Gold

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Introduction

Gold nanoparticle is gold at nano scale (few to few hundreds of manometers) with variation of shapes. It can be in aqueous form (colloidal gold nanoparticles) or in dried form. Aqueous form of gold nanoparticles (colloidal gold) is much more popular than the dried form. Colloidal gold is a sol or colloidal suspension of submicrometre-size nanoparticles of gold in a fluid, usually water. Due to the unique optical, electronic, and molecular-recognition properties of gold nanoparticles, they are the subject of substantial research, with applications in a wide variety of areas, including electron microscopy, electronics, nanotechnology and materials science. The range of applications for gold nanoparticles is growing rapidly and includes:

  • Electronics - Gold nanoparticles are designed for use as conductors from printable inks to electronic chips[1]. As the world of electronics become smaller, nanoparticles are important components in the chip design. Nanoscale gold nanoparticles are being used to connect resistors, conductors, and other elements of an electronic chip.
  • Photodynamic Therapy - Near-IR absorbing gold nanoparticles (including gold nanoshells and nanorods) produce heat when excited by light at wavelengths from 700 to 800 nm. This enables these nanoparticles to eradicate targeted tumors [2]. When light is applied to a tumor containing gold nanoparticles, the particles rapidly heat up, killing tumor cells in a treatment also known as hyperthermia therapy.
  • Therapeutic Agent Delivery - Therapeutic agents can also be coated onto the surface of gold nanoparticles[3] . The large surface area-to-volume ratio of gold nanoparticles enables their surface to be coated with hundreds of molecules (including therapeutics, targeting agents, and anti-fouling polymers).
  • Sensors - Gold nanoparticles are used in a variety of sensors. For example, a colorimetric sensor based on gold nanoparticles can identify if foods are suitable for consumption [4]. Other methods, such as surface enhanced Raman spectroscopy, exploit gold nanoparticles as substrates to enable the measurement of vibrational energies of chemical bonds. This strategy could also be used for the detection of proteins, pollutants, and other molecules label-free.
  • Probes - Gold nanoparticles also scatter light and can produce an array of interesting colors under dark-field microscopy. The scattered colors of gold nanoparticles are currently used for biological imaging applications [4]. Also, gold nanoparticles are relatively dense, making them useful as probes for transmission electron microscopy.
  • Diagnostics - Gold nanoparticles are also used to detect biomarkers in the diagnosis of heart diseases, cancers, and infectious agents [5]. They are also common in lateral flow immunoassays, a common household example being the home pregnancy test.
  • Catalysis - Gold nanoparticles are used as catalysts in a number of chemical reactions [5]. The surface of a gold nanoparticle can be used for selective oxidation or in certain cases the surface can reduce a reaction (nitrogen oxides). Gold nanoparticles are being developed for fuel cell applications. These technologies would be useful in the automotive and display industry.

Gold in its bulk form is well known to be chemically inert, non-toxic as well as a good candidate metal used for medical and clinical purposes such as anti-inflammatory agents to treat rheumatoid arthritis (Finkelstein et al. 1976). However, when the size of materials reduces to nano scale nano-gold's properties are very different from those properties of bulk-gold, which has raised many questions about its safety and concerns in the risk assessment for humans.

금 나노입자는 다양한 모양을 가진 나노 규모에서의 금이다. 이것은 수용액 상태(콜로이드 금 나노입자) 또는 건조된 상태로 존재할 수 있다. 수용액 상태의 금 나노입자(콜로이드 금)는 건조된 상태보다 더 대중적이다. 콜로이드 금은 보통 물같은 용액에서 서브마이크로미터 크기를 가지는 금 입자의 졸 또는 콜로이드 현탁액이다. 금 나노입자의 독특한 광학적, 전기적 그리고 분자인식의 특성 때문에 이것들은 전자현미경, 전자공학, 나노기술 그리고 재료 과학을 포함하는 다양한 분야에서 중요한 연구의 대상이 된다.

금 나노입자에 대한 적용범위는 빠르게 증가하고 있다.

