TWI794608B - Cellulose nanofibers with antibacterial activity against periodontal pathogens prepared by loading k-carrageenan oligosaccharides with surfactants and its preparation method - Google Patents

Cellulose nanofibers with antibacterial activity against periodontal pathogens prepared by loading k-carrageenan oligosaccharides with surfactants and its preparation method Download PDF

Info

Publication number
TWI794608B
TWI794608B TW109118634A TW109118634A TWI794608B TW I794608 B TWI794608 B TW I794608B TW 109118634 A TW109118634 A TW 109118634A TW 109118634 A TW109118634 A TW 109118634A TW I794608 B TWI794608 B TW I794608B
Authority
TW
Taiwan
Prior art keywords
cnf
mixed solution
preparation
cellulose
antibacterial activity
Prior art date
Application number
TW109118634A
Other languages
Chinese (zh)
Other versions
TW202146002A (en
Inventor
龔瑞林
安心亞 強生
繁平 孔
Original Assignee
龔瑞林
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 龔瑞林 filed Critical 龔瑞林
Priority to TW109118634A priority Critical patent/TWI794608B/en
Publication of TW202146002A publication Critical patent/TW202146002A/en
Application granted granted Critical
Publication of TWI794608B publication Critical patent/TWI794608B/en

Links

Images

Abstract

本發明係一種利用表面活性劑加載k-卡拉膠低聚醣製成對牙周病原體具抗菌活性之纖維素奈米纖維及其製法，該製法係對大豆豆漿渣中之纖維素進行再利用，先將其製成纖維素奈米纖維(Cellulose Nanofiber，簡稱CNF)，再進一步使用κ-卡拉膠低聚醣(ĸ - carrageenan oligosaccharides，簡稱CO)對CNF進行修飾，而製成本發明具抗菌活性之纖維素奈米纖維(以下簡稱CO-CNF)，以有效改善CNF之降解溫度、結晶度、溶脹性及生物相容性…等特性，且令其得以被應用於製作治療牙周炎所使用之奈米纖維膜。The present invention is a cellulose nanofiber with antibacterial activity against periodontal pathogens made by loading k-carrageenan oligosaccharides with a surfactant and a preparation method thereof. First make it into cellulose nanofiber (Cellulose Nanofiber, referred to as CNF), and then further use κ-carrageenan oligosaccharides (hereinafter referred to as CO) to modify CNF, and make the antibacterial activity of the present invention Cellulose nanofibers (hereinafter referred to as CO-CNF), to effectively improve the degradation temperature, crystallinity, swelling and biocompatibility of CNF, and make it be used in the production of periodontitis nanofiber membrane.

Description

利用表面活性劑加載k-卡拉膠低聚醣製成對牙周病原體具抗菌活性之纖維素奈米纖維及其製法Cellulose nanofibers with antibacterial activity against periodontal pathogens prepared by loading k-carrageenan oligosaccharides with surfactants and its preparation method

本發明係關於一種纖維素奈米纖維(Cellulose Nanofiber，簡稱CNF)之製法，尤指一種利用大豆豆漿渣(如：農業或食品加工業之廢棄物)中之纖維素，先萃取出纖維素奈米纖維(Cellulose Nanofiber，簡稱CNF)，再進一步使用κ-卡拉膠低聚醣(ĸ - carrageenan oligosaccharides，簡稱CO)對CNF進行修飾，而製作出本發明具抗菌活性之纖維素奈米纖維(以下簡稱CO-CNF)，以有效改善CNF之降解溫度、結晶度、溶脹性及生物相容性…等材料特性，從而令本發明之CO-CNF得以被應用於製作出治療牙周炎所使用之奈米牙周再生纖維膜。由於，牙周炎係屬於一種與微生物堆積有關之慢性炎症性疾病，而本發明之CO-CNF既經實驗證實確具對牙周病原體之抗菌活性，據此，它不僅能用以有效預防或改善牙周病症狀，尚能充分實現有效給藥之目的，從而在牙周炎之臨床治療過程中，對有效且精準地遞送藥物有著極大之潛力，而能大幅提升牙周炎之治療效果及效率，從而在牙齒保健及醫療產業中具有極高之利用價值。The present invention relates to a method for preparing cellulose nanofiber (Cellulose Nanofiber, referred to as CNF), especially a method for extracting cellulose nanofiber by using cellulose in soybean dregs (such as waste from agriculture or food processing industry). Rice fiber (Cellulose Nanofiber, referred to as CNF), and further use κ-carrageenan oligosaccharides (ĸ-carrageenan oligosaccharides, referred to as CO) to modify CNF, and produce the cellulose nanofiber with antibacterial activity of the present invention (hereinafter CO-CNF for short), in order to effectively improve the material properties of CNF such as degradation temperature, crystallinity, swelling and biocompatibility, etc., so that the CO-CNF of the present invention can be used to make periodontitis Nano periodontal regeneration fiber membrane. Because periodontitis is a chronic inflammatory disease related to microbial accumulation, and the CO-CNF of the present invention has been proved by experiments to have antibacterial activity against periodontal pathogens, accordingly, it can not only be used for effective prevention or Improving the symptoms of periodontal disease can still fully realize the purpose of effective drug delivery. Therefore, in the clinical treatment of periodontitis, it has great potential for effective and precise delivery of drugs, and can greatly improve the treatment effect and efficiency of periodontitis. It has extremely high utilization value in dental health care and medical industry.

按，在每個人之一生中一般均會擁有兩組牙齒；其中，第一組被稱之為「乳齒」（乳牙或奶齒），「乳齒」會在嬰兒出生約6個月後出現，兒童期間共計會長出20隻上下兩排平均分配之「乳齒」；俟4至13歲之青少年期間，將會陸續長出一組永久性的牙齒，稱為「恆齒」(或恆牙)，「恆齒」係形成在「乳齒」底下，以將「乳齒」推出頜骨後取而代之。新的一組「恆齒」共計有32隻，相應長大了的頜骨而加大。「恆齒」長出後，位於上下兩排牙齒兩端的4顆牙齒(即，所謂的第三大臼齒，或俗稱「智慧齒」)常會因頜骨位置不夠而不會長出，或在某些個案中根本不存在「智慧齒」，但是，一般人最多僅缺少其中一隻。此外，由於絕大部份人(無論白種人或黃種人)的頜骨均不夠大，故為了防止「智慧齒」因藏污納垢而造成蛀牙及發炎…等問題，及避免發生因「智慧齒」強行長出而迫使整副齒列變形而造成齒列不整齊的問題，許多牙醫師都會建議將「智慧齒」拔掉。雖然，乍看之下，牙齒頗類似於一般的骨骼，但是，事實上，牙齒並非一般骨頭，因為它不會生產紅血球，也沒有連接頜骨的關節，且遠較一般骨頭能承受更多且更大的磨損；倘若牙齒係一般的骨骼，那麼它將會因經常使用而極快地被磨平。By the way, everyone generally has two sets of teeth in their lifetime; among them, the first set is called "deciduous teeth" (deciduous teeth or milk teeth), and "deciduous teeth" will appear about 6 months after the baby is born. During this period, a total of 20 "deciduous teeth" that are evenly distributed in the upper and lower rows will grow; when the adolescents are between 4 and 13 years old, a set of permanent teeth will gradually grow, called "permanent teeth" (or permanent teeth), " The "permanent teeth" are formed under the "baby teeth" to replace the "baby teeth" after they are pushed out of the jawbone. The new set of "permanent teeth" has a total of 32, enlarged to correspond to the enlarged jaw. After the "permanent teeth" grow, the four teeth located at the two ends of the upper and lower rows of teeth (that is, the so-called third molars, or commonly known as "wisdom teeth") often do not grow due to insufficient jaw position, or in some There is no "wisdom tooth" in the case, but most people only lack one of them. In addition, since the jaws of most people (regardless of Caucasian or yellow race) are not large enough, in order to prevent "wisdom teeth" from causing tooth decay and inflammation due to dirt, and to avoid the occurrence of "wisdom teeth" Many dentists will recommend the extraction of "wisdom teeth". Although, at first glance, teeth are quite similar to ordinary bones, but in fact, teeth are not ordinary bones because they do not produce red blood cells, do not have joints connecting jaws, and can bear much more and more damage than ordinary bones. Greater wear and tear; if the tooth were ordinary bone, it would wear down extremely quickly with regular use.

一般言，請參閱第1圖所示，人類牙齒40構造主要包括齒冠41及齒根42兩部份，當人們張開嘴巴時，露出在牙齦43(包括牙肉及韌帶)外的牙齒40，只不過是其齒冠41的部份而已，齒冠41的部份係由最外層之琺瑯質(又稱牙釉質)411、中層之象牙質(又稱牙本質)412及最內層之齒髓組織413(又稱牙髓)共同組成，其中，琺瑯質411的厚度雖僅約1毫米，但其係由鈣質及磷肥質構成人體最堅硬的組織，若以摩氏硬度表(Mohs Hardness Scale)來表示，其硬度高達7～8度，雖略遜鑽石(摩氏硬度10度)一籌，但幾乎與水晶、黃玉硬度相當；象牙質412乃構成牙齒的主要身軀；齒髓組織413則為牙齒40的核心，包含神經線和血管；齒根42則係齒冠41的向下延伸而埋藏在牙齦43內且固定在顎骨(牙槽骨或齒槽骨)44上之部份。Generally speaking, please refer to Figure 1, the structure of human teeth 40 mainly includes two parts: the crown 41 and the root 42. When people open their mouths, the teeth 40 exposed outside the gums 43 (including gums and ligaments) , is only the part of its crown 41, the part of the crown 41 is composed of the outermost layer of enamel (also known as tooth enamel) 411, the middle layer of dentine (also known as dentin) 412 and the innermost layer of tooth Pulp tissue 413 (also known as dental pulp), among which, although the thickness of enamel 411 is only about 1 mm, it is composed of calcium and phosphate fertilizers to form the hardest tissue in the human body. If measured by Mohs Hardness Scale ) to indicate that its hardness is as high as 7 to 8 degrees, although it is slightly inferior to diamonds (10 degrees on the Mohs scale), but almost as hard as crystal and topaz; ivory 412 is the main body of teeth; pulp tissue 413 is It is the core of the tooth 40, including nerves and blood vessels; the root 42 is the part that extends downward from the crown 41 and is buried in the gum 43 and fixed on the jawbone (alveolar bone or alveolar bone) 44 .

按，牙周病係由黏附在牙齒表面的牙菌膜所引起之一種口腔疾病。請參閱第2圖所示，若人們口腔衛生習慣欠佳，牙菌膜50就會長期積聚在齒冠41底緣鄰近牙齦43部位的表面。牙菌膜50裏之細菌會分泌毒素，刺激牙齒40周圍的組織(如：牙齦43、牙周膜420及牙槽骨44…等)，而引致牙周病；請參閱第3圖所示，如果牙菌膜50積聚，牙齦43鄰近齒冠41的邊緣部位就會出現發炎症狀52，而造成輕微的牙周病（即，牙齦炎）；此時，若仍未對牙齒40施以更多的注意，且未給予更徹底的清潔，則齒冠41底緣鄰近牙齦43的部位及相鄰牙齒40間的部位53就會因長期積聚牙菌膜50，導致牙菌膜50內之細菌持續分泌毒素進一步刺激牙齦43，而引發牙齦43發炎症狀52；另，請參閱第4圖所示，牙菌膜50亦會被唾液（口水）鈣化，形成牙結石54，由於牙結石54的表面十分粗糙，因此導致更多牙菌膜50積聚，如此，惡性循環，將令牙齦43持續發炎，甚至有機會惡化成牙周病；嗣，若身體抵抗力下降，身體組織癒合能力欠佳，牙周組織包括牙齦43、牙周膜420及牙槽骨44等就會遭受破壞，而令情況惡化，形成嚴重的牙周病（即，牙周炎）；最後，復請參閱第4圖所示，若仍然沒未對口腔施予良好的護理診療，發炎的情況勢必持續惡化，導致原本緊附於牙根42表面的牙齦43與牙根42分離，而形成一個被稱為「牙周袋」的空間56。這空間56更易讓食物殘渣、牙菌膜50及牙結石54堆積於其中，而使牙周組織持續遭到破壞，最終導致牙周炎，此時，牙齦43邊緣甚至可能滲出膿液，長出牙瘡，牙齦43及牙槽骨44亦會隨之慢慢萎縮，導致部分牙根42外露，大幅增加了牙根42被蛀壞的機會。如果牙周炎繼續惡化，牙周組織包括牙齦43、牙周膜420及牙槽骨44將會受到嚴重破壞，導致牙槽骨44萎縮58，令牙齒40失去支撐，而變得鬆動及出現移位，最後，甚至會發生牙齒40脫落的結果。According to, periodontal disease is an oral disease caused by dental plaque adhered to the tooth surface. Please refer to FIG. 2 , if people have poor oral hygiene habits, dental plaque 50 will accumulate on the surface of the bottom edge of the crown 41 adjacent to the gum 43 for a long time. Bacteria in the dental plaque 50 will secrete toxins and stimulate the tissues around the teeth 40 (such as: gums 43, periodontal ligament 420, and alveolar bone 44, etc.), and cause periodontal disease; please refer to Figure 3, If dental plaque 50 accumulates, inflammation symptoms 52 will appear on the edge of the gum 43 adjacent to the crown 41, resulting in mild periodontal disease (i.e., gingivitis); Attention, and not give more thorough cleaning, then the part of the bottom edge of the crown 41 adjacent to the gum 43 and the part 53 between the adjacent teeth 40 will accumulate dental plaque 50 for a long time, causing the bacteria in the dental plaque 50 to persist. Secretion of toxins further stimulates the gums 43, causing inflammation symptoms 52 of the gums 43; in addition, please refer to Figure 4, the dental plaque 50 will also be calcified by saliva (saliva) to form dental calculus 54, because the surface of the dental calculus 54 is very Roughness, thus leading to more accumulation of dental plaque 50, in this way, a vicious cycle, will make the gums 43 continue to inflame, and even have the opportunity to deteriorate into periodontal disease; secondly, if the body's resistance decreases, the healing ability of the body tissue is not good, and the periodontal tissue Including gingiva 43, periodontal ligament 420 and alveolar bone 44, etc. will be damaged, and the situation will be aggravated, forming severe periodontal disease (that is, periodontitis); finally, please refer to Figure 4 again, if If good care and treatment are not given to the oral cavity, the inflammation will continue to worsen, causing the gingiva 43 that was originally attached to the surface of the root 42 to separate from the root 42, forming a space 56 called "periodontal pocket". This space 56 is more likely to allow food residues, dental plaque 50 and dental calculus 54 to accumulate in it, causing the periodontal tissue to be continuously damaged, eventually leading to periodontitis. With dental ulcers, the gums 43 and the alveolar bone 44 will also slowly shrink, resulting in the exposure of part of the tooth root 42, greatly increasing the chance of the tooth root 42 being decayed. If the periodontitis continues to worsen, the periodontal tissue including the gum 43, the periodontal ligament 420 and the alveolar bone 44 will be severely damaged, causing the alveolar bone 44 to atrophy 58, and the teeth 40 will lose their support and become loose and move. Bit, finally, can even occur as a result of 40 loss of teeth.

