: The most characteristic of mucin immunity is secreted IgA. The secreted IgA can prevent epithelial infection and remove the antigen that has passed through the epithelial cell. In order to measure the mucosal immune response to oral immunity, the microparticles were orally administered in the same manner as described above, and one stool was obtained one week after the final administration. The sIgA in the stool was measured by using an ELISA test. It is shown in 13. Liposomes, including OPCS-coated STs, were also significantly increased as compared to the administration groups of other microparticles.
[시험예 3] 면역후 살모넬라균 감염에 대한 저항성 조사Test Example 3 Investigation of Resistance to Salmonella Infection After Immunization
백신으로서의 효과를 측정하기 위하여 2주에 한번씩 2회 1011의 다양한 미세입자를 구강으로 투여한 후 5×105의 살아있는 살모넬라(SHJ2033) 균을 구강으로 투여하고 7일 후 Peyer's patch와 비장에서 세균의 수를 측정하였으며, 그 결과를 도 14에 나타내었다. OPCS로 포장된 미세입자를 처치 받은 동물 군의 Peyer's patch, 장간막 림프절 그리고 비장에서 ST의 증식이 유의하게 억제되었다. 이 결과로, 실시예 2에서 제조한 OPCS가 코팅된 리보좀이 백신 운반 체계로 매우 효과적임을 알 수 있었다.In order to measure the effect as a vaccine, oral administration of various microparticles of 10 11 twice a week, followed by 5 × 10 5 live Salmonella (SHJ2033) bacteria orally and 7 days later, Peyer's patch and spleen The number of was measured and the result is shown in FIG. The proliferation of ST was significantly inhibited in Peyer's patches, mesenteric lymph nodes and spleen of animals treated with OPCS-packed microparticles. As a result, it was found that the OPCS-coated ribosomes prepared in Example 2 were very effective as a vaccine delivery system.
상기에서 살펴본 바와 같이, 본 발명에서는 아그로박테리움 속에서 생산하는 세포외 다당류를 이용하여 항산성, 점막접착성 및 면역 증진성이 우수한 경구용 백신 운반용 미세입자를 제조할 수 있었다. As described above, in the present invention, by using extracellular polysaccharides produced in Agrobacterium, oral vaccine transport microparticles having excellent anti-acid, mucoadhesive and immune enhancing properties could be prepared.
▣ 참고문헌
▣ Reference
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