Photo-responsive drug delivery system based on photodynamic and photothermal effect

이준석 Pohang University of Science and Technology 2015 국내박사

Numerous types of chemotherapeutics have been developed in order to conquer the various diseases. In particular, chemotherapy is one of the most commonly implemented therapy for the conventional treatment of cancer. Nevertheless, a hydrophobic nature of the most of the anticancer drugs impedes an efficiency of chemotherapy. Moreover, severe side effect are manifested owing to the nonspecifically delivered drugs after systemic administration. For the reduction the side effect and enhancement of the efficacy, therefore, construction of an intelligent drug delivery system have been on the rise for biomedical application of chemotherapy. A brief overview of the emerging needs for the drug delivery system begins Chapter I, followed by grafting stimuli-responsive properties into drug delivery system. Among the various stimuli, an importance and the application of light, in particular, near infrared (NIR) window in a biomedical application is featured. Finally, the end of the chapter is focused on the recent progress of photodynamically and photothermally assisted drug delivery system. In Chapter II, a model study of long wavelength light responsive drug releasing platform based on the combination of the singlet oxygen sensitive linker (SOSL) and the photosensitizer (PS) is demonstrated. A long wavelength light responsive MSN (Pc@AP-E) was successfully rendered through the incorporation of PS into the pores of mesoporous silica nanoparticle (MSN) and the decoration of model drug on the surface of via SOSL. Upon an irradiation of the long wavelength light, singlet oxygen (1O2) was actively generated and it readily broke the SOSL to facilitate the release of the model drug from the surface of the carrier. Considering an excellent sensitivity of the Pc@AP-E against external stimulus and the active generation of 1O2, a potential as a photodynamically assisted drug releasing system had to be further investigated in advance. In the quest for the realization of the previous study for the biomedical application, an improved platform was developed and the detail is described in Chapter III. To provide better biocompatibility and stability in biological application, the real anticancer drug doxorubicin (DOX) was loaded in the PEGylated MSN which PEG was conjugated via SOSL. The release of the loaded drug was promoted by external irradiation of red light as expected. Surprisingly, the nano-carrier also exhibited an accelerated drug release at acidic pH condition, which is further favorable for the effective intracellular drug delivery. Moreover, the nano-carrier showed an enhanced cytotoxicity, especially, against chemo-resistant hepatocellular carcinoma (Hep3B) and colorectal cancer (HCT-8) cell line, as a result of the combination between photodynamic process and chemotherapy. In accordance to the high biocompatibility and photodynamically assisted drug release, our precisely designed platform has an immense potential to provide a combination of photodynamic and chemotherapy for the effective treatment of cancer. Beside photodynamic therapy, another technique commonly tried for the treatment of cancer with regard to the irradiation of NIR is photothermal therapy. Chapter IV and V present the development of photothermally assisted drug delivery platform mediated by the combination of photothermal agent and the thermo-sensitive phase change material (PCM). In particular, Chapter IV is mainly focused on the first introduction of PCM into the photo-responsive drug delivery system and its biomedical application. Among the various photothermal agents, gold nanorods (AuNRs) have attracted much attention by means of strong photothermal conversion and great biocompatibility. In order to combine the AuNRs and PCM, AuNR was covered by mesoporous silica shell (AuNR@mSiO2) to provide the stability and the reservoir for the drug. Through the facile fabrication method, several AuNR@mSiO2 formed a nano-assembly (DOX/PCM-AuNR@mSiO2) mediated by DOX loaded PCM. Photothermal conversion of AuNR core under the irradiation of NIR induced the phase transition of PCM, followed by the rapid release of the loaded drug. The nano-assembly exhibited an enhanced cytotoxicity against various cancer cells and the intracellular release of the DOX was easily monitored. Overall results reveal the broad applicability of PCM-based photothermally controllable drug delivery platform for an effective hydrophobic drug carrier in biomedical application. While the previous chapter is mainly focused on the comprehensive study of PCM-based photothermally assisted drug releasing platform, more fundamental studies for the investigation of the basic mechanism of PCM-based drug delivery platform is described in Chapter V. A PCM-based photothermally assisted drug releasing platform was formulated in a different manner. Single-layered graphene oxide (GO) was covered by mesoporous silica channel to form a honeycomb-like structure (GO@MS). Blend of DOX and PCM was loaded in the porous structure, thus the chocolate waffle-like nano-composite (DOX/PCM-GO@MS) was obtained. The nano-composite exhibited a similar photothermo-responsive releasing property in a same manner with previous nano-assembly and the even more than 50 times higher photo-induced cytotoxicity was observed. Moreover, intracellular uptake of GO material was successfully demonstrated by single cell Raman spectroscopy of trafficking GO. Additionally, the uptake inhibitor study implied that the main route of the endocytosis of PCM-coated material relies on the lipid-raft mechanism. Along with a series of studies for PCM-based photothermo-repsonsive system, photothermally assisted drug releasing platform possesses a feasibility for the spatiotemporally controllable drug delivery system.