  • 전기적 - 금 나노입자는 전자칩의 프린트 잉크에 대한 전도체로 사용하기 위해 만들어졌다. 전자적 세계가 작아지면서, 나노입자는 칩 설계에 중요한 요소이다. 나노크기의 금 나노입자는 저항, 전도체 그리고 전자칩의 다른 요소들과 연결하는데 사용된다.
  • 광역학적 치료 - 700에서 800 nm의 파장에서의 빛에 의해 여기되었을 때, NIR 흡수에서 금 나노입자(금 나노쉘 그리고 나노막대를 포함하는)는 열을 발생한다. 이러한 나노입자는 표적이 된 종양을 근절할 수 있다. 빛이 금 나노입자를 포함하는 종양에 적용될 때, 입 자는 빠르게 가열되고, 온열 요법으로 알려진 치료에서 종양세포를 죽인다.
  • 치료제의 전달 - 치료제는 금 나노입자의 표면에 코팅될 수 있다. 금 나노입자의 큰 surface area-to-volume ratio은 표면이 수백개의 분자로 코팅될 수 있게 한다(치료법, 목표제, 그리고 방오 고분자를 포함하는).
  • 센서 - 금 나노입자는 다양한 센서에 사용된다. 예를들어, 금 나노입자에 기초한 색채센서는 음식이 소비에 적합한지 아닌지를 구별할 수 있다. 다른 방법으로, 표면이 강화된 라만 분광법은 화학결합의 진동에너지 측정을 가능하게하는 기판으로 금 나노입자를 이용한다. 이것은 단백질, 오염물질 및 다른 label-free 분자의 검출에 사용될 수 있다.
  • 조사 - 금 나노입자는 빛을 산란하고 암시야 현미경에서 흥미로운 색상의 배열을 생성할 수 있다. 금 나노입자의 산란된 색은 생체 이미지 어플리케이션에 사용된다. 또한, 금 나노입자는 상대적으로 조밀해서 전자 투과 현미경과 같은 조사를 유용하게 한다.
  • 진단 - 금 나노입자는 심장질환, 암, 그리고 감염성 제제의 진단에서의 생물학적 지표를 검출하기 위해 사용된다. 그것들은 또한 가정 임신테스트를 예를들어서 측방 유동 면역에서 일반적이다.
  • 촉매 - 금 나노입자는 화학반응의 촉매로 사용된다. 금 나노입자의 표면은 선택적 산화를 위해 사용될 수 있고 또한 경우에 따라 표면은 반응(질소 산화물)을 줄일 수 있다. 금 나노입자는 연료 전지 어플리케이션으로 개발되고 있다. 이러한 기술들은 자동차 및 디스플레이 산업에 유용 할 것이다.

벌크 형태의 금은 화학적으로 불활성이고, 비 독성하다고 잘 알려져 있고, 류마티스 관절염을 치료하기 위한 항염증제와 같은 치료 목적으로 좋은 금속이라고 잘 알려져 있다. 그러나, 물질의 크기가 나노 크기로 줄어들게되면, 나노-금의 성질은 벌크-금의 성질과 매우 달라지고, 인간에 대한 위험성 평가에서의 안전에 대해 많은 의문을 제기되고 있다.

Manufacturer information

Number of articles related to gold nanoparticles cytptoxicity

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Manufacturers supplying Au NPs for cytotoxicity study

PChem properties

The properties of colloidal gold nanoparticles, and thus their applications, depend strongly upon their size and shape. Gold at the nanoscale can appear red, blue, green, or brown. These colors arise as a result from interaction of conduction band electrons in the metallic nanoparticles with the electric field vector of the incident light. Depending on the gold nanoparticle’s size, shape, and surrounding medium, a relatively narrow range of frequencies of incident light induce resonant conduction band electron oscillation. This resonance is called the localized surface plasmon resonance (LSPR), which occurs in the visible and near-infrared regime of the spectrum for gold nanoparticles, depending on their shape and size (Kellyet al. 2003). When the wavelength of light is optimum to satisfy the LSPR, extinction (sum of absorption and scattering) is observed from the nanoparticle. In the case of spherical nanoparticles, a single ‘‘plasmon’’ band is observed in the visible region. But, when the nanoparticles have an anisotropic shape such as a rod, two plasmon bands occur as a result of electron oscillation along the two axes.

콜로이드 금 나노입자, 그리고 그것들의 어플리케이션의 특성은 크기와 모양에 매우 의존한다. 나노규모의 금은 빨간색, 파란색, 초록색, 갈색을 나타낼 수 있다. 이러한 색깔들은 금속 나노입자 안에있는 전도 밴드 전자와 입사광의 전기장의 상호작용의 결과로 발생한다. 금 나노입자의 크기, 모양, 그리고 주변 배지에 의존하여, 상대적으로 입사광의 주파수의 좁은 범위는 공명전도밴드전자의 진동을 유도한다. 이 공명은 국소 표면 플라즈몬 공명(LSPR)이라고 불리고, 가시광선 및 근적외선 영역에서 발생하며 그것들의 모양과 크기에 의존한다(Kellyet al. 2003). 빛의 파장이 LSPR을 만족하는 최적조건이면, 흡광(흡수와 산란의 합)은 나노입자에서 관찰된다. 구형의 나노입자의 경우에, 단일 "플라즈몬" 밴드는 가시 영역에서 관찰된다. 그러나, 나노입자가 막대와 같이 이방성 모양을 가지면, 두개의 플라즈몬 밴드는 두 개의 축을 따라 전자진동의 결과로 나타난다.

  • Gold nanorods
A. Alkilany and C. Murphy, Journal of nanoparticle research: an interdisciplinary forum for nanoscale science and technology. 12, 2313 (2010).

Gold nanoparticles having a rod-like morphology (gold nanorods, AuNRs) are of particular interest because of their anisotropic shape. Due to their non-spherical geometry, these particles have both a transverse and longitudinal plasmon. The absorption profile includes two absorption bands: one due to light absorbed along the short axis (transverse) and the other due to absorption along the long axis (longitudinal). As the rod length increases, so does the longitudinal band red shift together with an increase in the extinction coefficient.  

막대 모양(금 나노막대, 등)을 가지는 금 나노입자는 그것들의 이방성 모양때문에 특히 관심이 있다. 그것들의 비 구형 모형 때문에, 이러한 입자들은 가로와 세로 플라즈몬을 모두 가진다. 흡수는 두 개의 흡수 밴드를 포함한다 : 하나는 짧은 축에 의한 (가로의) 약한 흡수이고, 다른 하나는 긴 축에 의한(세로의) 흡수이다. 막대의 길이가 길어질수록, 흡광계수의 증가와 함께 세로의 밴드는 red shift이동을 한다.    