查，牙周炎已被醫學界定論為口腔健康的“頭號殺手”，也被稱為繼癌症、心腦血管疾病之後，威脅人類身體健康的第三大殺手，據調查之統計數據顯示，在我國相關口腔病之患病率已高達97.6%，意即，在我們周遭有許多人正默默地忍受著口腔病的痛苦；誠如前述，依據研究顯示，口腔病患主要係由牙周組織直接或間接遭受感染所導致的疾病，因此，牙周炎的治療對我國乃至全球都是刻不容緩的重要議題。由於，牙周病係由牙菌斑引起的牙周組織的慢性破壞性疾病，如果得不到有效治療就會使牙齒鬆動或脫落，從而對人體健康產生不容忽視的負面影響。一般言，復請參閱第3及4圖所示，牙周病的治療方法可概分成「傳統治療法」（如：刮牙、拔牙…等）及「抗菌藥物治療法」（含全身及局部用藥）兩大類；雖然，該等治療方法能夠在不同程度上改善牙周炎的臨床症狀，但是「傳統治療法」在整體上的療效較低、難予根治且復發率較高，已逐漸式微；而「抗菌藥物治療法」雖可全身用藥，但到達局部的藥物濃度一般均不高，不僅難以實現預期的療效，造成大量藥物的浪費，尚可能因而引起如：肝腎損害、口腔及消化道菌群失調或抗藥性增強…等諸多藥物性負作用；反之，局部用藥則因用藥量較小，所衍生的藥物性負作用亦較小，能在牙周袋內短期達到較高濃度，而有效達成減少牙齦 43下牙菌斑的效果，其療效雖超過全身給藥治療的效果，且不致影響口腔或身體其他臟腑內的正常菌群，而具有較大的安全性，但是，這些局部用藥卻不易維持，且極易因喝水、漱口或刷牙而流失，一旦藥物流失即喪失治療作用，而容易導致牙齦炎或牙周炎反覆發作。According to the survey, periodontitis has been considered as the "number one killer" of oral health by medical definitions, and it is also known as the third killer that threatens human health after cancer and cardiovascular and cerebrovascular diseases. According to the statistics of the survey, in The prevalence of related oral diseases in my country has reached 97.6%, which means that many people around us are suffering from oral diseases silently; as mentioned above, according to research, oral diseases are mainly caused by periodontal tissue. Or suffer from diseases caused by infection indirectly. Therefore, the treatment of periodontitis is an urgent and important issue for our country and even the world. Because periodontal disease is a chronic destructive disease of the periodontal tissue caused by dental plaque, if it is not treated effectively, the teeth will loosen or fall off, which will have a negative impact on human health that cannot be ignored. Generally speaking, as shown in Figures 3 and 4, the treatment methods for periodontal disease can be roughly divided into "traditional treatment" (such as: tooth scaling, tooth extraction, etc.) and "antibacterial drug treatment" (including systemic and local treatment) Although these treatment methods can improve the clinical symptoms of periodontitis to varying degrees, the overall curative effect of "traditional treatment" is low, difficult to cure, and the recurrence rate is high, which has gradually declined ; and "antibacterial drug therapy" can be used systemically, but the local drug concentration is generally not high, not only difficult to achieve the expected curative effect, resulting in a lot of waste of drugs, but also may cause such as: liver and kidney damage, oral and digestive tract damage Bacterial flora imbalance or enhanced drug resistance... and many other adverse effects of drugs; on the contrary, because of the small dosage of topical medication, the derived drug adverse effects are also small, and can reach a higher concentration in the periodontal pocket in a short period of time, while Effectively achieve the effect of reducing dental plaque under the gums. Although its curative effect exceeds the effect of systemic drug treatment, it will not affect the normal flora in the oral cavity or other viscera of the body, and has greater safety. However, these topical drugs But it is not easy to maintain, and it is very easy to lose due to drinking water, gargling or brushing teeth. Once the medicine is lost, the therapeutic effect will be lost, which will easily lead to repeated attacks of gingivitis or periodontitis.

另，查「牙周引導組織再生技術」乃當今最新及最先進之牙周治療技術，其在牙醫臨床工作中已得到非常廣泛之應用，且能收到極為良好之療效。目前，復請參閱第3及4圖所示，在牙醫臨床上，施行「牙周引導組織再生技術」時所使用之「牙周再生片」係一種用於治療牙周炎的微孔膜片，植入後可達到物理性阻擋牙齦43結締組織細胞及上皮細胞與牙根42相接觸，以確保牙根42上殘餘之牙周膜420組織來源細胞及牙槽骨44細胞優先佔據植入面，並在其上生長及朝牙冠41方向爬行，從而達到生理性修復牙周膜420組織的功能。因此，在牙醫臨床實施上，通常均會要求牙周再生片不僅需具備良好的透氣性及保形性外，尚需具備良好的彈性及一定的力學性能，從而令其能充分發揮增強骨架的作用，以保證牙周再生片在臨床治療中除不致坍塌外，還能具有一定的阻擋作用，以有利於抗菌消炎，有效避免引起細菌感染或炎症。In addition, Zha's "periodontal guided tissue regeneration technology" is the latest and most advanced periodontal treatment technology. It has been widely used in clinical dentistry and can receive extremely good results. At present, as shown in Figures 3 and 4, the "Periodontal Regeneration Sheet" used by dentists to implement the "Periodontal Guided Tissue Regeneration Technique" is a microporous membrane used to treat periodontitis After implantation, it can physically prevent the connective tissue cells and epithelial cells of the gingiva 43 from contacting the tooth root 42, so as to ensure that the remaining periodontal ligament 420 tissue-derived cells and alveolar bone 44 cells on the tooth root 42 preferentially occupy the implant surface, and It grows on it and crawls toward the crown 41, so as to achieve the function of physiologically repairing the periodontal ligament 420 tissue. Therefore, in the clinical practice of dentists, periodontal regeneration sheets are usually required not only to have good air permeability and shape retention, but also to have good elasticity and certain mechanical properties, so that they can fully play the role of strengthening the skeleton. In order to ensure that the periodontal regeneration sheet will not collapse during clinical treatment, it can also have a certain blocking effect, which is beneficial to antibacterial and anti-inflammatory, and effectively avoid bacterial infection or inflammation.

一般言，目前市場上之牙周再生片，依其材質之不同主要分為「金屬牙周再生片」及「可降解牙周再生片」等兩大類；其中，由於「金屬牙周再生片」存在著必需以二次手術去除牙周再生片、易引發二次感染發炎及整體手術費用偏高…等弊端，而經常無法被病患接受；反之，使用生物降解材料製成之「可降解牙周再生片」，則因能有效避免二次手術取出牙周再生片之不便，而毫無疑問地具有較優異之應用前景。基於此，近年來，許多研究單位及人員已陸續針對各式「可降解牙周再生片」，進行了諸多研究、開發及探討，且已採用了一些生物可降解聚合物作為原料，製作出各該「可降解牙周再生片」，並獲致了一定成效，例如：在編號CN1775312A ； CN101607098A 及CN101791431A等中國發明專利中，發明人係採用靜電紡絲法…等工藝技術製備出「可降解牙周再生片」；另，在編號CN1775312A之中國發明專利中，沈新元…等發明人則利用可降解聚乳酸_羥基乙酸共聚物製成紗線，再通過採用熔融紡絲、編織及後塗層…等工藝技術製備出「牙周再生片骨架」；又，中國浙江大學之胡巧玲…等發明人則在編號CN101607098A之中國發明專利中，揭露了一種結合流涎成膜法及靜電紡絲法…等工藝技術，在殼聚糖基質膜上電紡一層具有多孔結構之羥基磷灰石/殼聚糖/聚乙烯醇無紡布膜的技術；最後，中國同濟大學之趙鵬…等發明人在編號CN101791431A之中國發明專利中，更以聚乳酸、聚己內酯、聚乳酸-羥基乙酸共聚物、膠原、改性殼聚糖等生物可降解材料作為原料，透過靜電紡絲…等工藝技術製備出薄膜材料，並透過超分子負載技術在該薄膜材料上負載多種生長因數。雖然，透過前述專利技術所製成之「可降解牙周再生片」均聲稱具有一定成效，但美中不足的是，各該「可降解牙周再生片」所採用之工藝製備技術及設備，不僅極為複雜且昂貴，導致其製作成本始終居高不下，而難以被普羅大眾所接收，而其所採用塗覆載藥之工藝技術，亦令所製成之各該「可降解牙周再生片」產品之抗菌及殺菌時效偏短、效果較差且極易發生二次感染…等問題，以致各該「可降解牙周再生片」產品始終無法被順利推廣應用至牙醫臨床上造福眾多之牙周病患。Generally speaking, the periodontal regeneration sheets currently on the market are mainly divided into two categories according to their materials: "metal periodontal regeneration sheets" and "degradable periodontal regeneration sheets". Among them, "metal periodontal regeneration sheets" There are disadvantages such as the need for secondary surgery to remove periodontal regeneration, easy to cause secondary infection and inflammation, and high overall surgical costs, etc., and are often unacceptable by patients; on the contrary, "degradable teeth" made of biodegradable materials "Periodontal regeneration film", because it can effectively avoid the inconvenience of taking out the periodontal regeneration film in a second operation, it undoubtedly has a better application prospect. Based on this, in recent years, many research institutes and personnel have successively carried out a lot of research, development and discussion on various "degradable periodontal regeneration tablets", and have used some biodegradable polymers as raw materials to produce various The "degradable periodontal regeneration sheet" has achieved certain results. For example, in Chinese invention patents such as CN1775312A; In addition, in the Chinese invention patent No. CN1775312A, inventors such as Shen Xinyuan used degradable polylactic acid-glycolic acid copolymer to make yarn, and then melt-spun, weaved and post-coated...etc. Process technology to prepare "periodontal regeneration film skeleton"; In addition, inventors such as Hu Qiaoling from Zhejiang University in China disclosed a process technology that combines salivation film-forming method and electrospinning method in the Chinese invention patent numbered CN101607098A , a technology of electrospinning a layer of hydroxyapatite/chitosan/polyvinyl alcohol non-woven fabric film with a porous structure on a chitosan matrix film; finally, Zhao Peng from Tongji University in China...etc. Among the Chinese invention patents, polylactic acid, polycaprolactone, polylactic acid-glycolic acid copolymer, collagen, modified chitosan and other biodegradable materials are used as raw materials, and film materials are prepared through electrospinning...etc. , and load various growth factors on the thin film material through supramolecular loading technology. Although the "degradable periodontal regeneration tablets" made through the above-mentioned patented technologies all claim to have certain effects, the fly in the ointment is that the preparation technology and equipment used in each of the "degradable periodontal regeneration tablets" are not only extremely Complicated and expensive, its production cost is always high, and it is difficult to be accepted by the general public, and the technology of coating and loading it adopts also makes the "degradable periodontal regeneration sheet" products made The antibacterial and sterilizing time is too short, the effect is poor, and secondary infection is easy to occur... etc., so that the "degradable periodontal regeneration sheet" products have not been successfully promoted and applied to dentists to benefit many periodontal patients. .