Polymer-functionalized graphene oxide for effective gene and drug delivery

김현우 Pohang University of Science and Technology 2015 국내박사

Nanomaterials give us interesting physicochemical and biological properties for biomedical application owing to their nano size, large surface area and unique ability to interact with the biological system. In particular, graphene oxide (GO) has been considered “the future promising material” as it features their unique mechanical, electronic, and optical properties and has been exploited in novel electronic, store energy and other fields. The GO is now expanding its region from electronic and chemical application to biomedical areas such as biomolecule sensing and drug/gene delivery. Therefore, in Chapter I, we describe synthesis, properties and biological application of GO, and discuss the recent studies on the toxicity of GO to provide some perspective on the possible risks to their future development in materials and biomedical fields. In Chapter II, we describe the development of GO-based efficient gene delivery carrier through installation of polyethylenimine (PEI), a cationic polymer, which has been widely used as a non-viral gene delivery vector. It was revealed that hybrid gene carrier fabricated by conjugation of low molecular weight (LMW) branched PEI (BPEI) to GO increased the virtual molecular weight of BPEI, and consequently improved the DNA binding and condensation, and transfection efficiency. Furthermore this hybrid material facilitated bioimaging due to its tunable and intrinsic optical properties. Considering the extremely high transfection efficiency comparable to high molecular weight (HMW) BPEI, high cell viability, and its application as a bioimaging agent BPEI-GO hybrid material could be extended to drug delivery and photo thermal therapy. In Chapter III, we mention about development of photothermally controlled gene/drug delivery carrier by conjugating low molecular weight BPEI and reduced graphene oxide (rGO) via hydrophilic polyethylene glycol (PEG) spacer. The PEG-BPEI-rGO nanocomposite can form stable nano-sized complex with plasmid DNA (pDNA) as confirmed by physicochemical studies. In vitro gene transfection study, PEG-BPEI-rGO shows higher gene transfection efficiency without observable cytotoxicity compared to unmodified controls in PC-3 and NIH/3T3 cells. In addition, Moreover, PEG-BPEI-rGO nanocomposite demonstrates enhanced gene transfection efficiency upon NIR irradiation which is attributed to accelerated endosomal escape of polyplexes augmented by locally induced heat. Moreover, Meanwhile, PEG-BPEI-rGO also plays a role as a nanotemplate for photothermally triggered cytosolic drug delivery by inducing endosomal disruption and subsequent drug release. PEG-BPEI-rGO has ability to load more amount of Doxorubicin (DOX) than unreduced PEG-BPEI-GO via π-π and hydrophobic interactions, showing high water stability. Loaded DOX could be efficiently released by glutathione (GSH) and photothermal effect of irradiated near IR (NIR) in vials as well as in cells. Importantly, PEG-BPEI-rGO/DOX complex was found to escape from endosome after cellular uptake by photothermally induced endosomal disruption and proton sponge effect, followed by GSH-induced DOX release into cytosol. Finally, it was concluded that more cancer cell death efficacy was observed in PEG-BPEI-rGO/DOX complex-treated cells with NIR irradiation rather than without. This study demonstrated the development of the potential of PEG-BPEI-rGO nanocarrier as to photothermally triggered cytosolic gene/drug delivery via endosomal disruption. Even though GO has attracted huge interest in the area of biomedical application due to its unique physicochemical properties, the issue of its long-term toxicity in the body remains unclear. In Chapter IV, we describe the rationally designed GO nanotemplate (ssFGO) with PEG and BPEI via disulfide linkage to control the biological delivery and elimination in the body sequentially. Polymer-shielded GO was uptaken selectively as drawing a distinction between target cells and macrophages, and bioreducible ssFGO filled the role of the designed function that performs in reducible cellular environment. In the blood circulation, the shielding effect of PEG in ssFGO reduced their non-specific uptake by macrophages which induce unwanted elimination, thus improving their accumulation into target cells. After cellular uptake, the ssFGO easily escaped from endosome by photothermally induced endosome disruption, followed by fast gene dissociation and de-PEGylation under intracellular reducible environment, thereby showing much higher gene transfection efficiency with low toxicity than FGO and control BPEIs. Moreover, after exocytosis, de-PEGylated ssFGO easily entrapped in macrophage, followed by enzymatic degradation. The degradation process was monitored by photoluminescence enhancement from degraded GO fragments. These results highlight new directions in the design of biodegradable and multifunctional GO based nanomedicine. Our precisely designed ssFGO suggests a new strategy in a certainly biodegradable manner which facilitates elimination in the body, thus may overcome current impediments to use of GO derivatives in delivery system.

Development of magnetically guided drug delivery system using nano-assembled particles