  • Gold nano sphere

Gold nanosphere have been utilized for centuries by artists due to the vibrant colors produced by their interaction with visible light. More recently, these unique optical-electronics properties have been researched and utilized in high technology applications such as organic photovoltaics, sensory probes, therapeutic agents, drug delivery in biological and medical applications, electronic conductors and catalysis. The optical and electronic properties of gold nanoparticles are tunable by changing the size, shape, surface chemistry, or aggregation state.

금 나노구체는 가시광선과의 상호작용에 의해 생성된 생동감 있는 색상 때문에 예술가들에 의해 수세기동안 사용되어져왔다. 최근에는 이러한 독특한 광학-전기 특성이 유기 태양전지, 치료제, 생물학적 및 의료적 약물전달, 전자 도체, 촉매와 같은 첨단기술 응용프로그램에 연구되고 사용되어왔다. 금 나노입자의 광학 그리고 전기적 특성은 크기, 모양, 표면화학 또는 응집상태를 변화시킴으로써 조정된다.

  • Gold nanoshell[6]
Gold nanoshell optical properties

Gold nanoshell is composed of a silica core around 100 nm and a thin shell of gold about few nanometers. The shell is formed by aging the gold clusters attached on the silicon core. When the size decreases, there is a red shift in absorption band. The red shift has been explained as the results of the hybridization of the plasmons of the inner sphere and outer cavity. The Surface Plasmon Resonance wavelength of gold nanoshells can be controlled by changing the shell thickness. Decreasing the thickness of the gold shell from 20 to 5 nm leads to Surface Plasmon Resonance red shift about 300 nm, which is attributed to the increased coupling between the inner and outer shell surface plasmons for thinner shell particles. Recently, DDA simulation shows that the Surface Plasmon Resonance frequency depends on the ratio of the shell-to-core thickness in a near-exponential relationship which is independent of the particle size, core and shell material and even surrounding medium.

금 나노껍질은 약 100 nm의 실리카 핵과 수 나노미터의 금의 얇은 껍질로 구성된다. 껍질은 실리콘 핵에 부착된 금 클러스터의 숙성에 의해 형성된다. 크기가 감소하면, 흡수 밴드는 red shift이동을 한다. red shift 이동은 내부 공간과 외부 공간의 플라즈몬의 혼성화 결과로 설명되었다. 금 나노껍질의 표면 플라즈몬 공명 파장은 껍질 두께의 변화에 의해 조절될 수 있다. 금 나노껍질의 20에서 5 nm까지의 두께 감소는 얇은 껍질 입자에서의 내부와 외부 껍질 플라즈몬 사이의 coupling을 증가시키도록 기여하는 300 nm에서 표면 플라즈몬 공명 red shift이동을 하게 한다. 최근에 DDA 모의실험은 입자 크기, 핵, 그리고 껍질 재료와 주변 매질에 무관한 가까운 지수관계에서 표면 플라즈몬 공명 주파수가 껍질-핵의 두께의 비율에 의존한다는 것을 보여준다.

  • Gold nanocage [6]
    Gold nanocage optical properties

Developed lately by Xia and co-workers , gold nanocages are a type of hollow and porous gold nanostructures which are formed by a galvanic replacement reaction between silver nanocubes and auric acid in aqueous solution. Simultaneous deposition of gold atoms and depletion of silver atoms results in gold nanoshells which then anneal to generate smooth hollow and porous structures. General size of the nanocages is around 50 nm edge width with few nanometers walls and holes for Surface Plasmon Resonance wavelength around 800 nm . By controlling the amount of auric acid solution, the Surface Plasmon Resonance of gold nanocages could be tuned to NIR region with specified wavelength. DDA calculation shows that the total light extinction of gold nanocages with Surface Plasmon Resonance around 800 nm is dominated by absorption, which makes them suitable for photothermal therapy.

Xia와 동료들에 의해 최근에 개발된 금 nanocage는 은 나노큐브와 수용액의 금산 사이의 갈바닉 치환반응에 의해 형성되는 속이 비었고 다공성의 금 나노구조 유형이다. 금 원자의 동시적 증착과 은 원자의 고갈은 금 나노껍질을 초래하고, 부드러운 속이 빈 다공성 구조를 생성하기 위해 강화시킨다. nanocage의 일반적인 크기는 800 nm의 표면 플라즈마 공명 파장에 대한 수 나노미터의 벽과 구멍이 있는 50 nm의 가장자리 폭을 가진다. 금 산 용액의 양을 조절함으로써, 금 nanocage의 표면 플라즈몬 공명은 규정된 파장의 NIR 영역으로 조정될 수 있다. DDA 계산은 흡광에 의해 지배되는 800 nm 주위의 표면 플라즈마 공명과 함께 금 nanocage의 전체 빛 흡광을 보여주고 광열 치료에 적합하게 한다.

  • PChem properties: size and charge

##We collected physico-chemical information of gold nano particles from 30 journal articles (from 2007 to 2015).