為了解決前述問題，亦有研究人員試圖將藥物直接載入至生物可降解聚合物中，製成片或膜等形狀，以期能因藥物的載入而據以有效殺死病原體，當然業界所期望者係一種具備藥物緩釋功能且能使患病區穩定地處於抗菌環境中之材料，從而能因此種材料有利於牙周患病組織細胞的再生。基於此一理念，韓國東國制藥株式會社之鄭鐘平…等發明人乃在編號CN1083355A之中國發明專利中，揭露了一種將藥物與聚酯內酯熔融混合，再透過擠壓成膜之製備技術；嗣，Zamani M， Morshed M， Varshosaz J及Jannesari M.…等發明人則研發出一種將甲硝唑苯甲酸及聚己內酯予以混合，再透過靜電紡將其製備成具有緩釋功能奈米纖維膜之創新技術(Controlled release of metronidazole benzoate from poly(ε-caprolactone)electrospun nanofibers for periodontal diseases)，且將該創新技術發表於2010年之European Journal of Pharmaceutics and Biopharmaceutics(75：179-185)期刊中，惟，由於其內己內酯基的載藥奈米纖維膜之力學強度及親水性能尚不夠理想，故其所製備之奈米纖維膜很容易因快速降解而導致載體崩坍。2011年，Bottino MC，Thomas V，Janowski GM...等發明人更研發設計出一種新穎之分級結構牙周再生膜(A novel spatially designed and functionally graded electrospun membrane for periodontal regeneration)，其主要係由聚(乳酸_己內酯)共聚物(PLCL)奈米纖維膜、羥基磷灰石與聚乳酸及蛋白質三元混合奈米纖維膜、甲硝唑與蛋白質混合奈米纖維膜等三層所組成，且該創新技術已被公開發表於2011年之Acta Biomaterialia 2011：216_224)期刊中，雖然，由於其內含大量親水性羥基之羥基磷灰石，不僅可誘導細胞生長，還可顯著提高材料之力學性能，而其膜層中之甲硝唑尚可抗菌及殺菌，但是，美中不足之處係其膜層中之羥基磷灰石降解困難，且期刊中僅簡單揭露了其具有對甲硝唑藥物之緩釋功能，至於其實際成效如何？則不得而知。 In order to solve the aforementioned problems, some researchers have tried to load drugs directly into biodegradable polymers and make them into sheets or films, in order to effectively kill pathogens due to the loading of drugs. Of course, the industry expects It is a material that has the function of slow release of drugs and can keep the diseased area in an antibacterial environment stably, so that this material can be beneficial to the regeneration of periodontal diseased tissue cells. Based on this idea, Zheng Zhongping of Korea Dongguk Pharmaceutical Co., Ltd. and other inventors disclosed a preparation technology of melting and mixing drugs and polyester lactones, and then extruding them into films in the Chinese invention patent No. CN1083355A ; Si, Zamani M, Morshed M, Varshosaz J and Jannesari M... and other inventors have developed a mixture of metronidazole benzoic acid and polycaprolactone, and then prepared it into a drug with slow-release function by electrospinning The innovative technology of rice fiber membrane (Controlled release of metronidazole benzoate from poly(ε-caprolactone) electrospun nanofibers for periodontal diseases), and the innovative technology was published in the European Journal of Pharmaceutics and Biopharmaceutics (75:179-185) in 2010 However, due to the insufficient mechanical strength and hydrophilic properties of the caprolactone-based drug-loaded nanofiber membrane, the prepared nanofiber membrane is easy to cause the carrier to collapse due to rapid degradation. In 2011, Bottino MC, Thomas V, Janowski GM...and other inventors developed and designed a novel hierarchical structure periodontal regeneration membrane (A novel spatially designed and functionally graded electrospun membrane for periodontal regeneration), which is mainly composed of polymer (Lactic acid-caprolactone) copolymer (PLCL) nanofiber membrane, hydroxyapatite, polylactic acid and protein ternary mixed nanofiber membrane, metronidazole and protein mixed nanofiber membrane, etc. And this innovative technology has been published in the 2011 Acta Biomaterialia 2011: 216_224) journal, although, because it contains a large number of hydrophilic hydroxyl hydroxyapatite, it can not only induce cell growth, but also significantly improve the mechanical properties of the material performance, and the metronidazole in the film layer can still be antibacterial and bactericidal, but the fly in the ointment is that the hydroxyapatite in the film layer is difficult to degrade, and the journal only briefly discloses that it has the effect of metronidazole drug Sustained release function, as for its actual effect? It is unknown.

誠如前述，儘管「生物可降解牙周再生片」已得到臨床上的應用，且獲致一定的成效，然而，其上仍存在一些尚待解決之問題及缺陷，例如：其所使用之純生物可降解聚合物普遍存在力學性能較差及疏水性能較強之問題，容易導致發生材料崩塌及細胞黏附困難...等缺陷；另，若通過塗層工藝將藥物載入至「生物可降解材料」中或採用疏水聚合物為載體，則藥物往往會富集在材料表面，極易出現藥物“早期突釋”之現象，而難以實現穩定緩釋藥物之預期療效。故，如何自眾多生物可降解原料中挑選出一種價廉物美之優質膳食蛋白質？並據以製作出結構強度充足之優質「可降解材料」，再透過簡易可行之製備技術，利用表面活性劑包封該優質「可降解材料」，以大幅增加其中之結晶度，令其所形成之聚合物基體具備更佳之結構強度，且能藉降低表面活性劑濃度而大幅增加其內細胞之存活率，並藉控制其孔隙率，有效提高其保水能力，以令其具有更優異之抗菌活性，從而令其因能有效減少細菌生長而具體實現防止生物膜(如：前述牙菌膜50)形成之能力。As mentioned above, although the "biodegradable periodontal regeneration sheet" has been clinically applied and achieved certain results, however, there are still some problems and defects that need to be solved, such as: the pure biological Degradable polymers generally have poor mechanical properties and strong hydrophobic properties, which can easily lead to defects such as material collapse and difficult cell adhesion; in addition, if the drug is loaded into the "biodegradable material" through the coating process In the medium or using hydrophobic polymers as carriers, the drug tends to be concentrated on the surface of the material, which is prone to the phenomenon of "early burst release" of the drug, and it is difficult to achieve the expected curative effect of the stable and sustained release drug. Therefore, how to choose a cheap and high-quality dietary protein from many biodegradable raw materials? And based on this, a high-quality "degradable material" with sufficient structural strength is produced, and then through a simple and feasible preparation technology, the high-quality "degradable material" is encapsulated with a surfactant to greatly increase the crystallinity of it, so that the formed The polymer matrix has better structural strength, and can greatly increase the survival rate of cells in it by reducing the concentration of surfactant, and effectively improve its water retention capacity by controlling its porosity, so that it has better antibacterial activity , so that it can effectively reduce the growth of bacteria and specifically realize the ability to prevent the formation of biofilm (such as: the aforementioned dental plaque 50).

另，在現今醫療市場上，針對體外藥物之持續釋放或控制釋放能力之優劣判斷，一般均係透過其誘導效率（Entrapment Efficiency， EE）、裝載能力（Loading Capacity， LC）等特性，來進行評估；其中，藥物及奈米藥物之質量比係被稱之為「藥物加載量」，而奈米藥物製備過程中之藥物利用率則係由藥物加載效率決定，可通過體外藥物之持續釋放或控制釋放能力來進行評估，故，透過EE、LC及體外藥物釋放率等因子即能有效評估奈米藥物持續釋放能力之優劣。In addition, in the current medical market, the judgment of the sustained release or controlled release of in vitro drugs is generally based on its induction efficiency (Entrapment Efficiency, EE), loading capacity (Loading Capacity, LC) and other characteristics to evaluate ; Among them, the mass ratio of drug and nano drug is called "drug loading", and the drug utilization rate in the preparation process of nano drug is determined by the drug loading efficiency, which can be controlled by sustained release or control of drug in vitro Therefore, factors such as EE, LC and in vitro drug release rate can effectively evaluate the pros and cons of sustained release ability of nano-drugs.

有鑑於前述生物可降解材料在牙周再生片的應用上所存在之諸多問題(如：力學性能較差、疏水性較強及細胞不易附著生長…等)，如何透過使用一種取得容易且價廉物美之優質膳食蛋白質作為原料，在無需使用特別昂貴之製程設備，即能以簡單、快捷且可控制性高之製備程序，大批量地生產製備出全生物降解材料，且令其得以被輕易應用於製作治療牙周炎之藥物緩釋奈米纖維膜，以使該奈米纖維膜不僅具備極佳之生物可降解性及生物相容性，尚具備較高之載藥量，能使活性藥物之有效釋放期持續長達20天以上，從而使得牙周病患者在接受治療期間，其牙腔患病區始終能處於一個殺菌及抗菌之環境，以有效避免其他同類產品所產生之二次感染…等問題；此外，業界更期望該全生物降解材料具有較理想之力學性能及較適宜之親水性能，以能有效避免一般奈米膜所存在之藥物早釋(或突釋)及易崩坍…等問題，同時，適宜之親水性能亦能令所據以製成之奈米纖維再生膜更利於正常牙周組織細胞的再生，從而大幅提升牙周再生之療效，即成為近年來相關生物降解材料業界始終汲汲營營、不斷探索、努力研究開發，以期能獲得突破之一共同議題，亦為本發明在後續內容中亟欲探討及克服之一重要課題。In view of the many problems of the aforementioned biodegradable materials in the application of periodontal regeneration sheets (such as: poor mechanical properties, strong hydrophobicity, and difficult attachment and growth of cells, etc.), how to obtain an easy, cheap and high-quality High-quality dietary protein as a raw material can produce fully biodegradable materials in large quantities through simple, fast and highly controllable preparation procedures without using particularly expensive process equipment, and it can be easily applied to Manufacture drug sustained-release nanofiber membrane for the treatment of periodontitis, so that the nanofiber membrane not only has excellent biodegradability and biocompatibility, but also has a high drug loading capacity, enabling effective release of active drugs The period lasts for more than 20 days, so that during the period of treatment for periodontal patients, the diseased area of the dental cavity can always be in a sterilizing and antibacterial environment, so as to effectively avoid secondary infections caused by other similar products...etc. In addition, the industry expects the fully biodegradable material to have better mechanical properties and more suitable hydrophilic properties, so as to effectively avoid the problems of early release (or sudden release) and easy collapse of drugs that exist in general nano-membranes. At the same time, suitable hydrophilic properties can also make the nanofiber regeneration membrane made of it more conducive to the regeneration of normal periodontal tissue cells, thereby greatly improving the curative effect of periodontal regeneration. It is a common issue of camping, continuous exploration, and hard research and development in order to obtain a breakthrough, and it is also an important issue that the present invention is eager to explore and overcome in the follow-up content.

有鑑於前述傳統「可降解牙周再生材料」在製備及使用上所存在之諸多問題及缺失，發明人經過長久努力研究與實驗，終於開發設計出本發明之一種「利用表面活性劑加載k-卡拉膠低聚醣製成對牙周病原體具抗菌活性之纖維素奈米纖維及其製法」，以期藉由本發明之提出，能對臺灣食品加工產業所遺留下來之大豆豆漿渣中價廉物美且品質優異之纖維素進行有效之再利用，且據以製作出品質優異、價格合宜且對牙周病原體具抗菌活性之纖維素奈米纖維(Cellulose Nanofiber，簡稱CNF)，從而能佳惠對優質「可降解牙周再生材料」產品有迫切需求之普羅大眾。In view of the many problems and deficiencies in the preparation and use of the aforementioned traditional "degradable periodontal regeneration materials", the inventors have finally developed and designed a kind of "using surfactant to load k- Carrageenan oligosaccharides are used to make cellulose nanofibers with antibacterial activity against periodontal pathogens and their preparation method", in order to improve the quality and cheapness of soybean milk dregs left over from Taiwan's food processing industry by proposing the present invention. The high-quality cellulose can be effectively reused, and the cellulose nanofiber (Cellulose Nanofiber, CNF for short) with excellent quality, low price and antibacterial activity against periodontal pathogens can be produced based on this, so that Jiahui can benefit from high-quality "can be Degradable periodontal regeneration materials" products are in urgent need of the general public.

本發明之主要目的，係提供一種「利用表面活性劑加載k-卡拉膠低聚醣製成對牙周病原體具抗菌活性之纖維素奈米纖維及其製法」，該製法係將食品加工產業在完成食品加工後所遺留下來之大豆豆漿渣中價廉物美且品質優異之纖維素，進行有效之再利用，以先自該等纖維素中萃取出纖維素奈米纖維(Cellulose Nanofiber，簡稱CNF)後，再進一步使用κ-卡拉膠低聚醣(ĸ - carrageenan oligosaccharides，簡稱CO)對CNF進行修飾，從而製作出本發明具抗菌活性之纖維素奈米纖維(以下簡稱CO-CNF)，以有效改善CNF之降解溫度、結晶度、溶脹性及生物相容性…等特性，令其得以被廣泛應用於製作治療牙周炎所需使用之奈米纖維膜。如此，由於牙周炎係屬於一種與微生物堆積有關之慢性炎症性疾病，而本發明之CO-CNF經實驗證實確具對牙周病原體之抗菌活性，不僅能據以有效預防或改善牙周病症狀，尚能充分實現有效給藥之目的，據此，本發明之CO-CNF材料即能在牙周炎之臨床治療過程中，有效且精準實現遞送藥物之能力，以大幅提升牙周炎之治療效果及效率，從而在牙齒保健及醫療產業領域中具有極高之利用價值。The main purpose of the present invention is to provide a kind of "cellulosic nanofibers with antibacterial activity against periodontal pathogens made by loading k-carrageenan oligosaccharides with surfactant and its preparation method". The cheap, good-quality and high-quality cellulose left over from the soybean milk residue after food processing is effectively reused to extract cellulose nanofibers (Cellulose Nanofiber, referred to as CNF) from the cellulose Afterwards, κ-carrageenan oligosaccharides (ĸ-carrageenan oligosaccharides, CO for short) were used to modify CNF to produce cellulose nanofibers with antibacterial activity (hereinafter referred to as CO-CNF) of the present invention to effectively Improve the degradation temperature, crystallinity, swelling and biocompatibility of CNF, etc., so that it can be widely used in the production of nanofiber membranes for the treatment of periodontitis. Thus, since periodontitis is a chronic inflammatory disease related to microbial accumulation, and the CO-CNF of the present invention has been proved by experiments to have antibacterial activity against periodontal pathogens, it can not only effectively prevent or improve periodontal disease Symptoms, the purpose of effective drug delivery can still be fully realized. Accordingly, the CO-CNF material of the present invention can effectively and accurately realize the ability to deliver drugs during the clinical treatment of periodontitis, so as to greatly improve the therapeutic effect and efficiency of periodontitis. , so it has a very high utilization value in the field of dental health care and medical industry.

為便  貴審查委員能對本發明之目的、技術特徵及其功效，做更進一步之認識與瞭解，茲舉實施例配合圖式，詳細說明如下：In order to facilitate your review committee to further understand and understand the purpose, technical features and effects of the present invention, the embodiments are hereby combined with the drawings, and the detailed description is as follows:

按，近年來，最有聚焦性之生質補強材料係一種取自於大自然之持續再生性原料，此種原料可為從木材分解而來之「纖維素奈米纖維」(Cellulose Nanofiber，簡稱CNF)，其也是目前市場上最令人矚目具備環境保護及資源永續特性之材料，以期能據以部份取代較高污染性之碳纖維補強材料。關於此種材料之未來發展性，根據Grand View Research Inc.之市場預估報告，全美奈米纖維市場有望在2024年達到6億美元，其中，請參閱第5圖所示，CNF材料年增長率最快，預估從2016年到2024年間將以29.6％之複合年增長率成長。至於日本之CNF製造廠商及企業亦正致力於提升CNF複合材料之實用性及應用性，以令該種材料可被廣泛地被應用至諸多領域(如：汽車生產及房屋建造…等)，從而使其成為近年來受到材料業界及學術界高度期待之一種創新的生質補強材料。In recent years, the most focused bio-reinforcing material is a sustainable renewable raw material from nature, which can be "Cellulose Nanofiber" (Cellulose Nanofiber, referred to as CNF), which is currently the most eye-catching material with environmental protection and resource sustainability characteristics in the market, in order to partially replace the more polluting carbon fiber reinforced materials. Regarding the future development of this material, according to the market forecast report of Grand View Research Inc., the nanofiber market in the United States is expected to reach 600 million US dollars in 2024. Among them, please refer to Figure 5 for the annual growth rate of CNF materials The fastest, it is estimated to grow at a compound annual growth rate of 29.6% from 2016 to 2024. As for Japanese CNF manufacturers and enterprises, they are also working to improve the practicality and applicability of CNF composite materials, so that this material can be widely used in many fields (such as: automobile production and housing construction...etc.), so that This makes it an innovative bio-reinforcing material highly anticipated by the material industry and academia in recent years.