전현정 Pohang University of Science and Technology 2016 국내석사

Conventional chemotherapy is a common technique for cancer treatment using small molecule based chemical substance. However, when small molecule drug circulates in the body, it affects to normal tissue as well as tumor tissue. Such unwanted accumulations lead to insufficient therapeutic effect as well as severe side effect such as vomit, hair loss, and body weight loss. In this aspect, nanomedicine has gained considerable attention in recent years as an alternative of conventional anticancer drug by improving the therapeutic efficiency and reducing side effects of anticancer drug. Conventional anticancer drugs are loaded in the nano-sized template that range 20-200 nm in size tend to accumulate selectively at tumor tissue region to exploit the effect by the enhanced permeability and retention (EPR) effect. Paclitaxel is a well-known traditional anticancer drug as a mitotic inhibitor on variable cancers including breast, lung and ovarian cancers. It is generally treated as a mixture of polyoxyethylate castor oil and ethanol (trade name is Taxol) because of its poor solubility in physiological condition. Its blend, however, gives rise to serious side effects due to the toxicity of solvating agents, thus the improvement of its solubility in aqueous condition has been required. To overcome the solubility problem, we employed herein host-guest interaction between βCD and PTX. By introducing βCD, solubility of paclitaxel was tremendously improved. Even more, multivalent interaction between βCD and paclitaxel was employed by polymer-conjugated βCD and polymer conjugated paclitaxel; this multivalent interaction between βCD and PTX improved to yield highly stable nano-assembly as well. Superparamagnetic iron oxide nanoparticles (SPIONs) have been utilized for various biomedical applications due to its paramagnetism which confers imaging ability and targeting ability. Especially, its targeting ability, i.e., magnetically guided delivery system has shown immense potential as a promising delivery system by improving accumulation in target site under the magnetic field. Therefore, the hybrid of the aforementioned polymer complex and the SPIONs is anticipated for improving the efficiency of drug delivery. This work demonstrates the development of magnetically guided drug delivery systems and its potential on efficient anticancer therapy. The magnetically guided drug delivery system was successfully developed by utilizing superparamagnetic iron oxide nanoparticle, β-cyclodextrin, and polymerized paclitaxel. Multivalent host-guest interactions between β-cyclodextrin conjugated superparamagnetic iron oxide nanoparticle and polymerized paclitaxel allowed to load the paclitaxel and the nanoparticle into the nano-assembly. Clustered superparamagnetic iron oxide nanoparticles in the nano-assembly permitted the rapid and efficient targeted drug delivery. Compared to the control groups, the developed nano-assembly showed the enhanced anticancer effects in vivo as well as in vitro. Therefore, the strategy of the use of superparamagnetic nanoparticles and multivalent host-guest interactions has a promising potential for developing the efficient drug delivery systems. 약물을 이용한 항암치료의 부작용을 줄이고 약물 전달과정 중에 생기는 약물의 손실을 줄이고자 약물 전달시스템이 활발히 연구되고 있다. 약물 전달 시스템이란 약물이 효험을 나타내기까지의 과정을 제어하는 방법으로, 약물의 부작용을 줄이면서 효험을 증대시키는 것을 목적으로 한다. 본 연구에서는 소수성 항암제인 파클리탁셀 (paclitaxel, PTX)의 체내 조건 내에서의 분산성 및 안정성을 높이고, 표적부위로의 전달효율을 향상시키고자 주인-손님 상호작용을 이용한 약물 전달시스템을 사용하였다. 주인 분자로는 β-사이클로덱스트린 (β-Cyclodextrin)을 사용하였으며 주인분자와 손님분자인 파클리탁셀을 각각 고분자와 결합한 후, 다가의 주인-손님 상호작용으로 나노 자기조립체를 형성하였다. 이를 통해 약물의 혈액 내 용해도를 증가시키고. 혈액 내에서도 전달체의 형태를 유지하며, 암 조직까지 약물을 효율적으로 전달시키고자 하였다. 또한 약물의 전달효율을 보다 향상시키고자 두 가지 전략을 사용하였다. 먼저 적정 나노크기로 만들어 enhanced permeability and retention (EPR) 효과를 이용하여 암으로의 선택적인 축적 효과를 증대시키고자 하였다. 또 다른 전략으로는 자기 유도 전달(magnetically guided delivery) 방법을 사용하였다. 자기 유도 방법이란 외부 자기장을 표적 부위로 가하여 약물 전달체의 표적 지향성을 향상시키는 것을 말하며, 그를 위하여 외부 자기장이 있을 때에만 자성을 띠는 성질을 지닌 초상자성 산화철 나노입자 (Superparamagnetic iron oxide nanoparticle, SPION)를 이용하였다. 주인-손님 상호작용을 이용한 시스템과 자기 유도 전달방법을 융합하고자 β-CD으로 개질화 된 초상자성 산화철 나노입자(βCD-SPION)와 고분자로 개질화 된 PTX (pPTX) 간의 다가의 주인 손님 상호작용을 이용하여 자기 조립구조인 pPTX/CD-SPION을 합성하였다. 그 후, 보다 발전된 약물 전달체로서의 합성이 성공적인지를 확인하기 위하여 다양한 분석을 진행하였다. 먼저 각 단계의 합성이 된 것을 확인하기 위하여 zeta potential을 분석하여 표면전위의 변화를 분석하였다. 또한 EPR효과를 적용시키기에 적합한 크기를 가진 다는 것과 pPTX/CD-SPION이 나노 자기조립체를 형성한다는 것을 DLS 및 TEM을 통하여 확인하였다. Serum stability test룰 통해 체내 조건에서도 안정하게 전달체의 형태를 유지할 수 있음을 밝혔으며, TGA 분석으로써 각 단계의 SPION의 질량비를 구하였으며 각 단계에서의 Fe의 질량비를 구할 수 있었다. SQUID 분석을 통하여 TCL-SPION, βCD-SPION, pPTX/CD-SPION의 자성을 분석하였고 그 결과 나노 자기조립체를 형성하였을 때 자성이 매우 강해진다는 것을 확인할 수 있었다. 물질분석 결과를 토대로 본 약물전달시스템의 성공적인 합성 및 자기 유도 전달에 이용될 수 있는 시스템임을 밝힐 수 있었고, 약물 전달체가 정상적으로 작용할 수 있을 지의 가능성을 보고자 in vitro 및 in vivo 실험을 진행하였다. In vitro 실험에서는 대조군인 PTX, pPTX, pCD/pPTX와 pPTX/CD-SPION의 세포 독성 비교를 통하여 외부에서 자기장이 가해질 때, 대조군에 비하여 단 시간의 자기장 노출만으로도 pPTX/CD-SPION의 세포 독성이 크게 증가하는 것을 확인하였다. 또한 개발된 시스템이 적혈구에 용혈현상을 거의 일으키지 않음을 hemolysis test을 통하여 확인하였다. 결장암세포주(CT26)를 가진 BALB/c 마우스를 이용하여 in vivo 실험을 진행하였고, pPTX/CD-SPION을 쥐에게 주입한 후에 자기장으로 약물전달체를 표적부위로 유도하였을 때, 다른 대조군보다도 뛰어나게 암의 성장을 저해하는 것을 알 수 있었다. 이러한 결과를 통해 본 연구에서 개발된 자기 유도 약물 전달 시스템은 안정한 구조와 자기 유도 약물 전달로 인하여 약물의 효과를 보다 증가시킬 수 있음을 알 수 있었으며, 본 시스템은 향후 항암치료 방법을 보다 발전시키는 것에 기여할 수 있을 것으로 기대한다.