##우리는 30개의 논문에서 금 나노입자의 물리화학적 정보를 얻었다.

##The core diameter of gold nanoparticles was in range of 1~150nm, the hydrodynamic diameter of of gold nanoparticles was in range of 5~320nm. Most of gold nanoparticles in QNTR DB has negative surface charge (green and cyan data in the below scatter plot). Gold nanoparticles with size below 100nm are the most popular subjects for the cytotoxicity study.

##금 나노입자의 지름은 1~150 nm이고, 금 나노입자의 유체 역학적 지름은 5~320 nm이다. QNTR DB에 있는 대부분의 금 나노입자는 음의 표면전하를 가진다.(아래의 점 그래프에서 초록색과 청록색)

Figure 1. PChem properties of Au NPs collected from QNTR DB (2016 04)

Cytotoxicity

Patra, H. K., & Dasgupta, A. K. (2012). Cancer cell response to nanoparticles: Criticality and optimality. Nanomedicine: Nanotechnology, Biology, and Medicine, 8(6), 842–852.

Effect of hydrodynamic size and zeta potential to the viability of cancer cells (A549, A375, HeLa, NCI H23) using MTT assay was investigated by Patra et. al. Small size of AuNPs (below 50nm) shows higher toxicity (viability below 60%) than the bigger AuNPs. AuNPs with surface charge around -10~-45 mV shows higher toxicity (viability below 60%) than the AuNPs with lower surface charge.

MTT assay를 이용한 암 세포(A549, A375, HeLa, NCI H23)의 생존률에서의 유체역학적 크기의 효과와 제타 전위는 Patra et. al.으로부터 연구되었다. 금 나노입자의 작은 크기(50 nm이하)는 큰 금나노입자보다 더 높은 독성(60% 이하의 생존률)을 보여준다.

Dose vs Viability AuNPs (QNTR DB 201604)

Figure show response of cell viability to the exposure concentration of AuNPs (Dose, ug/mL). 10 assay methods (Alamar Blue, CCK-8, Hoechst dye, LDH, Micromotion, MTS, MTT, Multisizer, Tryphan blue and XTT) and 30 cell lines were used for toxicity evaluation. There are totally 555 data points, each data points contains information of size, charge, cell line, assay method, exposure time, dose and cell viability. Most of the data show non-toxic effect of AuNPs (cell viability smaller than 50%). Few data show toxic effect of AuNPs (cell viability greater than 50%) which belong to Tryphan Blue, Multisizer and MTT assay methods.

그림은 금 나노입자의 노출 농도(ug/mL)에서의 세포 생존률을 보여준다. 10개의 방법(Alamar Blue, CCK-8, Hoechst dye, LDH, Micromotion, MTS, MTT, Multisizer, Tryphan blue and XTT)과 30개의 세포종류는 독성 평가에 사용되었다. 전체 555개의 데이터 포인트가 있고, 각각의 데이터 포인트는 크기, 전하, 세포종류, 방법, 노출시간, 복용량, 그리고 세포 생존률을 포함한다. 데이터의 대부분은 금 나노입자의 비 독성 효과를 보여준다(50%보다 적은 세포 생존률). 몇몇 데이터는 Tryphan Blue, Multisizer and MTT assay 방법을 사용하여 금 나노입자의 독성 효과를 보여준다(50% 보다 큰 세포 생존률).

3. Gold nanoparticle–physiological media interactions [7]

Applications of gold nanoparticles will require that the particles be introduced into a living system (at either the cellular level or at the organismal level). The bloodstream of an organism, the cytoplasm of the cell, and even the media in which cells grow are all complex aqueous mixtures of electrolytes, proteins, nutrients, metabolites, etc. What happens at the molecular level when nanoparticles are introduced into these systems? We expect that biological media–nanoparticle interactions precede the next biological steps (distribution, metabolism, elimination, etc.). Thus, understanding the chemical and physical interaction of nanoparticles with the biological media is essential to understanding and predicting the subsequent processes. The cellular growth media (for in vitro studies) contains serum proteins, essential amino acids, vitamins, electrolytes, and other chemicals (antibiotics, trace metals, etc.). These various components could interact with nanoparticles and change their physiochemical properties such as size and aggregation state, surface charge, and surface chemistry. The nanoparticles, especially if made in aqueous solution, have a surface charge to stabilize them against aggregation via electrostatic repulsion. The presence of electrolytes and the high ionic strength of the biological media can result in nanoparticle aggregation via electrostatic screening (Vesaratchanon et al.2007). Aggregation of nanoparticles could influence their ability to interact with or enter cells, and thus adds complexity to the system.

Protein adsorption to the nanoparticle surface can mediate the uptake of the nanomaterial via receptor mediated endocytosis (Conner and Schmid 2003). Therefore, different media with different protein compositions could result in different toxicity and uptake results. This is important when comparing results from different reports addressing the toxicity and uptake of nanoparticles using different methodologies.