在日前舉辦之Film Tech Osaka 2018專門技術演講中，更特別針對「纖維素奈米纖維」之天然環保特性作了許多詳實的介紹與討論；其中，京都市產業技術研究所之仙波健研究員亦針對CNF複合材料技術及其未來發展提出了成果發表，其成果發表顯示CNF植物纖維之重量可輕至僅為鋼鐵之1/5，然而其強度卻能高達約鋼鐵之8倍，且植物纖維之熱膨脹率僅為玻璃之1/50，具有極佳之低線熱膨脹係數等特性，可做為高強度之補強材料，被適當地添加至樹脂或其它材料中，以有效實現據以製作出高強度複合材料之目的。In the recently held Film Tech Osaka 2018 special technical lecture, he made a lot of detailed introductions and discussions on the natural environmental protection characteristics of "cellulose nanofibers". The CNF composite material technology and its future development have presented results. The results have shown that the weight of CNF plant fibers can be as light as 1/5 of steel, but its strength can be as high as about 8 times that of steel, and the thermal expansion of plant fibers The rate is only 1/50 of that of glass, and it has excellent characteristics such as low linear thermal expansion coefficient. It can be used as a high-strength reinforcing material, and it can be properly added to resin or other materials to effectively realize high-strength composites. Purpose of Materials.

此外，仙波健研究員亦針對CNF及其他補強材料進行比較，且將其比較結果揭示如下表所示： 補強用纖維 CNF 碳纖維 (PAN系) 芳綸纖維(Kevlar49) 玻璃纖維 密度(g/cm3 ) 1.5 1.82 1.45 2.55 彈性率(GPa) X 230 112 74 強度(GPa) X 3.5 3. 3.4 熱膨脹係數(ppm/K) 0.1 0 -5 5 價格 (日幣/公斤) 400 3，000 5，000 200~300 永續型資源 ◎ ─ ─ ─ 回收性 ◎ ∆：難燃 ◎ X：不燃 平滑性 ◎ X X X In addition, researcher Xian Bojian also compared CNF and other reinforcing materials, and revealed the comparison results as shown in the following table: fiber for reinforcement CNF Carbon fiber (PAN series) Aramid fiber (Kevlar49) glass fiber Density (g/cm 3 ) 1.5 1.82 1.45 2.55 Elasticity (GPa) x 230 112 74 Strength (GPa) x 3.5 3. 3.4 Coefficient of thermal expansion (ppm/K) 0.1 0 -5 5 Price (JPY/kg) 400 3,000 5,000 200~300 sustainable resources ◎ ─ ─ ─ recycling ◎ ∆: flame retardant ◎ X: non-combustible smoothness ◎ x x x

由上表所示可清楚得知，雖然CNF奈米纖維、碳纖維、尼龍纖維（Kevlar）與玻璃纖維（GF）同為市場上常用之補強用材料，然而在逐一比較各該補強材料在密度、彈性率、強度、可燃性及價格…等方面上之優劣後，顯然CNF奈米纖維具備優異之可燃性、不產生廢棄物、無難以銷毀問題及具備資源持續性…等優點，不僅如此，尚因其材料規格係屬於奈米等級，而令其極易與其它材料混合成複合材料，且在混合成各該複合材料後，其纖維亦不易浮出至各該複合材料之表面，從而令各該複合材料之外觀及觸感始終能保持預期之光滑及平整感；至於，在價格上，仙波健研究員亦表示，若CNF製程技術及設備能在未來獲得有效改善，而令CNF奈米纖維能開始實現量產化生產，其預估CNF奈米纖維之價格可因此而降低至僅約400日圓/kg，從而令CNF奈米纖維在各領域之應用均具有相當樂觀之競爭力。It can be clearly seen from the table above that although CNF nanofibers, carbon fibers, nylon fibers (Kevlar) and glass fibers (GF) are commonly used reinforcing materials in the market, when comparing the reinforcing materials one by one in terms of density, After considering the advantages and disadvantages of elastic modulus, strength, flammability and price, etc., it is clear that CNF nanofibers have excellent flammability, no waste, no problem of indestructibility, and resource sustainability. Not only that, but also Because the material specifications are at the nanometer level, it is very easy to mix with other materials to form composite materials, and after mixing into each composite material, its fibers are not easy to float to the surface of each composite material, so that each The appearance and touch of the composite material can always maintain the expected smooth and flat feeling; as for the price, the researcher Xian Bojian also said that if the CNF process technology and equipment can be effectively improved in the future, the CNF nanofiber can Started mass production, it is estimated that the price of CNF nanofibers can be reduced to only about 400 yen/kg, so that the application of CNF nanofibers in various fields has a very optimistic competitiveness.

有鑒於此，發明人乃思及有效利用臺灣食品加工產業在完成食品加工後所殘留之大量且優質的副產品（如，大豆豆漿渣）中之纖維素，且在據以萃取及製造出高品質之奈米纖維後，進一步使用κ-卡拉膠低聚醣(ĸ - carrageenan oligosaccharides，簡稱CO)對CNF進行修飾，而製作出本發明具抗菌活性之纖維素奈米纖維(以下簡稱CO-CNF)，以有效改善CNF之降解溫度、結晶度、溶脹性及生物相容性…等特性，從而令其得以被廣泛應用於製作治療牙周炎所需使用之奈米纖維膜。如此，由於牙周炎係屬於一種與微生物堆積有關之慢性炎症性疾病，而本發明之CO-CNF既經實驗證實確具對牙周病原體之抗菌活性，據此，不僅能據以被使用至牙周再生治療中，有效預防或改善牙周病症狀，尚能充分實現有效及精準給藥之目的，令本發明之CO-CNF材料能在牙周炎之臨床治療過程中，實現有效且精準遞送藥物之能力，以能大幅提升牙周炎之治療效果及效率，從而在牙齒保健及醫療產業中具有極高之利用價值。In view of this, the inventor thinks and effectively utilizes the cellulose in a large amount of high-quality by-products (such as soybean milk dregs) remaining after food processing in Taiwan's food processing industry, and extracts and manufactures high-quality cellulose based on it. After preparing nanofibers, κ-carrageenan oligosaccharides (CO for short) was used to modify CNF to produce cellulose nanofibers with antibacterial activity of the present invention (hereinafter referred to as CO-CNF) , to effectively improve the degradation temperature, crystallinity, swelling and biocompatibility of CNF, etc., so that it can be widely used in the production of nanofiber membranes for the treatment of periodontitis. Thus, since periodontitis is a chronic inflammatory disease related to microbial accumulation, and the CO-CNF of the present invention has been proved by experiments to have antibacterial activity against periodontal pathogens, accordingly, it can not only be used to In the periodontal regeneration treatment, it can effectively prevent or improve the symptoms of periodontal disease, and can still fully achieve the purpose of effective and precise drug delivery, so that the CO-CNF material of the present invention can achieve effective and precise drug delivery in the clinical treatment of periodontitis The ability to greatly improve the treatment effect and efficiency of periodontitis, so it has a very high value in dental care and medical industry.

本發明係提供一種利用表面活性劑加載k-卡拉膠低聚醣製成對牙周病原體具抗菌活性之纖維素奈米纖維及其製法，請參閱第6圖所示，該製法係包括下列步驟(按，以下各製程步驟均係發明人在歷經過多次製程實作，反覆分析及調整其中細節及條件後所歸納出之最佳步驟、數據及條件，因此，為簡化說明起見，本發明在後續描述中僅以最佳步驟、數據及條件為例，說明該製法；惟，本發明之該製法在實際施作時，不僅不侷限於此，其步驟、數據及條件，亦均能依實際需要，予以微調，只要其數據及條件之微調範圍在以下所列數據及條件之±5%範圍內，仍應能達成本發明之主要目的，而獲得所需之複合材料，亦為本發明在此欲主張保護的權利範圍；同時，本發明之該製法中所使用之原料亦不侷限於臺灣豆類食品加工產業加工後所殘留之優質副產品(如：大豆豆漿渣)中的纖維素，亦能依實際的需要，使用其它堅果類食品加工後所殘留之其它優質副產品(如：花生、綠豆或蠶豆...等腐渣)中的纖維素，予以取代，亦為本發明在此欲主張保護的權利範圍，合先陳明。 The present invention provides a cellulose nanofiber with antibacterial activity against periodontal pathogens made by loading k-carrageenan oligosaccharides with a surfactant and its preparation method. Please refer to Figure 6. The preparation method includes the following steps (Press, the following process steps are the best steps, data and conditions summarized by the inventor after repeatedly analyzing and adjusting the details and conditions after repeated process implementation. Therefore, for the purpose of simplifying the description, the present invention In the follow-up description, only the best steps, data and conditions are taken as an example to illustrate the method; however, the method of the present invention is not limited to this when it is actually implemented, and its steps, data and conditions can also be based on Actual needs, fine-tuning, as long as the fine-tuning range of the data and conditions is within the range of ±5% of the data and conditions listed below, the main purpose of the present invention should still be achieved, and obtaining the required composite material is also a part of the present invention The scope of rights to be protected here; at the same time, the raw materials used in the preparation method of the present invention are not limited to the cellulose in the high-quality by-products (such as soybean milk dregs) that remain after the processing of the soy food processing industry in Taiwan. According to actual needs, the cellulose in other high-quality by-products (such as: peanuts, mung beans, broad beans, etc.) remaining after processing other nut foods can be used to replace them, which is also what the present invention intends to advocate here The scope of rights to be protected shall be stated first.

復請參閱第6圖所示，在本發明之一較佳實施例中，據以製作本發明之對牙周病原體具抗菌活性之纖維素奈米纖維之方法，係包括下列步驟：(200)首先，自大豆豆漿渣的纖維素中萃取出纖維素奈米纖維(Cellulose Nanofiber，簡稱CNF)；(210)其次，使用κ-卡拉膠低聚醣(κ-carrageenan oligosaccharides，簡稱CO)對CNF進行修飾，以將CNF轉化成具抗菌活性之纖維素奈米纖維(以下簡稱CO-CNF)；及(220)最後，利用表面活性劑，包封該CO-CNF，即製作出本發明之對牙周病原體具抗菌活性之纖維素奈米纖維CO-CNF材料。 Referring back to Fig. 6, in a preferred embodiment of the present invention, the method for making the cellulose nanofibers with antibacterial activity against periodontal pathogens of the present invention comprises the following steps: (200) First, cellulose nanofibers (Cellulose Nanofiber, CNF for short) were extracted from the cellulose of soybean dregs; modified to convert CNF into cellulose nanofibers with antibacterial activity (hereinafter referred to as CO-CNF); and (220) finally, using surfactants to encapsulate the CO-CNF, that is, to produce the antibacterial Cellulose nanofiber CO-CNF material with antibacterial activity around pathogens.

如此，由於經後續實驗證實本發明之CO-CNF材料能有效改善CNF之降解溫度、結晶度、溶脹性及生物相容性...等特性，而令本發明之CO-CNF材料得以被應用於製作治療牙周炎所使用之奈米纖維膜，且能被據以使用於牙周再生治療之療程中，有效預防或改善牙周病症狀，更因其尚具備能緩釋藥物之特性，令其能有效實現對牙周炎患處充分且精準給藥之目的，從而能大幅提升牙周炎之治療效果及效率。In this way, because the CO-CNF material of the present invention can effectively improve the degradation temperature, crystallinity, swelling and biocompatibility of CNF through subsequent experiments, the CO-CNF material of the present invention can be applied In the manufacture of nanofiber membranes used in the treatment of periodontitis, and can be used in the course of periodontal regeneration treatment, effectively prevent or improve the symptoms of periodontal disease, and because it still has the characteristics of sustained release of drugs, It can effectively achieve the purpose of adequate and precise drug delivery to the affected area of periodontitis, thereby greatly improving the treatment effect and efficiency of periodontitis.