Stimuli-regulated functional delivery carrier to overcome cellular and systemic barriers

김진환 Pohang University of Science and Technology 2017 국내박사

Needs for delivery system which delivers biologically active molecules, referred to bio-therapeutics, into a target site without any side effect have been strongly demanded. Accordingly, various strategies and functional materials have been proposed as promising candidate for bio-therapeutics delivery. Especially, diverse nano-sized materials possessing distinct structural features have attracted huge attention along with the development of nanotechnology to achieve effective platform for the delivery of bio-therapeutics. Over the past decade, tremendous improvements have been achieved using such materials, however, various biological barriers still remained as huge challenges for the clinical translations. In this thesis, various design strategies and functional delivery carriers to overcome such biological barriers are presented. In Part I, a brief overview of bio-therapeutics delivery system and various biological barriers in delivery system would be described. In Part II, functional gene delivery carriers to overcome various cellular barriers would be presented. A brief background on gene delivery system using cationic polymer and the cellular barriers on polymeric gene delivery system would be given in this part. In Chapter I on Part II, a unique polymer architecture based on the interaction of phenylboronic acid (PBA) and sugar was prepared to facilitate gene release from polymers. For effective gene delivery, important requirements are target-specific cellular uptake of carrier, and maximum release of payloads at the target. To accomplish this, a crosslinked polymer architecture (PBA-PEG-CrossPEI) composed of PBA and sugar-installed polyethylenimine (PEI) and polyehtyleneglycol (PEG) was developed. By introducing PBA and galactose (sugar) moiety, the PBA-PEG-CrossPEI/gene polyplex maintained its stability in blood fluids that contain glucose, but selectively released gene contents at low pH (as occurs in endosome) and at concentration of ATP that are observed in cells. Furthermore, the PBA installation to the carrier favored its binding to sialylated glycoprotein in tumors; I exploited this characteristic to use this polyplex to target tumors. For therapeutic application, anti-angiogenic sFlt-1 plasmid was delivered by PBA-PEG-CrossPEI, and in vivo evaluation was conducted by evaluating inhibition of tumor growth. PBA-PEG-CrossPEI/sFlt-1 polyplex were very stable in the blood, had high anti-tumor efficacy after systematic administration, and degraded rapidly after entering cells. In Chapter II on Part II, a hybrid structure of cationic polymer and single layered MoS2 was studied to induce photothermally-triggered endosomal escape and subsequent fast gene release in cytoplasm. To achieve this, PEI and PEG modified single layered MoS2 nanocomposite (MoS2-PEI-PEG) via disulfide bond was prepared. Owing to the photothermal effect of single layered MoS2, facilitated endosomal escape was observed upon irradiation of near infrared (NIR) light to the polyplex. After endosomal escape, polyplex of MoS2-PEI-PEG encountered intracellular reductive environment, resulting in the effective gene release due to the detachment of PEIs and PEGs from MoS2. Furthermore, detailed mechanistic features of MoS2-PEI-PEG upon each stimulus were investigated by various blocking assays. In Part III, oligonucleotide-driven nanostructures to overcome various systemic barriers would be presented. The unique chemical properties of oligonucleotides in delivery system and some biological barriers upon systemic administration would be described in this part. In Chapter I on Part III, a pH-responsive DNA-mediated clustered nanoparticle was prepared to deliver the drug molecules by preventing non-specific accumulation of nanoparticles when administrated systemically. In the level of in vivo, the size of nanoparticle is significantly important when systematic injection undergoes. The smaller sized particles easily penetrate into non-tumorous region, while the larger sized ones raise many problems during blood circulation, so that it is necessary to control an appropriate size of the particles. However, they should be degraded into small molecules to prevent their chronic toxicity. In short, they must be disintegrated into smaller ones and play their own particular role after cluster made up of small particles uptake into the tumor cell. This study exploited DNA-grafted gold nanoparticle as a core. The cluster in a proper size via DNA hybridization using pH-responsive i-motif went into tumor region specifically more than non-tumorous cell and released drugs by decomposing due to pH change inside the cell. By doing such a disassembly process, effective and sustained tumor growth inhibition was achieved. In Chapter II on Part III, more advanced a dual pH-responsive DNA-mediated nanoshuttle was prepared to deliver anticancer drugs into whole tumor tissues homogeneously. As aforementioned in previous chapter, enhanced permeability and retention (EPR) effect of nanoparticle is quite promising for tumor extravasation, however, nanoparticle remains near the vascular structure because of high interstitial fluid pressure of 3-dimensional tumor, thus therapeutic effect of drug is limited to only peripheral region. That is, the concept of nanoshuttle (100-200 nm) to carry a bundle of small nanoparticles (< 15nm) is introduced to achieve tumor-specific accumulation and subsequent deep penetration by releasing small ones in response to tumor environment. To address this, two pH-sensitive DNA strands that respond to the pH changes in tumor microenvironment and intracellular endo/lysosomes are exploited. A dual pH-responsive hierarchized nanoshuttle was designed: DNA modified gold nanoparticles (i-motif and its complementary sequence, triplex binding sequence) were decorated in the large pores of newly synthesized silica nanoparticles modified with DNA (triplex sequence) by the hybridization with DNAs. In the double stranded i-motif and its complementary sequence, anticancer drug, doxorubicin (DOX) was loaded. By employing the strategy of dual pH-responsive gold nanoparticle release and subsequent drug release, homogeneous distribution of drug molecules in overall tumor tissues was monitored, and consequently higher anticancer effect was achieved.