금 나노입자의 적용은 입자가 생명체(세포 수준 또는 생명체) 내로 도입되는 것을 요구할 것이다. 생명체의 혈류, 세포의 세포질, 그리고 세포가 자라는 배양액은 전해질, 단백질, 영양소, 대사물 등의 혼합 용액이다. 나노입자가 이러한 시스템에 도입될 때, 분자수준에서는 어떤일이 일어날까? 우리는 생물학적 배양액과 나노입자의 상호작용이 다음 생물학적 단계(분포, 대사, 제거 등)를 앞설 것이라고 예상한다. 따라서, 생물학적 배양액과 나노입자의 화학적 그리고 물리적 상호작용을 이해하는 것은 다음의 과정을 이해하고 예측하기 위해서 필수적이다. in vitro 연구에서 세포 성장 배양액은 혈청 단백질, 필수 아미노산, 비타민, 전해질 그리고 기타 화학물질(항균제, 미량의 금속 등)을 포함한다. 이러한 다양한 구성요소는 나노입자와 상호작용할 수 있고 크기, 응집 정도, 표면 전하, 그리고 표면 화학과 같은 그들의 물리화학적 성질을 변화시킬 수 있다. 특히 수용액 안의 나노입자는 전기적 반발력을 통해 뭉침에 대한 그들 자신을 안정화시키는 표면 전하를 가진다. 전해질의 존재와 생물학적 배양액의 높은 이온강도는 정전기적 검사를 통해 나노입자가 응집 되게 할 수 있다 (Vesaratchanon et al.2007). 나노입자의 응집은 세포와 상호작용 하거나 세포에 들어가서 시스템을 복잡하게 할 수 있다. 나노입자 표면의 단백질 흡착은 endocytosis(세포 내 섭취)를 수용체로 하여 나노물질의 흡수를 매개할 수 있다(Conner and Schmid 2003). 그러므로, 다른 단백질 구성을 가지는 다른 배양액은 다른 독성과 흡수의 결과를 초래할 수 있다. 다른 방법론들을 이용하여 나노입자의 독성과 흡수를 다루는 다른 결과를 비교할 때 이것은 중요하다.

4. Methods to evaluate cellular toxicity of a gold nanoparticle [7]

There are many assays used to measure the cellular impact of a drug that can also be applied to measure the impact of nanoparticle exposure on cells. One common assay is the LDH assay, which is a colorimetric assay measuring the release of lactate dehydrogenase (LDH) into the culture media as an indicator of cellular membrane disruption (Marquis et al. 2009). A metabolic assay considered the ‘‘gold standard’’ for cytotoxicity is the MTT assay, which is a colorimetric assay that measures the enzymatic activity of cellular mitochondria. If cells properly metabolize the MTT dye, the cell culture will turn blue, allowing for simple absorbance measurements to be used to quantify cellular activity (Marquis et al. 2009).

Beyond these relatively simple measures of cell health, many standard assays for other indicators are generally available as commercial kits. These include ROS assays (monitoring oxidative stress by measuring the level of ROS, reactive oxygen species), and real-time polymerase chain reaction amplification and DNA micro-array analysis to examine the expression levels of genes that are, for example, related to stress in the cell. However, cell viability is a quite general term and each of these methods, which determine one or more cellular parameters, cannot be compared directly with the other as they basically measure different parameters. As a general advise, to avoid misinterpretations of the results, cytotoxicity should be verified with at least two independent assays. A further problem concerns with the occurrence of false-positive and false-negative results and cross-checking the data with alternative independent assays to ensure reliability of the results is certainly desirable and would help to avoid errors. For example, NPs with optical properties can alter the results from assays based on spectrophotometric measurements or NPs with high absorbance capacity and catalytic activity may interact with enzymes or substrates. A further example deals with the case of gold nanoparticle-treated cells, where the dead cells are imaged with the commonly used fluorescent propidium iodide [PI]. Normally, the fluorescent PI molecules cannot penetrate the cell membrane. However, in some experiments, the PI entered the cell during the endocytosis of the nanospheres and resulted in a false-positive toxicity result [36]. 

세포에 나노입자를 노출했을 때의 영향을 측정하는 것에 적용될 수 있는 약물의 세포에 미치는 영향을 측정하는데 사용되는 다양한 분석법이 있다. 하나의 공통적인 방법은 세포막 파괴의 지표로써 배양액으로의 젖산 탈수소 효소(LDH)의 방출을 측정하는 비색 분석법인 LDH 방법이다(Marquis et al. 2009). "금 기준"의 세포독성으로 고려되는 신진대사 방법은 미토콘드리아의 효소 활성을 측정하는 비색 분석법인 MTT 방법이다. 만약 세포가 MTT 시약과 적절히 대사작용을 하면, 세포 배양은 파란색으로 변할 것이고, 세포 활성을 정량화하는데 사용되는 간단한 흡광도 측정을 할 것이다(Marquis et al. 2009).    