在本發明之前述較佳實施例中，復請參閱第6圖所示，該製法係依下列步驟自大豆豆漿渣的纖維素中萃取出CNF： (201) 首先，將20 公克乾燥之豆漿渣(dried soybean curd residue)與250 毫升 5% 濃度之氫氧化鈉(NaOH)溶劑，予以均勻混合成一混合液，且在攝氏 120℃ 之溫度下，利用攪拌機對該混合液進行攪拌2小時，且在進行2次攪拌令該豆漿渣與該氫氧化鈉(NaOH)溶劑充分且均勻混合後，利用蒸餾水(distilled water)洗滌該混合液(本發明所謂之「洗滌」係指先以離心方式自該混合液中濾除原有的液體，再以蒸餾水沖洗該混合液中剩餘之殘渣)； (202) 嗣，在攝氏80℃之溫度下，使用250毫升1莫耳濃度之氯化氫( HCl)溶劑對該混合液中之殘渣進行殘渣清洗處理(本發明所謂之「殘渣清洗處理」係指先以離心方式自該混合液中濾除原有的液體，再將該混合液中剩餘之殘渣添加至該氯化氫( HCl)溶劑) 2小時； (203) 嗣，在攝氏50℃之溫度下，以250 毫升 5% 濃度之次氯酸鈉(NaClO) 溶劑對該混合液進行漂白處理(bleaching)，直到得到白色的混合溶液為止； (204) 使用蒸餾水對該白色混合溶液進行清滌處理後，在攝氏45℃之溫度下，添加250毫升 64% 濃度之硫酸( H2 SO4 )至該白色混合溶液中，且利用攪拌機對該白色混合溶液予以攪拌1~2小時，以進行水解處理(Hydrolysis)； (205) 嗣，添加250 毫升攝氏10℃溫度之冷水至該白色混合溶液來終止該水解反應； (206) 嗣，使用高速離心機以8000×g之轉速，對該白色混合溶液執行至少10分鐘之離心處理後，將該白色混合溶液以蒸餾水中透析(Dialysis)5天；及 (207)最後，使用均質機以11000轉/分之轉速對該白色混合溶液執行至少1 小時之均質化處理(homogenization)後，對該白色混合溶液執行冷凍乾燥，即能自該豆漿渣的纖維素中萃取得所需之CNF。In the foregoing preferred embodiment of the present invention, please refer to Fig. 6 again, the preparation method is to extract CNF from the cellulose of soybean dregs according to the following steps: (201) First, 20 grams of dried dregs (dried soybean curd residue) and 250 ml of 5% sodium hydroxide (NaOH) solvent were uniformly mixed to form a mixed solution, and at a temperature of 120°C, the mixed solution was stirred for 2 hours with a stirrer, and the Stir twice to make the soya-bean milk dregs and the sodium hydroxide (NaOH) solvent fully and uniformly mixed, and then utilize distilled water (distilled water) to wash the mixed solution (the so-called "washing" in the present invention means that the mixed solution is first extracted from the mixed solution by centrifugation). (202) Then, at a temperature of 80°C, use 250 ml of 1 molar hydrogen chloride (HCl) solvent to mix the mixture Residue in the liquid is carried out residue cleaning treatment (the so-called " residue cleaning treatment " of the present invention refers to first centrifuging from the mixed solution to filter out the original liquid, and then adding the remaining residue in the mixed solution to the hydrogen chloride (HCl ) solvent) for 2 hours; (203) Then, at a temperature of 50°C, the mixed solution was bleached with 250 ml of 5% sodium hypochlorite (NaClO) solvent until a white mixed solution was obtained; (204) After cleaning the white mixed solution with distilled water, add 250 ml of 64% sulfuric acid (H 2 SO 4 ) to the white mixed solution at a temperature of 45° C. The white mixed solution was stirred for 1-2 hours to carry out hydrolysis treatment (Hydrolysis); (205) Next, add 250 ml of cold water at a temperature of 10°C to the white mixed solution to terminate the hydrolysis reaction; (206) Next, use high speed Centrifuge the white mixed solution at a speed of 8000×g for at least 10 minutes, then dialyze the white mixed solution in distilled water for 5 days; and (207) finally, use a homogenizer at 11000 rpm After performing homogenization on the white mixed solution at a speed of 1/min for at least 1 hour, the white mixed solution is freeze-dried, and the desired CNF can be extracted from the cellulose of the soya-bean dregs.

在本發明之前述較佳實施例中，復請參閱第6圖所示，該製法係依下列步驟，利用κ-卡拉膠低聚醣(κ-carrageenan oligosaccharides，簡稱CO)對該CNF進行修飾處理：(211)首先，將1公克之κ-卡拉膠低聚醣(CO)溶解在100毫升(ml)之蒸餾水中，形成CO水溶液後，將CO水溶液添加至100毫升濃度為30mg/ml之CNF水溶液中，形成CO-CNF混合溶液，並以攪拌機在攝氏80℃之溫度下，對該CO-CNF混合溶液進行攪拌至少30分鐘；及(212)嗣，在攝氏80℃之溫度下，利用攪拌機以800rpm之轉速對該CO-CNF混合溶液進行強烈攪拌至少3小時，且在強烈攪拌過程中，將50毫升1莫耳濃度之氯化鉀(KCl)溶劑添加至該CO-CNF混合溶液中，俟該CO-CNF混合溶液完全膠凝後，由於膠凝反應後該CO-CNF混合溶液中會形成有固形物，故，透過離心機以8000×g之轉速，對該CO-CNF混合溶液執行至少10分鐘之離心處理後，即能收集該等固形物，並對該等固形物進行超聲波處理(Ultrasonic treatment)，且將所獲得之樣品用蒸餾水洗滌並予冷凍乾燥後，即能獲得該CO-CNF。在本發明之前述較佳實施例中，請參閱第7圖所示，係κ-卡拉膠低聚醣連接CNF而形成低聚醣CO-CNF之示意圖。 In the aforementioned preferred embodiment of the present invention, please refer to Figure 6, the preparation method is as follows, using κ-carrageenan oligosaccharides (κ-carrageenan oligosaccharides, referred to as CO) to modify the CNF : (211) First, dissolve 1 gram of κ-carrageenan oligosaccharide (CO) in 100 milliliters (ml) of distilled water to form a CO aqueous solution, then add the CO aqueous solution to 100 milliliters of CNF with a concentration of 30 mg/ml In the aqueous solution, a CO-CNF mixed solution is formed, and the CO-CNF mixed solution is stirred for at least 30 minutes at a temperature of 80°C with a stirrer; and (212) then, at a temperature of 80°C, using a stirrer The CO-CNF mixed solution was vigorously stirred at a speed of 800rpm for at least 3 hours, and during the vigorous stirring, 50 ml of 1 molar concentration of potassium chloride (KCl) solvent was added to the CO-CNF mixed solution, After the CO-CNF mixed solution is completely gelled, solids will be formed in the CO-CNF mixed solution after the gelation reaction, so the CO-CNF mixed solution is processed through a centrifuge at a speed of 8000×g. After at least 10 minutes of centrifugation, the solids can be collected, and the solids are subjected to ultrasonic treatment (Ultrasonic treatment), and the obtained samples are washed with distilled water and freeze-dried to obtain the CO -CNF. In the aforementioned preferred embodiments of the present invention, please refer to FIG. 7 , which is a schematic diagram of κ-carrageenan oligosaccharides linking CNF to form the oligosaccharide CO-CNF.

在本發明之前述較佳實施例中，復請參閱第6圖所示，該製法尚包括利用表面活性劑(surfactant)，依下列步驟，包封該CO-CNF：(221)首先，將50毫升濃度為3.2mg/mL之該CO-CNF溶液，透過超音波(Ultrasound)溶解於蒸餾水中，形成CO-CNF混合溶液備用；(222)以非離子性表面活性劑(如：Tween 80)做為表面活性劑，將不同濃度之Tween 80(50、100、200mg)溶解於2毫升之甲醇(Methanol)中，形成各該表面活性劑溶液備用；(223)在攝氏60℃之溫度下，利用攪拌機以800rpm之轉速對該CO-CNF混合溶液進行強烈攪拌時，將200微升(μL)不同濃度油相之各該表面活性劑溶液分別添加至水相之該CO-CNF混合溶液中，繼續攪拌20分鐘； (224)嗣，將8毫升濃度為1.87mg/mL之亞甲基雙丙烯醯胺(N,N’-Methylenebisacrylamide，簡稱MBAA)作為穩定劑，添加至該CO-CNF混合溶液中且持續攪拌2小時；及(225)最後，經超聲處理1小時後，即能分別獲得經各該不同濃度表面活性劑包封之奈米顆粒材料50SNPs、100SNPs及200SNPs。 In the aforementioned preferred embodiment of the present invention, please refer back to Figure 6, the preparation method also includes the use of surfactants (surfactant) to encapsulate the CO-CNF according to the following steps: (221) First, the 50 The CO-CNF solution with a milliliter concentration of 3.2 mg/mL was dissolved in distilled water by ultrasonic waves (Ultrasound) to form a CO-CNF mixed solution for use; (222) made with a non-ionic surfactant (such as: Tween 80) As a surfactant, dissolve Tween 80 (50, 100, 200 mg) of different concentrations in 2 ml of methanol (Methanol) to form each surfactant solution for later use; (223) at a temperature of 60°C, use When the stirrer was vigorously stirring the CO-CNF mixed solution at a speed of 800rpm, add 200 microliters (μL) of the surfactant solutions of different concentrations in the oil phase to the CO-CNF mixed solution in the water phase, and continue Stir for 20 minutes; (224) Next, add 8 ml of methylenebisacrylamide (N,N'-Methylenebisacrylamide, MBAA for short) with a concentration of 1.87 mg/mL as a stabilizer to the CO-CNF mixed solution and keep stirring for 2 and (225) Finally, after ultrasonic treatment for 1 hour, 50SNPs, 100SNPs and 200SNPs of nanoparticle materials encapsulated by the surfactants with different concentrations can be obtained respectively.

嗣，發明人使用動態光散射(Dynamic Light Scattering，簡稱DLS)法對各該CNF及CO-CNF之粒徑、ZETA電位及多分散度指數(polydispersity，簡稱PDI)等特徵逐一進行分析後，獲得下表-1所示之動態光散射分析結果：

Figure 109118634-A0305-02-0016-1
Then, the inventors used the dynamic light scattering (Dynamic Light Scattering, referred to as DLS) method to analyze the particle size, zeta potential and polydispersity index (polydispersity, referred to as PDI) and other characteristics of each of the CNF and CO-CNF one by one, and obtained The dynamic light scattering analysis results shown in Table-1 below:
Figure 109118634-A0305-02-0016-1

該動態光散射分析結果顯示，在前述DLS分析過程中，由於多普勒拓寬作用，分子會在溶液中不斷運動，故透過對散射強度之檢測，可得到粒子之平均尺寸；且其結果清楚顯示，CNF及CO-CNF之平均尺寸分別為263.60±12.27nm及330.66±16.44nm；其中，CO-CNF尺寸之增加係由於親水基團(羥基及硫酸鹽基團)較多所致，表示其在DLS測量條件下對水分子較具親和力；Zeta電位係指粒子分散在液體中時之表面電荷，它受pH值、離子強度及樣品濃度之影響，且這種帶負電荷之顆粒會通過靜電相互作用，而產生隨後之內吞作用，並會進一步透過中和膜之電荷及隨後之內吞作用進行內化。 The results of the dynamic light scattering analysis show that during the aforementioned DLS analysis process, due to the Doppler broadening effect, the molecules will continue to move in the solution, so the average size of the particles can be obtained through the detection of the scattering intensity; and the results clearly show , the average sizes of CNF and CO-CNF are 263.60±12.27nm and 330.66±16.44nm respectively; among them, the increase of CO-CNF size is due to more hydrophilic groups (hydroxyl and sulfate groups), which means that it is in Under DLS measurement conditions, it has a higher affinity for water molecules; Zeta potential refers to the surface charge of particles dispersed in a liquid, which is affected by pH value, ionic strength and sample concentration, and such negatively charged particles will interact with each other through static electricity. function, resulting in subsequent endocytosis, and will be further internalized by neutralizing the charge of the membrane and subsequent endocytosis.

嗣，發明人尚透過測量CNF及CO-CNF之多分散指數PDI，來分析其顆粒之均勻性，由於發明人所測量到之CNF及CO-CNF的PDI值均為0.4左右，據此，前述DLS分析已清楚表明，該等CNF及CO-CNF顆粒之PDI均已達奈米等級、中度單分散及帶負電荷之特性。而經各該表面活性劑包封後之奈米顆粒材料50SNPs、100SNPs及200SNPs的粒子徑度、zeta電位及PDI結果則如下表-2所示；其中，50SNPs、100SNPs及200SNPs粒子之平均尺寸分別為242.60±16.44 nm、203.60±9.76nm及232.90±2.58nm。本發明所製備之藥物負載顆粒CO-CNF、50SNPs、100SNPs及200SNPs係採用油包水乳化法(W/O emulsion)製備。Zeta電位係表示材料表面電荷之參數，其中，CO-CNF之Zeta電位為42.10±0.70，加入該表面活性劑後，其電荷會發生變化，且顯示載藥之奈米顆粒50SNPs、100SNPs及200SNPs比載體CO-CNF更穩定；PDI係用以檢驗顆粒均勻性之指標。PDI值越高，表示均勻性越差，加載表面活性劑後之奈米顆粒50SNPs、100SNPs及200SNPs之PDI值提高到0.2以上，表示比載體CO-CNF更均勻(PDI為0.4)。 Next, the inventors also analyzed the uniformity of the particles by measuring the polydispersity index PDI of CNF and CO-CNF. Since the PDI values of CNF and CO-CNF measured by the inventors were both about 0.4, the aforementioned DLS analysis has clearly shown that the PDI of these CNF and CO-CNF particles has reached the nano-scale, moderately monodisperse and negatively charged characteristics. The particle diameter, zeta potential and PDI results of the nanoparticle materials 50SNPs, 100SNPs and 200SNPs encapsulated by each of the surfactants are shown in the following table-2; wherein, the average size of the 50SNPs, 100SNPs and 200SNPs particles are respectively 242.60±16.44 nm, 203.60±9.76nm and 232.90±2.58nm. The drug-loaded particles CO-CNF, 50SNPs, 100SNPs and 200SNPs prepared in the present invention are prepared by water-in-oil emulsification (W/O emulsion). Zeta potential is a parameter indicating the surface charge of the material. Among them, the Zeta potential of CO-CNF is 42.10±0.70. After adding the surfactant, its charge will change, and it shows the ratio of 50SNPs, 100SNPs and 200SNPs of the drug-loaded nanoparticles The carrier CO-CNF is more stable; PDI is an index used to test the uniformity of particles. The higher the PDI value, the worse the uniformity. The PDI value of the nanoparticles 50SNPs, 100SNPs and 200SNPs after loading the surfactant increased to more than 0.2, which means that it is more uniform than the carrier CO-CNF (PDI is 0.4).