Functionalized bioreducible polyethyleneimine with peptide-based targeting for efficient gene delivery

이두환 Pohang University of Science and Technology 2016 국내박사

생명공학의 발전과 인간게놈프로젝트의 완성을 통하여 유전자 치료를 이용한 난치병의 치료는 여러 연구자로부터 큰 관심을 받아왔다. 초기의 연구자들은 유전자 치료를 위하여 바이러스를 이용한 전달 시스템을 개발하였지만, 안전과 관련된 여러 문제점이 발견되었다. 이를 대신하여 리포좀, 전기 충격, 덴드리머, 고분자와 같은 여러 구조적 형태를 보이는 비바이러스성 전달체가 개발되었다. 그중에서도 양이온성 고분자의 일종인 가지형 폴리에틸렌이민(BPEI)은 개질화가 쉽고 뛰어난 유전자 전달 능력을 갖추고 있으므로 전도유망한 비바이러스성 전달체로 간주하고 있다. 하지만 BPEI는 분자량이 증가함에 따라 유전자 전달 효율과 독성이 같이 증가하기 때문에 이를 극복하기 위하여 연구자들은 이황화 결합 (disulfide linkage)를 BPEI의 사슬에 도입하는 시도를 하였다. 본 연구에서는 세포 수준 및 동물 수준에서 유전자 전달 능력의 향상을 위한 여러 가지 전략을 이용하여 환원형 BPEI (BPEI-SS)를 기반으로 하는 효율적인 유전자 전달체를 개발하였다. Chapter I에서는 유전자 치료에 대한 개요와 유전자 전달 시스템에 대하여 간략하게 설명하였고, 고분자를 이용한 유전자 전달 시스템에 대한 기본적인 배경지식 및 BPEI-SS와 이를 합성하는 방법에 대하여 알아보았다. 가교결합을 통하여 증가한 유전자 복합화 능력과 이황화 결합의 분해로 인한 쉬운 DNA의 방출을 통하여 환원형 BPEI는 높은 유전자 전달 효율을 보인다. 이 전략은 고분자의 구조와 세포 내 특수한 환원환경에 초점을 두어 BPEI의 유전자 전달 효율을 증가시켰다. Chapter II에서는 고분자-유전자 복합체와 세포막의 상호작용에 대하여 주목하였다. 세포막 표면에는 당단백질(glycoprotein)이나 인지질(phospholipid)로부터 carboxy, sulfate, phosphate와 같은 작용기가 존재하고, 구아니딘 (guanidine group)과 이좌배위 수소결합 (bidentate hydrogen bonding)으로 강한 결합을 할 수 있다. 구아니딘은 세포막 통과 펩타이드 (cell penetrating peptide)의 서열 중 주요한 아르기닌 (arginine)의 작용기이며, 세포막 통과 펩타이드는 세포 외부에서 내부로의 특정 전달체의 전달을 도와주는 역할을 한다. 이 점을 이용하여 해당 연구에서는 BPEI-SS에 구아니딘을 도입하여 세포막과 고분자-유전자 복합체간의 상호작용을 증가시켜 유전자 전달 효율을 높이고자 하였다. 구아니딘이 도입된 BPEI-SS (GBPEI-SS)는 구아니딘에 의한 일차 아민의 전하의 비편재와 환원능력을 통하여 낮은 세포독성을 보였다. 또한, 대조군보다 구아니딘에 의한 늘어난 세포 내 이입과 이황화 결합의 분해로 인한 효율적인 유전자의 방출로 인하여 향상된 유전자 전달 효율을 보였다. Chapter II에서는 세포 수준에서 BPEI-SS의 유전자 전달 효율의 향상에 대한 연구를 수행하였다. 이에 Chapter III에서는 생체 내 (in vivo)에서 BPEI-SS의 유전자 전달 효율을 증가시키는 데 초점을 맞췄다. 하지만 BPEI-SS는 강한 양이온성을 갖고 있으므로 생체 내에 주입되면 혈장 내의 혈장 단백질과 상호작용을 하여 전달 효율이 감소한다. 이러한 양이온성 고분자와 혈장 단백질 간의 직접적인 상호작용을 막기 위하여 폴리에틸렌글라이콜(polyethylene glycol, PEG)를 도입하였다. 또한, PEG 사슬 끝에 고분자-유전자 복합체를 우리가 원하는 표적 세포에 전달할 수 있게 해주는 RPM이라는 표적 펩타이드를 도입하였다. RPM 펩타이드는 침습성 대장암 세포에 과발현된 인테그린 수용체를 표적할 수 있다. 마지막으로 생체 내에서의 고분자의 거동을 관찰하기 위하여 적외선 영역의 형광을 방출하는 IR820을 도입하였다. 표적 펩타이드가 도입된 BPEI-SS는 그렇지 않은 BPEI-SS에 비하여 높은 세포 내 이입과 유전자 전달 효율을 세포 상에서 그리고 생체 내에서 보였다. PEG와 표적 리간드를 BPEI-SS에 도입하여 생체 내에서도 증대된 표적 효과와 유전자 전달 효율을 확인하였다. Chapter V에서는 표적 펩타이드의 구조에 주목하였다. 표피세포성장인자수용체(Epithermal growth factor receptor, EGFR)은 사람의 표피세포를 기원으로 하는 모든 암종에 과발현되어 있으므로 최근 유전자나 약물 전달시스템의 개발에 있어 많은 연구자들이 EGFR을 표적하는 전략을 사용한다. EGFR은 리간드의 결합이 없어도 이합체의 형태로 존재한다. 이러한 독특한 EGFR의 특징을 이용하여, 우리는 두 가지 종류의 표적 펩타이드를 구현하였다. 하나는 기존의 표적 펩타이드(GE11)이며 다른 하나는 두 개의 표적 펩타이드가 구부러진 형태로 묶인 펩타이드(bGE11)이다. 본 연구에서는 구조적 특징이 다른 두 가지 표적 펩타이드를 갖는 BPEI-SS를 합성하고 평가하였다. 표적 펩타이드가 도입된 고분자들은 종류에 상관없이 높은 유전자 전달 효율, 표적 지향성, 그리고 낮은 독성을 보였다. 그중에서도 bGE11 펩타이드가 도입된 BPEI-SS는 GE11 펩타이드가 도입된 고분자보다도 생체 내에서 더 높은 표적 지향성을 보였다. 리간드 구조의 변화로 인한 표적 효율의 향상을 통하여 본 전략이 표적 지향성 전달 시스템에서 유전자 전달 효율을 증가시키는 하나의 방법이 될 수 있음을 확인하였다. Through progression of bioengineering and completion of human genome project, treatments of heredity disorder by gene therapy have drawn tremendous interest from many researchers. Early researchers had developed virus as gene carriers to deliver therapeutic genes to patients. However, there were severe problems related with safety issue. Instead of viral vectors, non-viral vectors have been developed with various kinds of formulations; liposome, electroporation, dendrimer and polymer. Among them, branched polyethyleneimine (BPEI), one of the cationic polymers, has been considered as one of the promising non-viral vectors due to its exceptional transfection efficiency and easy modification. In the use of BPEI, however, the fact that its transfection efficiency and cytotoxicity are proportional to increase of its molecular weight is issued. To address the impediment, researchers introduce bioreducible linkage (disulfide bond) to the backbone of PEI. In this thesis, advanced strategies to enhance transfection efficiency in vitro and in vivo level based on bioreducible PEI were presented. In Chapter I, we describe a brief overview of gene therapy and role of gene delivery system. Then simple background of polymeric gene delivery system was also explained. Finally, introduction of bioreducible polyethyleneimine (BPEI-SS) and various synthetic methods of BPEI-SS were featured. Enahnced gene condensation ability through crosslinking and easy DNA release via cleavage of disulfide linkage lead to high transfection efficiency of BPEI-SS. In other words, this strategy is only focused on structure of carriers and intracellular events. In Chapter II, we were concentrated on the increase of the interaction between plasma membrane and polyplex. In plasma membrane, there are carboxy, sulfate, and phosphate group from glycoprotein and phospholipid, which well interact with guanidine group through bidentate hydrogen bonding. The guanidine group is found at a residue of arginine, a major amino acid in sequence of cell penetrating peptides that help molecular cargos to penetrate into cells. Therefore, in this study, we introduce guanidine group to BPEI-SS (GBPEI-SS) to enhance interaction between polyplex and plasma membrane. GBPEI-SS showed negligibly significant toxicity due to its bioreducibility and delocalizing of the positive charge of the primary amine in BPEI by guanidine group. Compared with BPEI-SS without guanidine group, GBPEI-SS showed enhanced transfection efficiency owing to increased cellular uptake and efficient pDNA release by cleavage of disulfide bond. This system is very efficient for delivering pDNA into cells, thereby achieving high transfection efficiency and low cytotoxicity. Up to the Chapter II, we focused on enhancing transfection efficiency of BPEI-SS in vitro level. In Chapter III, we further changed our view to apply BPEI-SS for in vivo application. For one thing we had to take a careful look on in vivo experiment, however, was the serum stability of it since the highly positively charged BPEI-SS interacts well with serum protein in bloodstream. Therefore, we functionalized BPEI-SS with polyethylene glycol (PEG) which prevents the direct interaction between cationic polymer and anionic serum proteins. In addition, we introduced RPM peptide, targeting peptide, at the end of PEG chain to deliver polyplex to specific site. The peptide can bind with integrin receptor overexpressed in invasive colorectal cancer (CRC) which is one of the leading causes of cancer-related deaths worldwide. Finally, IR820 which emits NIR fluorescence is also conjugated to BPEI-PEG-RPM to visualize polymers in vivo. The synthesized RPM-conjugated gene carrier formed a compact polyplex with pDNA that had low toxicity. Furthermore, the RPM-conjugated polymer not only had higher cellular uptake in invasive colon cancer than the non-targeted polymer, but also showed enhanced transfection efficiency in invasive colon cancer cells in vitro and in vivo. Introduction of PEG and targeting ligand to BPEI-SS showed enhanced targeting ability and transfection efficiency in vivo level. In Chapter IV, we focused on the structure of targeting ligands. Epithermal growth factor receptor (EGFR) has been considered as one of the promising targets for gene and drug delivery system because of its overexpression in human cancers from epithelial origin. EGFR is used to exist as preformed dimers in the absence of ligands. Inspired by theis unique phenomenon of EGFR, we designed the targeting peptide in two different types. One is normal peptide (GE11) and the other is branched peptide (bGE11) which has two ligands in one peptide. In this study, two kinds of BPEI-SS-PEG-peptide which have structurally different targeting ligands were synthesized and evaluated. Our results demonstrated that GE11 or bGE11-tethered gene delivery carriers showed efficient gene condensing ability, enhanced transfection efficiency and targeting ability with low cytotoxicity. Interestingly, bGE11-tethered polymer showed the higher targeting ability to EGFR-overexpressed cancer cells in vivo than the GE11-tethered polymer. Therefore, this branched structure of targeting ligand has the potential for providing the novel strategy to design efficient targeted delivery system.