이렇게 비교적 간단한 측정 외에도, 상용되는 kit와같이 다른 지표로 많은 표준 분석법을 사용할 수 있다. 이것들은 예를들어, ROS 분석법(ROS, 활성 산소종의 수치를 측정함으로써 산화 스트레스를 확인하는 것), 실시간 중합 효소 연쇄반응 증폭, 세포의 스트레스와 관련이 있는 유전자의 발현을 검사하는 DNA micro 배열이 있다. 그러나, 세포 생존률은 꽤 일반적인 명칭이고, 하나 또는 하나이상의 세포 매개변수를 결정하는 각각 이러한 방법들은 기본적으로 각자 다른 매개변수를 측정하기 때문에 즉시 서로 비교될 수 없다. 일반적인 조언으로, 결과의 오해를 방지하기 위해 세포독성은 적어도 두개의 독립적인 방법으로 검증되어야 한다. 거짓-양성, 거짓-음성의 결과를 고려하고, 결과의 신뢰성을 확실하게 하기 위해 독립적인 방법으로 데이터를 교차 검사하는 또 다른 문제점은 확실히 바람직하고 오류를 방지하는데 도움이 된다. 예를들어, 광학특성이 있는 나노입자는 분광 측정에 기초한 분석 결과 또는 나노입자의 높은 흡수 용량을 바꿀 수 있고 촉매활성은 효소 또는 기질과 상호작용할 것이다. 또 다른 예는 죽은 세포가 일반적으로 사용되는 형광 요오드화 프로피듐(PI)으로 이미지화 되는 금 나노입자로 처리된 세포의 경우를 다룬다. 일반적으로, 형광 PI 분자는 세포막을 투과할 수 없다. 그러나, 일부 실험에서, PI는 나노입자의 새포내섭취(endocytosis) 동안 세포를 통과하고 거짓-양성 독성의 결과를 나타낸다.    

5. The effect of size and shape of Gold nanoparticles toxicity [8]

The table below summaries some AuNPs in vitro toxicity studies that examined size, type of cell culture and their biological effects.

아래의 표는 크기, 세포 종류, 그리고 생물학적 효과에 대한 in vitro 독성 연구에서 금 나노입자에 대해 요약한다.

Yah, C. S. (2013). The toxicity of gold nanoparticles in relation to their physiochemical properties. Biomedical Research (India), 24(3), 400–413.

Synthesized AuNPs come in a variety of sizes and shapes ranging from 1 nm to 500 nm: some as rods, spheres, tubes, wires, ribbons, plate, cubic, hexagonal, triangular, tetrapods, etc . The small size and their ‘needle- like’ penetrating ability into cells have also made AuNPs excellent carriers in biomedical and molecular biology techniques . This needle like feature as reported by De Jong et al have ease the absorption, penetration, circulation and distribution of AuNPs in bio-systems as a size dependent factor. These findings were similar to those earlier reported by Connor et al who found that AuNPs of approximately 18 nm in diameter could penetrate the cells without cell injury and toxicity. A study by Tsoli et al also demonstrated that AuNPs of approximately 1 nm in diameter could penetrate the cell and nuclear membranes and attach to DNA without cell injury and cell death. The mechanism of entry into cells without cell injury has not been elucidated, but it seems the small nanosize plays a major role. The small size of the AuNPs therefore, facilitates their incorporation into biological systems for subsequent probing and modifica- tion . These unique features of AuNPs have led them to various chemical properties transducing into dissimilar cellular studies where some are reported either as toxic or non toxic. Some display size dependent toxicity due to the presence of coated surface ligands , while others because of their large surface area to volume ratio provide platforms for increase surface particle activity . This therefore, contributes an easy flexible pathway of penetration and reactivity in biological system than bulk gold material.

In terms of size, De Jong et al found that 10 nm AuNP when administered to experimental animals can circulate more within 24h than other sizes. The mechanism of this 10 nm AuNP widespread has not been elucidated. Apart from the fact that AuNPs circulations in the system are highly size dependent, earlier findings by Hauck et al also showed that other sizes such 50 nm AuNPs when exposed within 30 min can be the most abundant cellular AuNPs in the a system.

The interactions of AuNPs with biological systems are often related to their physiochemical characteristics which enable them to be internalized within cells, a situation which is not possible for larger particles. This is one of the reasons why AuNPs may be toxic than larger particles when compared on a mass dosage. This emphasizes lies in the importance of their dimension, the large surface area to volume ratio which enables them applicable in biomedical systems.

Other studies of size-dependent cytotoxicity have been demonstrated in triphenylphosphine stabilised AuNPs using four cell lines such as tissue fibroblasts (L929), epithelial cells (HeLa), macrophages (J774A1) and melanoma cells (SK-Mel-28) . Data obtained from these studies shown that cellular response is size depend- ent. For example 1.4 nm AuNP was observed as the most toxic responsible for rapid cell death by necrosis as compare to 15 nm which was shown to be non-toxic . This suggests that “larger” NPs are non-toxic in vitro. Furthermore, other in vitro studies on AuNPs of 20 and 100 nm in diameters have been shown to have no apparent effect on viability of human retina microvascu- lar endothelial cells . Although some studies have shown AuNPs not having an effect on cell viability, it is important to note that genotoxicity can occur without cytotoxicity and may result in genetic damage and transcription alterations which are not phenotypically expressed. A study on the effect of 5nm to 20 nm AuNPs on MRC-5 human fetal lung fibroblast cells have showed no influence on the viability of MRC-5 treated cells [ - ]. However, cell proliferation was inhibited which was linked to downregulation of cell cycle genes. More so, oxidative DNA damage has been observed in conjunc- tion with a downregulation of DNA repairs. Fur- thermore, other reports have revealed that AuNPs of 2-4 nm, 5-7 nm and 20-40 nm are non-toxic to MRC-5 cells however when they were ≥ 10 ppm induced apoptosis and up-regulated the expressions of pro-inflammatory genes interlukin-1 (IL-1), interlukin-6 (IL-6) and tumor necrosis factor (TNF-alpha) .