Figure 109118634-A0305-02-0017-2
Figure 109118634-A0305-02-0017-2

嗣，發明人尚透過電子顯微設備(如：穿透式電子顯微鏡(Transmission electron microscope，簡稱TEM)、掃描式電子顯微鏡(Scanning Electron Microscope，簡稱SEM)...)對該CNF及CO-CNF奈米顆粒進行了形態學的分析，其結果如第8圖所示；其中，CNF具有較長之長度及較短之直徑；CNF之平均直徑(D)係37.89±3.07nm，長度(L)約504.97±82.19nm。長寬比(L/D)約為15.62±1.90；CO-CNF會形成網絡結構，CO會以球狀顆粒之形式出現在CNF上，其顆粒之平均直徑為316±0.16nm。 Next, the inventors still use electron microscopic equipment (such as: transmission electron microscope (Transmission electron microscope, TEM for short), scanning electron microscope (Scanning Electron Microscope, SEM for short)...) for the CNF and CO-CNF Nanoparticles were analyzed morphologically, and the results are shown in Figure 8; among them, CNF has a longer length and a shorter diameter; the average diameter (D) of CNF is 37.89±3.07nm, and the length (L) About 504.97±82.19nm. The aspect ratio (L/D) is about 15.62±1.90; CO-CNF will form a network structure, CO will appear on CNF in the form of spherical particles, and the average diameter of the particles is 316±0.16nm.

另，發明人還針對(a)表面活性劑素SCR、(b)CO-CNF、(c)50SNPs、(d)100SNPs及(e)200SNPs及進行了傅立葉變換紅外光譜(Fourier-transform infrared spectroscopy，簡稱FTIR)分析，其FTIR光譜如第9圖所示；其中，表面活性劑素SCR係由蛋白質、脂肪、纖維及碳水化合物等不同成分組成之複雜混合物；在3369cm-1處之寬峰及在2925cm-1處之尖峰分別表示SCR中-OH及CH之拉伸振動；1539cm-1，1246cm-1(木質素之芳環振動)、1161cm-1 （COC半纖維素及木質素之不對稱拉伸）之尖峰係表示半纖維素及木質素之存在，而1748 cm-1 之尖峰則係表示吸收帶之存在；而與SCR一樣，在 CO-CNF中也觀察到-OH（3350 cm-1 之寬峰）及CH（2910 cm-1 ）之特徵峰；在1640 cm-1 及1426 cm-1 處之尖峰表示纖維素之吸收及結晶帶；在899 cm-1 處之C-H糖苷形變，1061 cm-1 處之尖峰則表示C-O價振動。同時，發明人還注意到，代表木質素及半纖維素之特徵峰在CO-CNF中不存在，這清楚地表明了它們已被去除；在CO及CO-CNF中，發明人均觀察到-OH及C-H之拉伸振動；在CO中，發明人係在1377 cm-1 處觀察到硫酸鹽之拉伸，而在1126 cm-1 處則觀察到糖苷鍵之形成；在CO-CNF中所形成之糖苷鍵(1125 cm-1 )則表明了CO及CNF係由糖苷鍵連接；另，在CO-CNF中，發明人尚觀察到CO-CNF之O=S=O(1266 cm-1 )及-O-SO3(848 cm-1 )之不對稱拉伸；此外，通過傅立葉變換紅外光譜FTIR分析，發明人亦發現木質素及半纖維素已被去除；最後，CO-CNF中之糖基變形則清楚表示出CO及CNF係透過糖基鍵而相互連接在一起的。In addition, the inventors also carried out Fourier-transform infrared spectroscopy (Fourier-transform infrared spectroscopy, Abbreviated as FTIR) analysis, its FTIR spectrum is shown in Figure 9; wherein, the surfactant SCR is a complex mixture of different components such as protein, fat, fiber and carbohydrates; the broad peak at 3369cm -1 and the The peak at 2925cm -1 represents the stretching vibration of -OH and CH in SCR respectively ; The sharp peak of ) indicates the existence of hemicellulose and lignin, and the sharp peak of 1748 cm -1 indicates the existence of absorption band; and the same as SCR, -OH (3350 cm -1 The broad peak of CH (2910 cm -1 ) and the characteristic peak of CH (2910 cm -1 ); the sharp peaks at 1640 cm -1 and 1426 cm -1 represent the absorption and crystallization bands of cellulose; the CH glycoside deformation at 899 cm -1 , 1061 The sharp peak at cm -1 represents the CO valence vibration. At the same time, the inventors also noticed that the characteristic peaks representing lignin and hemicellulose did not exist in CO-CNF, which clearly indicated that they had been removed; in both CO and CO-CNF, the inventors observed -OH and the stretching vibration of CH; in CO, the inventors observed the stretching of sulfate at 1377 cm -1 and the formation of glycosidic bonds at 1126 cm -1 ; the formation of CO-CNF The glycosidic bond of CO-CNF (1125 cm -1 ) indicates that CO and CNF are connected by glycosidic bond; in addition, in CO-CNF, the inventors have observed that O=S=O (1266 cm -1 ) and -Asymmetric stretching of O-SO3 (848 cm -1 ); In addition, through Fourier Transform Infrared Spectroscopy FTIR analysis, the inventors also found that lignin and hemicellulose have been removed; finally, the glycosyl deformation in CO-CNF It clearly shows that CO and CNF are connected to each other through glycosyl bonds.

按，X射線衍射（X-ray Diffraction，簡稱 XRD）檢測分析係一種用於分析材料結晶性及無定形性之檢測技術，能用來準確地分析不同界面上原子排列之定量信息，以藉由所檢測到之較高結晶度來表示聚合物基體結構具備更好之強化性。有鑑於此，發明人亦針對CNF及CO-CNF進行了X射線衍射檢測分析，以期通過XRD檢測，具體分析CNF及CO-CNF之結晶特性，其檢測結果如第10圖所示，其中，在CNF之衍射圖中所呈現22°及16°處之兩個特徵尖峰可能與纖維素I之結晶結構有關。CNF及CO-CNF之結晶指數分別約為70%和85%。這種結晶度的增加係由於分子間及分子內之羥基形成了分子間及分子內之H鍵，據此，應可理解為，在CNF中加載CO後，減少了無定形區域，從而增加了CO-CNF之結晶度。Press, X-ray Diffraction (X-ray Diffraction, referred to as XRD) detection and analysis is a detection technology used to analyze the crystallinity and amorphousness of materials, which can be used to accurately analyze the quantitative information of atomic arrangement on different interfaces, by The higher crystallinity detected indicates better reinforcement of the polymer matrix structure. In view of this, the inventor also carried out X-ray diffraction detection and analysis on CNF and CO-CNF in order to analyze the crystallization characteristics of CNF and CO-CNF through XRD detection. The detection results are shown in Figure 10. Among them, The two characteristic peaks at 22° and 16° in the diffraction pattern of CNF may be related to the crystal structure of cellulose I. The crystalline indices of CNF and CO-CNF are about 70% and 85%, respectively. This increase in crystallinity is due to the formation of intermolecular and intramolecular H bonds between intermolecular and intramolecular hydroxyl groups. Accordingly, it should be understood that after loading CO in CNF, the amorphous region is reduced, thereby increasing Crystallinity of CO-CNF.

一般言，在對材料進行細胞活性測試時，均係透過細胞活存率分析( (3-(4，5-Dimethylthiazol-2-yl)-2，5-diphenyltetrazolium bromide ，簡稱MTT assay) 測定法，予以評估，有鑑於此，發明人亦分別針對CO-CNF、50SNPs、100SNPs及200SNPs逐一進行了MTT分析測定，以期通過評估培養之細胞在用藥物處理時之代謝活性來檢測材料之毒性，其檢測結果如第11圖所示；其中，CO-CNF與其他組相比，顯示出最高之細胞存活率，且顯示細胞存活率係隨著表面活性劑濃度之增加而降低，當濃度高達100μg/L時，所有組都顯示出80%以上之細胞存活率；反之，當濃度達200μg/L時，則顯示100SNPs之細胞存活率會突然降低；當在400μg/L之濃度下，則顯示200SNPs之細胞存活率不到50%；當在200μg/L時，則顯示100SNPs之細胞存活率為最低。綜上所述，可明顯看出，表面活性劑之添加係以濃度依賴性之方式降低了細胞之存活率。 Generally speaking, when testing the cell activity of the material, it is through the cell viability analysis ((3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, referred to as MTT assay) assay method, To be evaluated, in view of this, the inventor also carried out MTT analysis and determination for CO-CNF, 50SNPs, 100SNPs and 200SNPs one by one, in order to detect the toxicity of the material by evaluating the metabolic activity of the cultured cells when they are treated with drugs. The results are shown in Figure 11; among them, CO-CNF showed the highest cell survival rate compared with other groups, and showed that the cell survival rate decreased with the increase of surfactant concentration, when the concentration was as high as 100μg/L When the concentration reached 200μg/L, the survival rate of cells showing 100SNPs suddenly decreased; when the concentration reached 400μg/L, the cell survival rate showing 200SNPs The survival rate was less than 50%; when it was at 200μg/L, the cell survival rate of 100SNPs was shown to be the lowest. In summary, it can be clearly seen that the addition of surfactants reduces the concentration of the cells in a concentration-dependent manner. survival rate.

按，保水能力(Water Holding Capacity，簡稱WHC)之檢測，主要係據以評估材料之孔隙率，畢竟，材料之持水能力主要係取決於其孔隙率及其相關之化學及物理結構特性，有鑑於此，發明人乃特別針對CNF及CO-CNF奈米顆粒分別以離心法檢測及分析其保水能力，其檢測結果如第12圖所示，其中，清楚顯示在室溫(即，攝氏23℃)及體溫(即，攝氏37℃)狀態下，CO-CNF具有較強之保水能力；尤其是，在23℃時，CNF之保水能力約為13g/g，當溫度增加至37℃時，其保水能力卻僅稍增加到接近15g/g；反之，CO-CNF之保水能力WHC則隨著溫度之升高而有著較為明顯的增加。在23℃及37℃時，CO-CNF之保水能力WHC分別為22及25g/g，據此，在這兩種溫度下，CO-CNF之保水能力WHC顯然均高於CNF之保水能力。 According to the test of Water Holding Capacity (WHC), it is mainly used to evaluate the porosity of the material. After all, the water holding capacity of the material mainly depends on its porosity and its related chemical and physical structural characteristics. In view of this, the inventors specifically tested and analyzed the water retention capacity of CNF and CO-CNF nanoparticles by centrifugation. The test results are shown in Fig. ) and body temperature (ie, 37°C), CO-CNF has a strong water retention capacity; especially, at 23°C, the water retention capacity of CNF is about 13g/g, when the temperature increases to 37°C, its The water retention capacity only increased slightly to close to 15g/g; on the contrary, the water retention capacity WHC of CO-CNF increased significantly with the increase of temperature. At 23°C and 37°C, the water retention capacity WHC of CO-CNF is 22 and 25 g/g respectively. Accordingly, at these two temperatures, the water retention capacity WHC of CO-CNF is obviously higher than that of CNF.

查，一般在對材料進行抗菌活性檢測時，均係利用瓊脂孔擴散法評估抗菌活性，其作法係在令瓊脂孔中之抗菌劑通過瓊脂培養基擴散，以抑制微生物之生長。有鑑於此，發明人亦特別針對去氧羥四環素(Doxycycline)、CO-CNF、50SNPs、100SNPs及200SNPs分別以瓊脂孔擴散法檢測及分析其抗菌活性能力，其檢測結果如第13及14圖所示，其係藉選擇使用Porphyromonas gingivalis及Streptococcus mutans兩種不同之口腔病原體(其檢測結果分別如第13及14圖所示)來評估各該樣品(即，CO-CNF、50SNPs、100SNPs及200SNPs)之抗菌活性；其中，藥物四環黴素之濃度為10mg/mL；表面活性劑之濃度為10 mg/mL；各該樣品之濃度則分別為20mg/mL濃度之CO-CNF；20mg/mL濃度之50SNP；20mg/mL濃度之100SNP及20mg/mL濃度之200SNP；其抗菌活性檢測檢測數據則如下表-3所示，其中清楚顯示，S.mutans口腔病原體對各該樣品之敏感度較高；雖然，在這兩種口腔病原體中沒有觀察到CO-CNF組之抑制區，但是，卻能清楚觀測到這兩種口腔病原體之抑制區會隨著表面活性劑濃度之增加而增加，200SNPs比CO-CNF、50SNPs及100SNPs表現出更高之抑制區。在P.gingivalis口腔病原體中，200SNPs在四環黴素旁邊表現出較好之活性，且會隨著表面活性劑濃度之增加，而令其奈米粒子之活性略有增加；此外，下表-3所列之抗菌活性檢測數據更清楚顯示，200SNPs與四環黴素相比，對兩種口腔病原體均有較好之抗菌活性。 Generally, when the antibacterial activity of materials is tested, the agar hole diffusion method is used to evaluate the antibacterial activity. The method is to make the antibacterial agent in the agar hole diffuse through the agar medium to inhibit the growth of microorganisms. In view of this, the inventors also specifically tested and analyzed the antibacterial activity of Doxycycline (Doxycycline), CO-CNF, 50SNPs, 100SNPs and 200SNPs by the agar well diffusion method, and the test results are shown in Figures 13 and 14. It is shown that each of the samples (i.e., CO-CNF, 50SNPs, 100SNPs and 200SNPs) was evaluated by choosing to use two different oral pathogens, Porphyromonas gingivalis and Streptococcus mutans (the results of which are shown in Figures 13 and 14, respectively). antibacterial activity; among them, the concentration of the drug tetracycline is 10 mg/mL; the concentration of the surfactant is 10 mg/mL; the concentration of each sample is 20 mg/mL concentration of CO-CNF; 20 mg/mL concentration 50SNP of 20mg/mL concentration; 100SNP of 20mg/mL concentration and 200SNP of 20mg/mL concentration; the antibacterial activity detection data are shown in the following table-3, which clearly shows that S.mutans oral pathogens are more sensitive to each sample; Although no zone of inhibition of the CO-CNF group was observed in these two oral pathogens, it was clearly observed that the zone of inhibition of these two oral pathogens increased with the increase in surfactant concentration, and 200SNPs were more than CO -CNF, 50SNPs and 100SNPs showed higher inhibition zones. In the P.gingivalis oral pathogen, 200SNPs showed better activity next to tetracycline, and with the increase of surfactant concentration, the activity of its nanoparticles increased slightly; in addition, the following table- The antibacterial activity test data listed in 3 more clearly show that 200SNPs has better antibacterial activity against two oral pathogens than tetracycline.