Study of nitric oxide and dna responsive hydrogel adhesives

조재룡 Pohang University of Science and Technology 2018 국내석사

Hydrogel refers to a jelly-like substance in which a water-soluble polymer forms a three-dimensional network structure through physical or chemical bonds and contains a large amount of water. Since hydrogels are easy to make and have good biocompatibility, many research are being conducted in various fields such as tissue engineering, contact lenses, 3D printing, drug delivery system, and so on. Hydrogels can also be used as adhesives. Hydrogel-based adhesives are hypoallergenic than conventional chemical adhesives, and show low immune response. However, there is still a drawback that it is difficult to control the adhesion. Therefore, in order to overcome this drawback, hydrogels sensitive to various stimuli are being studied recently. In this study, we confirmed use of hydrogels sensitive to specific stimuli as interfacial adhesives. Nitric oxide (NO) and DNA sequences were used as the stimuli. In the case of NO-responsive adhesives, we showed change of adhesives when NO gas was injected or placed in saturated NO solution. In addition, tensile strength of NO-responsive adhesives was measured depending on the concentration and the presence or absence of NO. In the case of DNA-responsive adhesives, storage and loss modulus of the hydrogel made from DNA as a crosslinking agent were measured. In addition, we showed that it responds only to a specific DNA sequence using a complementary sequence and a random sequence. Also, DNA-responsive hydrogel was shown to be sensitive to temperature. Finally, we confirmed that DNA-responsive hydrogel can be used as adhesives. This work demonstrates the development of stimuli-responsive adhesives and its potential on specific substance-responsive visible detector and bio-medical adhesives. For example, concentration of nitric oxide is high in automobile exhaust pipe. Therefore, as a simple adhesive, it can help to measure the degree of nitric oxide by changing the adhesives when attached to automobile exhaust pipe. In the case of DNA-responsive adhesives, it can be transformed into special adhesives that can detect specific substances by changing the DNA sequence. For instance, it can be applied to simple detection of substances that are distributed in a specific disease patient. 하이드로젤 (Hydrogel)은 수용성 고분자가 물리적 혹은 화학적 결합을 통해 3차원 망목 구조를 형성하여, 많은 양의 물을 함유하고 있는 젤리 모양의 물질을 말한다. 하이드로젤은 가공이 용이하고 생체적합성이 좋기 때문에 콘택트 렌즈, 수술 조직 밀봉제, 경피 패치 등 여러 분야에서 연구가 진행되고 있다. 하지만, 하이드로젤은 여전히 분해 속도를 조절하기 힘들기 때문에 담지된 약물의 방출 속도를 조절하거나 적절한 시간에 하이드로젤이 붕괴되어 사라지게 하기 힘들다는 단점들이 존재한다. 따라서 이를 극복하기 위해, 최근에는 여러 자극에 감응하는 하이드로젤이 연구되고 있는 중이다. 본 연구에서는 특정 자극에 감응하는 하이드로젤을 이용하여 계면 접착제로서의 사용 가능성을 확인해보았다. 자극이 되는 물질로는 일산화질소, 그리고 특정 DNA 염기서열을 이용하였다. 일산화질소는 혈관 확장, 세포 자멸, 신경 치료, 염증 유발 등 우리 인체 내에서 다양한 기능과 연관되어 있다. 또한, 류마티스 관절염, 골 관절염과 같은 관절염을 유발하는 원인으로도 알려져 있다. 최근 본 연구실에서는 일산화질소에 감응하는 하이드로젤 개발에 성공을 하였고, 일산화질소에 감응하여 하이드로젤이 부풀어 오르는 것을 보여주었다. 이를 응용하여, 본 연구에서는 일산화질소에 감응하는 하이드로젤을 계면 접착제로써 사용하여, 떨어져 있는 두 물질을 연결해보았다. 그리고, 연결된 물질이 일산화질소 가스를 주입하거나 혹은 일산화질소가 녹아있는 용액에 두었을 때 어떤 변화를 보이는 지 관찰을 하였다. 그 결과, 일산화질소가 존재할 때, 계면 접착제가 반응하면서 접착제가 분해되면서 연결된 물질이 떨어지는 것을 확인할 수 있었다. 또한, 접착제의 인장 강도를 접착제에 사용된 가교제의 농도에 따라, 일산화질소 용액에 노출된 시간에 따라 달라지는 변화를 측정하였다. 그 결과, 가교제의 농도가 높을수록, 인장 강도 (Tensile Stress)는 세지고, 반대로 인장 변형률 (Tensile Strain)은 줄어드는 것을 확인할 수 있었다. 또한, 일산화질소 용액에 노출되는 시간이 길어질수록, 인장강도 및 인장변형률이 줄어드는 것을 확인하였다. 또 다른 자극원으로는 DNA 염기서열을 이용하였다. 우선, DNA 염기서열에 감응하는 하이드로젤을 만들기 위하여, 가교제는 아크릴아마이드가 연결되어 있는 DNA 염기서열을 이용하였다. 이렇게 DNA를 이용하여 하이드로젤을 만들면 여러 가지 장점이 존재한다. 우선, 기존의 하이드로젤의 가교제는 화학 물질을 이용하지만, DNA는 인체 내에도 존재하듯이 낮은 세포 독성과 면역 반응을 보이므로 생체 적합성에서 더 뛰어나다. 또한, ATP, RNA, 단백질 등의 특정 물질과 결합할 수 있게 염기서열을 구상하면, 원하는 물질에 감응하는 하이드로젤을 만들 수 있게 된다. 본 연구에서는 그 가능성을 확인해보기 위해, 간단하게 이루어진 DNA 염기서열을 이용하여 하이드로젤을 만들어보았다. 그리고, 만들어진 하이드로젤에 상보적인 염기서열, 비상보적인 염기서열이 녹아있는 용액을 넣어주었을 때 어떤 변화를 보이는 지 관찰하였다. 그 결과, 상보적인 염기서열에만 감응하여 하이드로젤의 붕괴가 일어나는 것을 확인할 수 있었다. 또한, 하이드로젤의 온도를 높여주었을 때 어떤 변화가 일어나는 지도 확인을 해보았다. 그 결과, 온도가 높아지면 부분적으로 혼성화가 풀리면서 하이드로젤의 모양이 변형되는 것을 확인할 수 있었다. 마지막으로, DNA에 감응하는 하이드로젤을 계면 접착제로써 사용하여, 떨어져 있는 두 물질이 연결되는 지 확인을 해보았다. 이러한 결과를 통해 본 연구에서는 일산화질소 그리고 DNA에 감응하는 하이드로젤이 계면 접착제로써 사용이 가능한 것을 알 수 있었고, 이 시스템은 특정 물질을 감지하는 가시적인 센서나 의료용 접착제로써 응용이 될 수도 있을 것이다. 예를 들어, 화학 공장이나 자동차 배기관에서 나오는 대표적인 오염물질로 일산화질소가 있다. 배기관이나 공장에 일산화질소에 감응하는 접착제를 붙여두면, 간단하게 오염 물질인 일산화질소의 유무, 일산화질소의 분포 정도를 확인할 수 있을 것이다. DNA 감응형 접착제의 경우, 염기서열에 변화를 주면, 원하는 물질에 감응하는 접착제로 응용이 가능하다. 예를 들어, 특정 질병을 앓고 있는 환자에 많이 분포하는 물질에 감응하는 염기서열을 이용하여 접착제를 만들면, 환자의 샘플을 채취하여 접착제에 넣어주었을 때 변화를 관찰함으로써, 별다른 기구 없이 환자의 질병 진단을 할 수 있을 것으로 기대된다.