Furthermore, the influence of AuNP toxicity has also been shown to vary due to the different particle shapes. Among the shapes, rods shaped AuNPs have been re- ported to demonstrate more toxicity than their spherical counterparts. Research on gold nanorods has shown that they are more toxic to human keratinocyte cells (HaCaT) as compared to spherical gold nanomaterials. The mechanisms of less toxicity of spherical AuNPs com- pared to nanorods are yet to be demonstrated; however, they are all engulfed on their surface properties. Studies investigating the cytotoxicity and cellular uptake of gold nanorods on human breast adenocarcinoma cell line (MCF- 7) also reported loss of mitochondrial integrity in cells treated with nanorods as compared to spherical shapes. Li et al also showed that naked AuNPs (20 nm in diameter) when taken up by MRC-5 human lung fibroblast in vitro can induce autophagy (degrada- tion of a cell's own components via lysosomal machin- ery) concomitant with oxidative stress, stimulating up- regulation of antioxidants, stress response genes and pro- tein expression. Other studies have also shown nanorods toxicity to be highly associated with surface layer used for the synthesis of nanorods such as CTAB. There- fore the association of surface stabilizers and functional ligands chemistry or composition should not be over- looked. Also as the application of AuNPs are increasing in medicine to diagnose and treat diseases detailed data on the possible toxic effect of various sizes, shapes and ligands of the AuNPs are needed because the current available information are limited and inconsistent.

합성된 금 나노입자는 1 nm에서 500 nm까지 다양한 크기와 모양을 가진다(rods, spheres, tubes, wires, ribbons, plate, cubic, hexagonal, triangular, tetrapods). 작은 크기와 '바늘과 같은' 세포로의 침투 능력은 금 나노입자를 생물의학적 그리고 분자 생물학 기술에의 훌륭한 운반체로 만든다. De Jong et al에 의해 보고된 바늘과 같은 모양은 바이오 시스템에서 크기에 의존되는 요소로써 금 나노입자의 흡수, 침투, 순환 그리고 분포가 쉽다. 이러한 결과는 이전에 18 nm의 금 나노입자가 세포의 상처나 독성 없이 세포에 침투할 수 있다는 것을 발견한 Connor et al에 의해 보고된 것과 유사했다. Tsoli et al에 의한 연구는 1 nm의 금 나노입자가 세포와 핵 세포막에 침투하고 세포 손상과 죽음 없이 DNA에 부착할 수 있다는 것을 보여주었다. 세포의 손상 없이 세포로 들어가는 경로는 밝혀지지는 않았지만, 아마도 작은 나노크기가 주된 역할을 할 것이다. 따라서 작은 크기의 금 나노입자는 이후의 조사와 수정을 위해 생물학적 시스템에의 도입을 가능하게한다. 이러한 금 나노입자의 다양한 특징은 비슷하지 않은 세포 연구에서 독성 또는 비독성 중의 하나로 보고된 다양한 화학적 성질의 변환을 이끌었다. 몇몇은 코팅된 표면 리간드 때문에 크기에 의존적인 독성을 보여주고, 다른 것은 표면 입자 활성을 증가시키기 위해 큰 단위 체적당 표면적 비율이 플랫폼을 제공하기 때문이다. 그러므로, 이것은 큰 금 물질보다 침투의 쉽고 유연한 경로와 생물학적 시스템에의 반응성을 기여한다.

크기 측면에서. De Jong et al은 10 nm 금 나노입자가 실험동물에 투여될 때 24시간 안에 다른 크기의 입자보다 더 순환할 수 있다는 것을 찾았다. 10 nm 금 나노입자의 확산의 경로는 밝혀지지 않았다. 시스템 안에서 금 나노입자의 순환이매우 크기 의존적이라는 사실로부터, Hauck et al에 의한 이전의 연구결과도 30분 안에 노출되었을 때 50 nm 금 나노입자와 같은 다른 크기가 시스템 안에서 가장 많은 금 나노입자가 될 수 있다는 것을 보여줬다.

생물학적 시스템에서 금 나노입자의 상호작용은 종종 세포 안으로 들어갈 수 있게하는 물리화학적 성질과 연관된다.

6. The effects of surface charge on the toxicity of gold nanoparticles.

Surface charge which is measured by zeta potential is one of the major physical characteristic influencing AuNPs toxicity. The application of zeta potential provides useful information on the stability of colloid nanomaterials. It is thus, essential to always state whether the zeta potential of colloid NPs is positively or negatively charged. Surface charges determine the prop- erties and functions of NPs. AuNPs have charged (nega- tively or positively) surfaces which make them highly reactive and receptive to surface modifications due to either cations or anions interaction, thus, creating a net surface charge. Based on surface charges, AuNPs can promote protein refolding through electrostatic inter- actions between the exposed charged residues on the un folded protein and the oppositely charged ligands on the AuNPs. The overall high negative charge of the NP-protein complex prevents the proteins from aggregating; the NP thereby promotes refolding which can be used to refold proteins in a chemical denatured state.