Figure 109118634-A0305-02-0020-3
Figure 109118634-A0305-02-0020-3

按，細菌之多細胞群落會產生生物膜，該生物膜係包裹在由多醣、蛋白質及DNA所組成之基質中。有鑑於此，發明人乃利用結晶紫測定法對生物膜進行了定量分析，復請參閱第13及14圖所示，且檢測出在P.gingivalis口腔病原體中，200SNP能表現出較其他各組具有更高之生物膜抑制作用，其次為四環黴素，其生物膜抑製作用約為40%。四環黴素對S.mutans口腔病原體之抑制作用超過50%，奈米顆粒對各該口腔病原體之抑制活性大小則依次為200SNPs>100SNPs>50SNPs>CO-CNF。據此，顯然從上述抗菌活性檢測結果可清楚看出，各該奈米顆粒CO-CNF、50SNPs、100SNPs或200SNPs分別具有減少細菌生長的特性，從而均能用以防止生物膜(如：前述之牙菌膜50)的形成。 Accordingly, multicellular colonies of bacteria produce biofilms that are encapsulated in a matrix composed of polysaccharides, proteins, and DNA. In view of this, the inventors conducted a quantitative analysis of the biofilm using the crystal violet assay, as shown in Figures 13 and 14, and detected that in the P.gingivalis oral pathogen, 200SNP can show a higher It has a higher biofilm inhibitory effect, followed by tetracycline, and its biofilm inhibitory effect is about 40%. The inhibitory effect of tetracycline on S.mutans oral pathogens was more than 50%, and the inhibitory activity of nanoparticles on each oral pathogen was 200SNPs>100SNPs>50SNPs>CO-CNF. Accordingly, it can be clearly seen from the above-mentioned antibacterial activity test results that each of the nanoparticle CO-CNF, 50SNPs, 100SNPs or 200SNPs has the characteristics of reducing bacterial growth respectively, thereby all can be used to prevent biofilm (such as: the aforementioned Plaque formation 50).

按，在對體外藥物持續釋放或控制釋放能力進行評估時，一般均係將藥物及奈米藥物之質量比稱之為藥物加載量，而在奈米藥物製備過程中，其藥物之利用率則係由藥物加載效率予以決定，且可透過體外藥物之誘導效率(Entrapment Efficiency，簡稱EE)、裝載能力(Loading Capacity，簡稱LC)及體外藥物釋放率等因子來評估奈米藥物之持續釋放或控制釋放能力。有鑑於此，發明人尚特別針對CO-CNF之EE及LC進行檢測，其檢測結果詳如第15圖所示，其中加載表面活性劑之CO-CNF的EE和LC分別為53.15±2.36%和36.72±1.24%；嗣，透過透析法評估CO-CNF中表面活性劑劑之體外藥物釋放情況，試驗結果發現，在1小時觀察到表面活性劑之快速釋放，可能係由於物理夾帶藥物之釋放，之後，CO-CNF則有效控制了藥物之釋放，而令約68%之藥物在隨後之24h時被穩定地釋放。 By the way, when assessing the sustained or controlled release of drugs in vitro, the mass ratio of the drug to the nano-drug is generally referred to as the drug loading, and in the preparation process of the nano-drug, the utilization rate of the drug is It is determined by the drug loading efficiency, and the sustained release or control of nano-drugs can be evaluated by factors such as in vitro drug induction efficiency (Entrapment Efficiency, EE), loading capacity (Loading Capacity, LC) and in vitro drug release rate. release ability. In view of this, the inventors specifically tested the EE and LC of CO-CNF, and the test results are shown in Figure 15. The EE and LC of CO-CNF loaded with surfactants were 53.15±2.36% and 53.15±2.36% respectively. 36.72 ± 1.24%; Then, the in vitro drug release of the surfactant agent in CO-CNF was evaluated by dialysis. The test results found that the rapid release of the surfactant was observed in 1 hour, which may be due to the release of physically entrapped drugs. Afterwards, CO-CNF effectively controlled the release of the drug, and about 68% of the drug was released stably in the next 24 hours.

據上論結，本發明採用農業或食品加工業之廢棄物(如：大豆豆漿渣)中之優質纖維素作為原料，利用成熟便捷之製備程序及設備，自該物美價廉之優質原料中，萃取出纖維素奈米纖維(Cellulose Nanofiber，簡稱CNF)，再進一步使用κ-卡拉膠低聚醣(κ-carrageenan oligosaccharides，簡稱CO)對CNF進行修飾，而製作出本發明具抗菌活性之纖維素奈米纖維(以下簡稱CO-CNF)，以有效改善CNF之降解溫度、結晶度、溶脹性及生物相容性...等材料特性，從而令本發明之CO-CNF得以被應用於製作出治療牙周炎所需使用之奈米牙周再生纖維膜。由於，牙周炎係屬於一種與微生物堆積有關之慢性炎症性疾病，而本發明之CO-CNF既經實驗證實確具對牙周病原體之抗菌活性，據此，它不僅能用以有效預防或改善牙周病症狀，尚能充分實現有效給藥之目的，從而在牙周炎之臨床治療過程中，對有效且精準地遞送藥物有著極大之潛力，而能大幅提升牙周炎之治療效果及效率，從而在牙齒保健及醫療產業中具有極高之利用價值。 According to the above conclusion, the present invention adopts the high-quality cellulose in the waste (such as: soybean soymilk dregs) of agriculture or food processing industry as raw material, utilizes mature and convenient preparation procedure and equipment, from this high-quality and cheap high-quality raw material, Extract cellulose nanofibers (Cellulose Nanofiber, referred to as CNF), and then further use κ-carrageenan oligosaccharides (abbreviated as CO) to modify CNF, and produce the cellulose with antibacterial activity of the present invention Nanofibers (hereinafter referred to as CO-CNF), to effectively improve the material properties of CNF such as degradation temperature, crystallinity, swelling and biocompatibility, so that the CO-CNF of the present invention can be applied to produce Nano periodontal regenerated fiber membrane required for the treatment of periodontitis. Because periodontitis is a chronic inflammatory disease related to microbial accumulation, and the CO-CNF of the present invention has been proved by experiments to have antibacterial activity against periodontal pathogens, accordingly, it can not only be used for effective prevention or Improving the symptoms of periodontal disease can still fully realize the purpose of effective drug delivery. Therefore, in the clinical treatment of periodontitis, it has great potential for effective and precise delivery of drugs, and can greatly improve the treatment effect and efficiency of periodontitis. It has extremely high utilization value in dental health care and medical industry.

此外，本發明在採用化學機械方法，萃取出CNF後，尚能透過CO對CNF進行修飾，以藉加載不同濃度劑量(如：50mg、100mg及200mg)之表面活性素，製成不同之奈米纖維材料50SNPs、100SNPs及200SNPs；嗣，分別對各該奈米纖維材料進行表徵分析，獲得CNF及CO-CNF之平均尺寸分別為263 nm和330 nm，且由顯微鏡研究結果顯示，CNF直徑較短、長度較長，而與CO交聯後所形成之CO-CNF則呈內含珠粒之網絡狀，且因CO之添加有效改善了CNF之降解溫度、結晶度及溶脹性，從而令所形成之CO-CNF材料具有受控之藥物釋放特性，令其藥物之包封率及載藥量分別達到53.15±2.36％和36.72±1.24％，此外，由於各該材料尚具有抗氧化之活性，因此不僅具備能防止生物膜形成之能力，尚具備減少代謝活性及促進氧化應激來抑制牙周病原體(例如：變形鏈球菌及牙齦卟啉單胞菌)生長之能力。據此，本發明藉由成功地提取CNF，並以CO對其進行修飾而製成CO-CNF，不僅能有效改善CNF之物理特性及參數，尚令所形成之CO-CNF能因加載有表面活性素，而對牙周病原體具有潛在之抗菌活性，從而令所獲得之CO-CNF材料在生物醫學應用領域中成為一具有巨大潛力之生物材料，且因此而令其在醫療產業上具備極大之經濟價值。In addition, after the CNF is extracted by the chemical mechanical method, the present invention can modify the CNF by CO, so that different concentrations of surfactin (such as: 50mg, 100mg and 200mg) can be loaded to make different nanoparticles. Fiber materials 50SNPs, 100SNPs and 200SNPs; Next, the nanofiber materials were characterized and analyzed, and the average size of CNF and CO-CNF were 263 nm and 330 nm, respectively, and the results of microscope research showed that the diameter of CNF was shorter , the length is longer, and the CO-CNF formed after cross-linking with CO is in the form of a network containing beads, and the addition of CO can effectively improve the degradation temperature, crystallinity and swelling of CNF, so that the formed The CO-CNF material has controlled drug release characteristics, so that the encapsulation efficiency and drug loading of the drug can reach 53.15±2.36% and 36.72±1.24%, respectively. In addition, because each of the materials still has antioxidant activity, so Not only has the ability to prevent biofilm formation, but also has the ability to reduce metabolic activity and promote oxidative stress to inhibit the growth of periodontal pathogens (such as: Streptococcus mutans and Porphyromonas gingivalis). Accordingly, the present invention successfully extracts CNF and modifies it with CO to make CO-CNF, which not only can effectively improve the physical properties and parameters of CNF, but also enables the formed CO-CNF to be capable of being loaded with surface active factor, and has potential antibacterial activity against periodontal pathogens, so that the obtained CO-CNF material becomes a biomaterial with great potential in the field of biomedical applications, and thus makes it have great potential in the medical industry. Economic Value.

按，以上所述，僅為本發明最佳之一具體實施例，惟本發明之技術特徵並不侷限於此，任何熟悉該項技藝者在本發明領域內，可輕易思及之等效變化或修飾，皆可涵蓋在以下本案之專利範圍。According to, the above description is only one of the best specific embodiments of the present invention, but the technical characteristics of the present invention are not limited thereto, any equivalent changes that can be easily conceived by anyone familiar with the art in the field of the present invention Or modification, can be covered in the following patent scope of this case.

[習知] 40:牙齒 41:齒冠 411:琺瑯質 412:象牙質 413:齒髓組織 42:牙根 420:牙周膜 43:牙齦 44:牙槽骨 50:牙菌膜 52:發炎症狀 53:部位 54:牙結石 56:空間 58:萎縮 [本發明] 200、210、220、201~207、211~212、221~225:步驟[knowledge] 40: Teeth 41: crown 411: Enamel 412: Dentin 413:Dental pulp tissue 42: tooth root 420: Periodontal membrane 43: Gums 44:Alveolar bone 50: Plaque 52: Symptoms of inflammation 53: parts 54: dental calculus 56: space 58: Atrophy [this invention] 200, 210, 220, 201~207, 211~212, 221~225: steps

第1圖係人類牙齒之結構示意圖；第2圖係人類齒列的外觀及剖面示意圖；第3圖係罹患牙齦炎之人類齒列的外觀及剖面示意圖；第4圖係罹患牙周炎之人類齒列的外觀及剖面示意圖；第5圖係纖維素奈米纖維材料自2016年到2024年預估之複合年增長率趨勢示意圖；第6圖係本發明製造方法之流程示意圖；第7圖係本發明將CNF轉化成CO-CNF及以κ-卡拉膠低聚醣對CO-CNF進行包封之示意圖；第8圖(a)係本發明對CNF、CO-CNF及CO-CNF進行檢測之透射電子顯微圖及(b)掃描電子顯微鏡(SEM)圖；第9圖(a)、(b)、(c)、(d)及(e)係分別代表表面活性劑素SCR、CO-CNF、50SNPs、100SNPs及200SNPs之傅立葉變換紅外光譜(FTIR)分析示意圖；第10圖係CNF及CO-CNF之X射線衍射(XRD)分析示意圖；第11圖係CO-CNF、50SNPs、100SNPs和200SNPs之細胞活力分析示意圖；第12圖係CNF及CO-CNF之保水能力評估分析示意圖；第13圖係P.gingivalis之生物膜抑制作用的奈米顆粒定量分析示意圖；第14圖係S.mutans之生物膜抑制作用的奈米顆粒定量分析示意圖；及第15圖係誘導效率(Entrapment Efficiency，EE)、裝載能力(Loading Capacity，LC)及體外藥物釋放率之釋放譜示意圖。Figure 1 is a schematic diagram of the structure of a human tooth; Figure 2 is a schematic diagram of the appearance and cross-section of a human dentition; Figure 3 is a schematic diagram of the appearance and cross-section of a human dentition suffering from gingivitis; Figure 4 is a schematic diagram of a human dentition suffering from periodontitis Figure 5 is a schematic diagram of the estimated compound annual growth rate trend of cellulose nanofiber materials from 2016 to 2024; Figure 6 is a schematic flow chart of the manufacturing method of the present invention; Figure 7 is a schematic diagram of the present invention Schematic diagram of converting CNF into CO-CNF and encapsulating CO-CNF with κ-carrageenan oligosaccharides; Figure 8 (a) is the transmission electron detection of CNF, CO-CNF and CO-CNF in the present invention Micrograph and (b) Scanning Electron Microscope (SEM) picture; Figure 9 (a), (b), (c), (d) and (e) represent surfactant SCR, CO-CNF, Schematic diagram of Fourier transform infrared spectroscopy (FTIR) analysis of 50SNPs, 100SNPs and 200SNPs; Figure 10 is a schematic diagram of X-ray diffraction (XRD) analysis of CNF and CO-CNF; Figure 11 is a schematic diagram of cells of CO-CNF, 50SNPs, 100SNPs and 200SNPs Schematic diagram of activity analysis; Figure 12 is a schematic diagram of water retention capacity assessment analysis of CNF and CO-CNF; Figure 13 is a schematic diagram of nanoparticle quantitative analysis of P.gingivalis biofilm inhibition; Figure 14 is a biofilm of S.mutans Schematic diagram of nanoparticle quantitative analysis of inhibition; and Figure 15 is a schematic diagram of the release profile of induction efficiency (Entrapment Efficiency, EE), loading capacity (Loading Capacity, LC) and in vitro drug release rate.