Enzyme-responsive polymeric micelles by controlled depolymerization for drug delivery system

조석희 Pohang University of Science and Technology 2018 국내석사

약물을 이용한 항암치료의 부작용을 줄이고 약물의 전달 효율을 높이고자 고분자 마이셀을 이용한 약물 전달 시스템이 활발히 연구되어왔다. 고분자 마이셀이 특정 자극으로 마이셀의 붕괴를 유도하고 약물을 방출하는 시스템을 개발하여, 약물의 부작용을 줄이면서 전달 효율을 높이는 것을 목적으로 한다. 본 연구에서는 생체적합성이 우수한 친수성인 폴리에틸렌글라이콜(polyethyleneglycol, PEG)과 소수성인 폴리카프로락톤(polycaprolactone, PCL)을 기반으로 한 양친매성 블록 공중합체를 자기 조립 시스템을 이용하여 고분자 마이셀을 형성하여 약물 전달체로써 사용하고자 하였다. 또한, 고분자 마이셀에 암에 대한 선택성을 높이기 위해 암에서 과발현하는 NAD(P)H:quinone oxidoreductase-1(NQO1) 효소에 감응하는 quinone propionic acid (QPA)를 접목하고, 효율적인 붕괴를 유도하기 위해서 해중합(depolymerization) 시스템을 폴리카프로락톤에 도입하고자 하였다. 이를 통해 고분자 마이셀이 enhanced permeability and retention (EPR) 효과를 이용하여 암 조직에 축적된 후 NQO1 효소에 감응하여 해중합이 유도하고, 고분자 마이셀이 붕괴되면서 약물을 방출시키는 시스템을 고안하였다. 먼저 NQO1 효소에 감응하는 QPA 그룹을 합성하고, QPA 그룹을 해중합 시스템이 도입된 카프로락톤 전구체에 접목했다. 다음 카프로락톤 전구체를 최종적인 카프로락톤 단량체로 합성하고, 폴리에틸렌글라이콜을 개시제로 사용하여 개환중합을 통해 양친매성 블록공중합체 (PEG-b-PCL-QPA)로 중합하였다. 이 때 QPA 대신 염화벤조일 (benzoyl chloride, Bz)를 접목시켜 대조군 블록 공중합체 (PEG-b-PCL-Bz)를 중합하였다. 합성한 두 고분자를 겔 투과 크로마토그래피 (gel permeation chromatography, GPC)와 핵자기 공명 (nuclear magnetic resonance, NMR)을 통해 분석하였다. 또한 PEG-b-PCL-QPA 고분자가 선택적으로 NQO1 효소에 감응하여 해중합을 유도한다는 것을 GPC를 통한 분자량 감소와 NMR을 통한 구조 변화를 관찰하여 확인하였다. 양친매성인 PEG-b-PCL-QPA 고분자와 PEG-b-PCL-Bz 고분자가 자기조립 시스템을 통해 고분자 마이셀을 형성하다는 것을 동적 광산란법 (dynamic light scattering, DLS)과 투과 전자현미경 (transmission electron microscopy, TEM), 임계 마이셀 농도 (critical micelle concentration, CMC) 측정을 통해 확인하였다. PEG-b-PCL-QPA 고분자 마이셀이 선택적으로 NQO1 효소에 감응하여 붕괴된다는 것을 DLS를 통한 마이셀 크기 변화와 TEM을 통한 마이셀 형태 변화를 통해 확인하였다. 또한, 형광체를 담지한 마이셀의 형광 분석을 통해 시간에 따른 마이셀의 붕괴 거동을 관찰하였다. 다음 독소루비신 (doxorubicin, DOX) 약물을 사용하여 고분자 마이셀의 약물 담지 능력에 대해 관찰하고, 약물을 담지한 PEG-b-PCL-QPA 고분자 마이셀이 선택적으로 NQO1 효소에 의해 약물의 방출이 가속화된 것을 확인하였다. 고분자 마이셀이 세포에서의 거동을 관찰하기 위해 NQO1 효소를 과발현하는 암세포 (A549 cell)와 과발현하지 않는 암세포 (H596 cell)를 이용하여 in vitro 실험을 진행하였다. 형광현미경을 통해 세포 내 환경에서도 NQO1 효소에 의해 PEG-b-PCL-QPA 고분자 마이셀이 약물의 방출이 가속화된 것을 확인하였다. 다음 세포 독성 실험을 통해 약물을 담지하지 않은 고분자 마이셀의 경우 높은 생체적합성을 나타내는 것을 확인하였고, 약물을 담지한 마이셀의 경우 PEG-b-PCL-Bz 고분자 마이셀보다 PEG-b-PCL-QPA 고분자 마이셀이 NQO1 효소를 과발현하는 세포에서 높은 세포 독성이 확인되었다. 이러한 결과를 통해 본 연구에서 개발된 고분자 마이셀을 이용한 약물 전달 시스템은 선택적으로 NQO1 효소에 의해 해중합을 유도하고 마이셀 붕괴를 통하여 효율적인 약물 전달을 하는 것을 알 수 있었다. 따라서 본 시스템은 고분자 마이셀을 이용한 약물 전달 시스템의 발전에 기여할 수 있을 것으로 기대한다. Chemotherapy is a technique of anticancer therapy using chemical substance as a drug. However, the anticancer drugs have serious side effects owing to non-specific interaction with normal cells, as well as tumor cells. In this aspect, self-assembled polymeric nanoparticles have obtained considerable attention as anticancer drug carriers because of improving the therapeutic efficiency and reducing side effects of anticancer drugs. However, traditional polymeric carriers with nondegradable polymer exhibits insufficient drug release in the cancer cells. To accomplish the efficacy of the drug, it is necessary to develop controlled dissociation and triggered drug release of polymeric carriers. The disintegration of the drug carriers has been induced by utilizing specific-stimuli responsiveness. In particular, enzymes over-expressed in cancer are recognized and used as crucial targets in the improvement of carriers for anticancer therapy. The activity or high concentration of a specific enzyme in cancer has been utilized in diagnosis of cancer, as well as cancer-targeted drug delivery system. NAD(P)H:quinone oxidoreductase-1 (NQO1) is one of over-expressed enzymes in cancer cells. The NQO1 activity in certain cancer cells is raised up to 50 times than in normal cells. In addition, quinone propionic acid (QPA) has been employed as a trigger of NQO1 for anticancer such as prodrug, probe and liposome. Although QPA was also used to the polymeric micelle for drug delivery, the strategy was not enough to dissociate the micelles and release drug with NQO1. Recently, depolymerization have been reported to completely decomposed a polymer chain. Moreover, the depolymerizable polymer by self-immolation was induced to the polymeric carrier for drug delivery. However, the self-immolative polymer is not sufficient to verify biocompatibility for in vivo and in vitro. Depolymerization of biocompatible polycaprolactone (PCL) by intramolecular cyclization was reported. Therefore, polymeric micelles constituted QPA trigger and depolymerzable PCL is anticipated to enhanced the efficiency of drug delivery. This work demonstrates that the development of enzyme-responsive drug delivery systems. The enzyme-responsive polymer (PEG-b-PCL-QPA) by depolymerization was successfully synthesized by ring-open polymerization. PEG-b-PCL-QPA was depolymerized by cascade cyclization with NQO1 enzyme. In addition, the polymeric micelle of PEG-b-PCL-QPA was formed by self-assembly system and disintegrated by NQO1. it is allowed to load the hydrophobic drug. Compared to the control group (PEG-b-PCL-Bz), enzyme-responsive micelle showed efficient drug release and enhanced anticancer effects in vitro. Therefore, the strategy of NQO1 enzyme responsive polymeric micelle by depolymerization has promising potential for improving the efficient drug delivery system.