It is important to note that modifications of NP surfaces may cause undesirable ionic interactions with biological systems, due to changes in surface charges. Many AuNPs are stabilized with surface charges to prevent aggregation via electrostatic repulsion, playing a significant role in toxicity of the NP. Aggregated AuNPs have modified surface charges which intend influence changes of cellular environment and thus altering the cellular behaviour and cellular toxicity.

Other studies have shown that surfaces charges of NPs enhance their uptake into cells. For example findings by Chithrani et al using incubated citric acid coated AuNPs have shown that NPs surface charges can poten- tial influence their uptake by mammalian cell line HeLa. Furthermore, He et al also found that although the surface charges play a significant role in phygocytoses they also aid phagocytic clearance due to the NPs small size and high diffusible nature. The interaction of AuNPs with serum proteins therefore alter the physiochemical properties of the NP, which can intend affect uptake and target drug delivery processes. Other factors such as ionic strength (charges) of AuNPs can also affect their biocompatibility, thereby interfering with the biokinetics of the cells, resulting in a reduction in cell viability.

However, citrate stabilized AuNPs toxicity test using MTT assay have shown that 20 nm AuNPs at a concen- tration of 300 µM have no significant effect on cell hu- man dermal fibroblast-fetal which was similar to earlier findings by Connor et al. In other study puri- fied and citrate sterilized AuNPs have shown rather milder cytotoxicity in A549 and NCIH441 cells as com- pared to the particles with excess citrate. This indicates that functionalized side chain can interfere with the ac- tivities of the AuNPs depending on the shape and surface charge. This, therefore, indicates that further more in vitro studies on cell viability concerning charge AuNPs properties are required to ascertain their toxicity.

Implication for in vivo / Cytotoxicity mechanism

The effect of AuNPs shows that the smaller the AuNP the higher the probability of it to cause toxicity as well as bind easily on cellular surfaces. For example 1.4 nm AuNP in diameter was found to bind with DNA and affect genes (mutation) as comparable to their larger ccounterparts.

The mechanisms of biodistribution of AuNPs so far described are via endocytotic-exocytotic activity and to a lesser extend by paracellular transport (transport of molecules around cells and via tight junctions of epithe- lial cells). Such mechanisms are due to differ- ences in the surface properties of the AuNPs, the type of animals used and the route of exposures.

Gosens et al believes that single AuNPs can pose greater health effects than their agglomerates and aggre- gates counter parts. Because of the agglomerates and aggregates relative larger sizes, they are restricted from translocating easily across membranes as compared to single nanogold particles. Although, when Gosens et al intratracheally instilled AuNPs agglomerates and spherical single dose of 1.6 mg/kg AuNPs (50 nm or 250 nm) into rat lungs, both particles gave mild pulmonary inflammation at the same dosage. Meanwhile, earlier reports by Mühlfeld et al and Sadauskas et al showed that when AuNPs are inhaled and deposited in the lungs, only a small fraction (both single and agglom- erates) can be phagocytozed with a small part translo- cated across the alveolar epithelium. Nevetheless, the nanosize factor is a major significant feature in determin- ing the deposition, translocation, distribution and fate of AuNPs. These facilitate the crossing of the blood brain barrier by AuNPs, which accumulate in neural tissues as well as in the placenta and fetus. Earlier re- ports by Takahashi and Matsuoka reported the up- take of colloidal AuNPs of 5 and 30 nm after maternal intravenous injection in rats. Other studies by Lee et al and Myllynen et al have also showed the inter- nalization of 10-30 nm PEGlyated AuNPs in the placen- tal cells which are comparable to immunoglobulins that cross the placenta (IgG). The findings from these studies also showed AuNPs with sizes up to 240 nm crossing the human placental barrier without affecting the viability of the placental explants. Other findings by Sadauskas et al, however, showed that AuNPs of 2 and 4 nm when injected intravenously or intraperitoneally respectively did not seem to penetrate either the placenta barrier or the blood - brain barrier but were found in the macro- phages and Kupffer liver cells. Information from litera- ture envisages size as the most significant physical prop- erty responsible for inducing AuNP toxicities

Conclusion

Although gold in the bulk form is non-toxic, nano gold has shown some negative effects on cells. Despite the promising future of gold nanoparticles in different biomedical fields, there are many fundamental toxic issues that need to be addressed. Size, shape and surface are important factors which determine the toxicity level of gold nanoparticles. Among them, gold nanoparticles with hydrodynamic size below 50 nm, negative surface charge (-40~-10mV) and rod-shape showed the most significant toxicity to cancer cells. However, the toxicity depend much on many other conditions such as assay methods, cell lines and surface coating materials. Further more, a point deserves attention is "in vitro toxicity evaluation are representative enough of in vivo prediction".


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[6] Brown, S. D.; Nativo, P.; Smith, J.-A.; Stirling, D.; Edwards, P. R.; Venugopal, B.; Flint, D. J.; Plumb, J. A.; Graham, D.; Wheate, N. J. J. Am. Chem. Soc., 2010, 132, 4678-4684.

[9] Peng, G.; Tisch, U.; Adams, O.; Hakim, M.; Shehada, N.; Broza, Y. Y.; Bilan, S.; Abdah-Bortnyak, R.; Kuten, A.; Haick, H. Nature Nanotech., 2009, 4, 669-673

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