200、210、220、201~207、211~212、221~225:步驟200, 210, 220, 201~207, 211~212, 221~225: steps

Claims (9)

一種利用表面活性劑加載k-卡拉膠低聚醣製成對牙周病原體具抗菌活性之纖維素奈米纖維的製法，該製法包括下列步驟：自大豆豆漿渣的纖維素中萃取纖維素奈米纖維(CNF)；使用κ-卡拉膠低聚醣(CO)對該纖維素奈米纖維(CNF)進行修飾，以製作出具抗菌活性之纖維素奈米纖維(CO-CNF)；及利用表面活性劑，包封該CO-CNF，製作出對牙周病原體具抗菌活性之奈米纖維材料。 A method for preparing cellulose nanofibers with antibacterial activity against periodontal pathogens by using k-carrageenan oligosaccharides loaded with surfactants, the preparation method comprising the following steps: extracting cellulose nanofibers from cellulose of soybean dregs fiber (CNF); using κ-carrageenan oligosaccharide (CO) to modify the cellulose nanofiber (CNF) to produce cellulose nanofiber (CO-CNF) with antibacterial activity; and utilizing surface activity agent, encapsulating the CO-CNF, and producing a nanofibrous material with antibacterial activity against periodontal pathogens. 如請求項1所述之製法，該製法係依下列步驟，自該大豆豆漿渣的纖維素中萃取出該纖維素奈米纖維(CNF)：首先，將20公克乾燥之該豆漿渣與250毫升5%濃度之氫氧化鈉(NaOH)溶劑，予以均勻混合成一混合溶液，且在攝氏120℃之溫度下，利用一攪拌機對該混合溶液予以攪拌2小時，並在進行2次攪拌令該豆漿渣與該氫氧化鈉(NaOH)溶劑均勻混合後，利用蒸餾水洗滌該混合溶液；嗣，在攝氏80℃之溫度下，使用250毫升1莫耳濃度之氯化氫(HCl)溶劑對該混合溶液中之殘渣進行殘渣清洗處理2小時；嗣，在攝氏50℃之溫度下，以250毫升5%濃度之次氯酸鈉(NaClO)溶劑對該混合溶液進行漂白處理，直到獲得白色的混合溶液為止；嗣，使用蒸餾水對該白色混合溶液進行清洗處理後，在攝氏45℃之溫度下，添加250毫升64%濃度之硫酸(H2SO4)溶劑至該白色混合溶液中，且利用攪拌機對該白色混合溶液予以攪拌1-2小時來進行水解作業；嗣，添加500毫升且溫度為攝氏10℃的冷水至該白色混合溶液來終止水解反應； 嗣，使用高速離心機以8000×g之轉速，對該白色混合溶液執行至少10分鐘之離心處理後，將該白色混合溶液以蒸餾水中透析5天；及最後，使用一均質機以11000轉/分的速度對該白色混合溶液執行至少2小時之均質處理後，對該白色混合溶液執行冷凍乾燥，即能自該豆漿渣中萃取出該等CNF奈米纖維顆粒。 The preparation method as described in claim 1, the preparation method is to extract the cellulose nanofiber (CNF) from the cellulose of the soybean dregs according to the following steps: first, mix 20 grams of dried soy dregs with 250 ml 5% sodium hydroxide (NaOH) solvent was uniformly mixed into a mixed solution, and at a temperature of 120°C, the mixed solution was stirred for 2 hours with a stirrer, and the soybean milk dregs were stirred twice. After uniformly mixing with the sodium hydroxide (NaOH) solvent, wash the mixed solution with distilled water; then, at a temperature of 80°C, use 250 ml of 1 molar hydrogen chloride (HCl) solvent to remove the residue in the mixed solution Carry out residue washing treatment for 2 hours; then, at a temperature of 50° C., bleach the mixed solution with 250 ml of 5% sodium hypochlorite (NaClO) solvent until a white mixed solution is obtained; then, use distilled water to After cleaning the white mixed solution, add 250 ml of 64% sulfuric acid (H2SO4) solvent to the white mixed solution at a temperature of 45°C, and stir the white mixed solution with a mixer for 1-2 hours To carry out the hydrolysis operation; then, add 500 ml of cold water with a temperature of 10° C. to the white mixed solution to terminate the hydrolysis reaction; Then, after centrifuging the white mixed solution for at least 10 minutes at a speed of 8000 × g using a high-speed centrifuge, dialyze the white mixed solution in distilled water for 5 days; and finally, use a homogenizer at 11000 rpm After homogenizing the white mixed solution for at least 2 hours at a speed of 10 minutes, the white mixed solution is freeze-dried to extract the CNF nanofiber particles from the soybean milk dregs. 如請求項2所述之製法，該製法係依下列步驟，利用κ-卡拉膠低聚醣(CO)對該等CNF奈米纖維顆粒進行修飾處理：首先，將1公克之κ-卡拉膠低聚醣溶解在100毫升(ml)的蒸餾水中，形成κ-卡拉膠低聚醣水溶液後，將κ-卡拉膠低聚醣水溶液添加至100毫升濃度為30mg/ml的CNF水溶液中，形成CO-CNF混合溶液，並以攪拌機在攝氏80℃之溫度下，對CO-CNF混合溶液進行攪拌30分鐘；及嗣，在攝氏80℃之溫度下，利用攪拌機以800rpm轉速對CO-CNF混合溶液進行強烈攪拌至少3小時，且在強烈攪拌過程中，將50毫升1莫耳濃度之氯化鉀(KCl)溶劑添加至CO-CNF混合溶液，俟CO-CNF混合溶液完全膠凝後，通過離心機以8000×g之轉速，對該CO-CNF混合溶液執行至少10分鐘的離心處理後，收集固體，並對其進行超聲波處理，且將獲得的樣品用蒸餾水清滌並予冷凍乾燥後，即能獲得該CO-CNF。 As the preparation method described in claim 2, the preparation method is to use κ-carrageenan oligosaccharides (CO) to modify the CNF nanofiber particles according to the following steps: first, 1 gram of κ-carrageenan low After the polysaccharide was dissolved in 100 milliliters (ml) of distilled water to form an aqueous solution of κ-carrageenan oligosaccharides, the aqueous solution of κ-carrageenan oligosaccharides was added to 100 ml of CNF aqueous solution with a concentration of 30 mg/ml to form CO- CNF mixed solution, and the CO-CNF mixed solution was stirred for 30 minutes with a stirrer at a temperature of 80°C; Stir for at least 3 hours, and during vigorous stirring, add 50 ml of 1 molar potassium chloride (KCl) solvent to the CO-CNF mixed solution. After the CO-CNF mixed solution is completely gelled, pass it through a centrifuge to After centrifuging the CO-CNF mixed solution at a speed of 8000×g for at least 10 minutes, the solid was collected and subjected to ultrasonic treatment, and the obtained sample was washed with distilled water and freeze-dried to obtain The CO-CNF. 如請求項3所述之製法，該製法係依下列步驟，利用該表面活性劑，包封該CO-CNF：首先，將50毫升濃度為3.2mg/mL之該CO-CNF溶液，通過超音波溶解於蒸餾水中，形成CO-CNF混合溶液備用； 以非離子性表面活性劑做為表面活性劑，將50mg、100mg與200mg之各該非離子性表面活性劑分別溶解於2毫升之甲醇溶劑中，形成不同濃度的各該表面活性劑溶液備用；在攝氏60℃之溫度下，利用攪拌機以800rpm轉速對該CO-CNF混合溶液進行強烈攪拌時，將200微升(μL)不同濃度油相之各該表面活性劑溶液分別添加至水相之CO-CNF溶液中，繼續攪拌20分鐘；嗣，將8毫升濃度為1.87mg/mL之穩定劑，添加至該CO-CNF混合溶液且持續攪拌2小時，其中，該穩定劑為亞甲基雙丙烯醯胺；及最後，經超聲處理1小時後，即能獲得經不同濃度表面活性劑包封之奈米顆粒材料50SNPs、100SNPs、200SNPs。 As the preparation method described in claim 3, the preparation method is to use the surfactant to encapsulate the CO-CNF according to the following steps: first, 50 milliliters of the CO-CNF solution with a concentration of 3.2 mg/mL is passed through ultrasonic waves Dissolve in distilled water to form a CO-CNF mixed solution for later use; With non-ionic surfactant as surfactant, 50mg, 100mg and 200mg of each of the non-ionic surfactants were dissolved in 2 ml of methanol solvent respectively to form each of the surfactant solutions of different concentrations for subsequent use; At a temperature of 60°C, when the CO-CNF mixed solution was vigorously stirred with a stirrer at a speed of 800 rpm, 200 microliters (μL) of the surfactant solutions in the oil phase with different concentrations were added to the CO-CNF in the water phase. In the CNF solution, continue to stir for 20 minutes; then, add 8 ml of a stabilizer with a concentration of 1.87 mg/mL to the CO-CNF mixed solution and continue to stir for 2 hours, wherein the stabilizer is methylene bisacryl amine; and finally, after ultrasonic treatment for 1 hour, nanoparticle materials 50SNPs, 100SNPs, and 200SNPs encapsulated by different concentrations of surfactants can be obtained. 如請求項4所述之製法，其中，該非離子性表面活性劑係Tween 80。 The method according to claim 4, wherein the nonionic surfactant is Tween 80. 如請求項5所述之製法，其中，該CNF及該CO-CNF之平均尺寸分別為263.60±12.27nm及330.66±16.44nm。 The method according to claim 5, wherein the average size of the CNF and the CO-CNF is 263.60±12.27nm and 330.66±16.44nm, respectively. 如請求項6所述之製法，其中，該CNF及該CO-CNF之PDI值為0.4。 The method according to claim 6, wherein the PDI value of the CNF and the CO-CNF is 0.4. 如請求項7所述之製法，其中，該50SNPs、100SNPs及200SNPs係採用油包水乳化法(W/O emulsion)製備。 The method of claim 7, wherein the 50SNPs, 100SNPs and 200SNPs are prepared by water-in-oil emulsion (W/O emulsion). 如請求項8所述之製法，其中，該CNF及CO-CNF之結晶指數分別為70%及85%。The manufacturing method as described in Claim 8, wherein the crystallization indices of the CNF and CO-CNF are 70% and 85% respectively.
TW109118634A 2020-06-03 2020-06-03 Cellulose nanofibers with antibacterial activity against periodontal pathogens prepared by loading k-carrageenan oligosaccharides with surfactants and its preparation method TWI794608B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
TW109118634A TWI794608B (en) 2020-06-03 2020-06-03 Cellulose nanofibers with antibacterial activity against periodontal pathogens prepared by loading k-carrageenan oligosaccharides with surfactants and its preparation method

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
TW109118634A TWI794608B (en) 2020-06-03 2020-06-03 Cellulose nanofibers with antibacterial activity against periodontal pathogens prepared by loading k-carrageenan oligosaccharides with surfactants and its preparation method

Publications (2)

Publication Number Publication Date
TW202146002A TW202146002A (en) 2021-12-16
TWI794608B true TWI794608B (en) 2023-03-01

Family

ID=80783684

Family Applications (1)

Application Number Title Priority Date Filing Date
TW109118634A TWI794608B (en) 2020-06-03 2020-06-03 Cellulose nanofibers with antibacterial activity against periodontal pathogens prepared by loading k-carrageenan oligosaccharides with surfactants and its preparation method

Country Status (1)

Country Link
TW (1) TWI794608B (en)

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
期刊 Zarina S, et al. BioResources "Biodegradable composite films based on k-carrageenan reinforced by cellulose nanocrystal from kenaf fibers", BioResources, 10(1) 256-271, 2015.

Also Published As

Publication number Publication date
TW202146002A (en) 2021-12-16

Similar Documents

Publication Publication Date Title
US11970789B2 (en) Filaments comprising an active agent nonwoven webs and methods for making same
Kim et al. Carbon nanotube incorporation in PMMA to prevent microbial adhesion
Khan et al. Tinidazole functionalized homogeneous electrospun chitosan/poly (ε-caprolactone) hybrid nanofiber membrane: Development, optimization and its clinical implications
Mei et al. Improved biological characteristics of poly (L-lactic acid) electrospun membrane by incorporation of multiwalled carbon nanotubes/hydroxyapatite nanoparticles
EP2462265B1 (en) Filaments comprising an active agent, nonwoven webs and methods for making same
RU2541275C2 (en) Filaments, containing non-aromatised active agent, non-woven cloths and methods of obtaining thereof
Nobre et al. Adhesion of hydroxyapatite nanoparticles to dental materials under oral conditions
RU2543892C2 (en) Production of films from nonwoven webs
CN106492283B (en) A kind of mineralising guide tissue regeneration film and its preparation method and application
Wu et al. Multifunctional chitin-based barrier membrane with antibacterial and osteogenic activities for the treatment of periodontal disease
Seo et al. Enhanced mechanical properties and bone bioactivity of chitosan/silica membrane by functionalized-carbon nanotube incorporation
TW201242626A (en) Composite particle, manufacturing method and application thereof
Zhang et al. Bilayered PLGA/wool keratin composite membranes support periodontal regeneration in beagle dogs
CN104248775A (en) Hydroxyapatite-chitosan compound gel and preparation process and application thereof
TWI794608B (en) Cellulose nanofibers with antibacterial activity against periodontal pathogens prepared by loading k-carrageenan oligosaccharides with surfactants and its preparation method
JPH10502554A (en) Alginate fibers, their production and use
Vaishali et al. Nanotechnology in periodontics: a review
Alipour et al. Fabrication of a novel fibrous mat based on gliadin/ethylcellulose incorporated with triamcinolone for treatment of oral ulcers
CN114775266A (en) Zinc oxide-loaded antibacterial blood-like ultrashort fiber and preparation method and application thereof
CN106924090A (en) A kind of preparation and application of the immobilized lysozyme toothpaste of multistage hole bioactive glass
CN107106729A (en) Bioactivity wound dressing and tooth coating based on the amorphous calcium polyphosphate with morphogenic activity
CN111956595A (en) A dental mouthpiece for preventing or treating dental caries and periodontal disease
CN105534725B (en) A kind of comprehensive function type toothpaste containing nanometer diamond alkene
Chen et al. A Biomimetic Smart Nanoplatform as “Inflammation Scavenger” for Regenerative Therapy of Periodontal Tissue
CN116284872B (en) Nano composite hydrogel and preparation method and application thereof