Nitric oxide releasing/responsive materials for bioapplication

박정홍 Pohang University of Science and Technology 2018 국내박사

일산화질소는 생물학적으로 매우 의미 있는 가스분자이다. 1998년 이래로 심혈관계의 혈관확장, 항박테리아 효과 그리고 면역반응과 같은 일산화질소의 생물학적 기능의 정확한 매커니즘을 이해하기 위해서, 많은 연구들이 활발히 진행되었다. 이를 토대로 하여, 일산화질소를 전달하려는 시도로 발전되어 왔으며, 추후에 혈관확장제, 상처치유제, 항암제, 항생제 등으로 응용될 것 이다. 일산화질소는 적절한 양으로 조절될 경우 위와 같이 좋은 방향으로 사용할 수 있지만, 그렇지 않을 경우 심각한 질병을 야기하는 경우도 있다. 실제 일산화질소는 암, 류마티스관절염, 심혈관질병 등의 병인화와 관계가 깊다. 그래서 일산화질소 관련 질병들을 치료하기 위한 하나의 전략으로서 일산화질소 감응형 물질을 개발한다면, 질병 주변의 환경을 인식하여 불특정한 독성과 일산화질소를 줄일 수 있을 뿐만 아니라 효율적인 치료약물전달물질로써 응용될 수 있을 것이라고 기대하였다. 하지만 일산화질소의 반감기는 매우 짧기 때문에 감응성 물질을 설계하는 것은 매우 힘들다. 본 학위논문에서는 일산화질소를 방출시킬 수 있는 고분자와, 일산화질소에 감응할 수 있는 하이드로젤/금나노입자를 서술한다. Nitric oxide is a very meaningful gas molecule in biology. Studies on the basic biological functions of NO such as cardiovascular vasodilation, antibacterial activity and immune response have been actively researched to understand the precise mechanisms. Based on the understanding of NO, researchers have tried to acquire the function by delivering NO to the desired region. Through these efforts, the NO releasing materials are expected to be promising candidates for bio-therapeutics such as vasodilator, wound treatment agent, anticancer drug, and antibacterial agent. In recent, a new directional study of NO has begun because NO is not always a good molecule in our body unless it is properly regulated. In fact, NO is closely linked to the pathogenesis of cancer, rheumatoid arthritis, and cardiovascular diseases, so the development of NO-responsive material is essential for establishing a therapeutic strategy for the treatment of NO-related diseases. However, designing NO-responsive materials is a big challenge because NO is a temporally transient radial. In the thesis, NO releasing biocompatible polymer and NO responsive gel/gold nanoparticles for bioapplication are presented.

Integrated operation visibility for smart energy iot-cloud services over smartX-mini palyground

김승룡 Gwangju Institute of Science and Technology 2017 국내석사

(A) protein-protein interaction modulating method and its applications using Cucurbit[n]urils

이홍희 Pohang University of Science and Technology 2016 국내박사

As the increase in life expectancy has become the worldide phenomena, riak of degenerative diseases has increased. According to the World Health Organization report, patients with dementia are currently estimated at 35.6 million, and this number will double by 2030 and more than triple by 2050. The most common form of dementia is Alzheimer’s disease, which is characterized by amyloidosis. Amyloidosis is a group of diseases associated with fibril-like aggregate formation, such as Alzheimer’s disease, Parkinson’s disease, spongiform encephalopathy, and type II diabetes. However, there is no commercial drug available on the market for treating amyloidosis. Amyloid fibrillation is an energetically highly favored process, and fibers are condensed over time because of the self-catalytic characteristic of aggregates. Furthermore, many amyloidogenic proteins have intrinsically disordered and fluctuating structures under physiological conditions. The structural instability also impedes the conventional “lock and key” approach in drug development. In this thesis, a new inhibitory strategy for the amyloid protein fibrillation is suggested, based on the fundamental understanding of protein-protein interaction and the modulation of the interaction with a supramolecule, cucurbit[n]uril. This thesis focuses on 1) fundamental understanding of binding property of cucurbit[n]urils (CB[n], n = 6 and 7) to amino acid residues and 2) monitoring the inhibitory effect of CB[n] for human insulin fibrillation and discussing the inhibition mechanism based on the structural, thermodynamic and kinetic characteristics. 3) The method is further expanded to inhibition of beta-amyloid fibrillation using CB[7] and explanation of the mechanism. 단백질 사이의 상호작용은 선택성을 기반으로 하는 특성때문에, 생물학적 기능과 밀접한 연관성을 가지고 있다. 단백질 상호작용은 결합상수에 따라 상시적 및 일시적 상호작용으로 구분할 수 있는데, 상시적 상호작용은 높은 결합상수를 가져(Ka > 109) 역반응이 일어나기 어려운 반면, 일시적 상호작용은 그 보다 낮은 결합상수를 가지고 (Ka ~ 106 을 기준으로) 초 단위로 복합체를 유지한다. 아밀로이드 섬유화 현상은 특수한 단백질-단백질 상호작용 중 하나로서, 에너지적으로 매우 우세한 반응이며, 동시에 핵 형성을 통한 자가-촉매적인 반응으로 인해 시간에 따라 축적되는 성질을 지니고 있다. 알츠하이머 병은, 단백질들이 섬유형태의 침전물을 만드는 것을 특징으로하는 아밀로이드성 질환의 일종으로, WHO의 보고에 따르면, 치매 환자는 현재 35.6 백 만명 정도이며, 환자 수는 2030년에는 그 두 배로, 2050년까지는 세 배로 증가할 것이라 전망하고 있다. 이 외에도 파킨슨병, 광우병, 및 2형 당뇨 등 아밀로이드성 질환의 대표적인 예시이다. 이러한 중요성에도 불구하고, 현재 아밀로이드성 질환에 대해 상용화된 약물은 전무한 실정이다. 에너지적으로 우세한 반응의 특성에 더해 많은 아밀로이드성 단백질은 체내와 동등한 조건에서 특정한 2차 및 3차구조를 갖지 못하는 비정형적 구조의 특성을 갖고 있다. 이는 일반적인 약물개발 방법인 자물쇠-열쇠 컨셉을 기반으로 하여 열역학적인 우세를 점하는 리간드를 개발하기 어렵게 만든다. 따라서 섬유화를 저해하기 위한 새로운 방법론이 필요하며, 본 논문에서는 그 접근법의 하나로서 거대분자를 이용한 아밀로이드 단백질 사이의 상호작용을 저해하는 방법을 제시하고자 한다. 본 논문에서 거대분자의 종류 중 하나인 쿠커비투릴[n]을 이용하여, 먼저 1) 아미노산 잔기와 인슐린 및 인슐린 변형체와 쿠커비투릴[n] 사이의 상호작용을 연구하고 이 특성을 파악한 뒤, 2) 인슐린과 3) 아밀로이드의 섬유화 반응을 쿠커비투릴[n]이 저해하는 기작에 대해 자세히 논하고자 한다. 쿠커비투릴 [n] 의 결합에 의한 에너지적 이득에 더해, 쿠커비투릴[n]이 단백질보다 복합체를 이루는 속도가 빠른 것을 이용한 속도론적 측면에서의 접근을 이용하는 전략을 제시함으로서, 아밀로이드성 섬유화를 저해하기 위한 새로운 전략을 제시하는데서 그 의의를 가진다.