- 1 Cellular uptake pathway
- 2 Nanoparticle uptake
- 3 References
Cellular uptake pathway
나노 물질에 의한 세포 독성 (Cytotoxicity) 은 세포 안에서 나타나는 독성으로, 이러한 독성은 나노 입자가 세포 내로 들어간 방향 ( Entrance route)과 세포 내의 위치에 의해 결정된다.  따라서, 나노물질이 uptake에 대해 이해하는 것은 나노 물질에 따른 독성을 평가하는데 있어 중요하다.
세포-나노입자의 상호작용을 통한 pathway 에 영향을 미치는 요인은 여러가지가 있다. 그 중 나노물질의 표면에 따라 pathway가 결정되는 것이 가장 크다.
나노 입자가 생체환경에서 들어갔을 때, 나노 입자의 표면에 단백질이 흡착을 하고 그 때문에 나노 입자의 표면이 개질화가 된다. (즉, 나노입자의 표면성질이 바뀜.) ; '나노입자-단백질 코로나(corona)의 형성'
나노입자-단백질 코로나는 in vivo 내에서 입자의 hydrodynamic 크기, 응집에 대한 resistance, 전하, 표면성질을 나타내는데 중요한 역할을 한다. 대부분의 경우 나노물질들에서의 세포반응은 나노물질 자체와의 반응보다는 흡착된 단백질과 같은 생물분자들에 의해 직접적으로 나타난다. 
Particles can cross the membrane only if they are at most 10 nm to 30 nm in size. 나노입자가 10-30 nm의 크기인 경우, 세포막을 가로지를 수 있다. 세포막은 세포 내부와 외부의 환경을 분리해주는 '장벽(barrier)'의 역할을 한다. 나노 입자가 이 장벽을 넘어 세포 내로 들어가는 메카니즘 중 한가지가 바로 Endocytosis 이다. 이 endocytosis는 주로 바이러스 (virus)에 의해 이용되고, 바이러스와 비슷한 크기를 가지는 나노입자 또한 세포 내로 이러한 메카니즘을 통해 들어갈 수 있다고 알려져왔다.  ※ 바이러스 크기 20-400 nm 
Spherical particles were rapidly and uniformly internalized because of their symmetry. This effect of shape was independent of particle size in the size ranges studied (0.1–100% of the volume of the macrophage).
The only difference observed related to particle size was the extent of internalization, which was only observed with particles in which the volume of the particle was larger than the volume of the cell. 
Several forms of endocytosis are distinguished, based on the substance to be internalized.
The binding and activation of membrane receptors and subsequent protein expression strongly depend on nanoparticle size. Although all nanoparticles within the 2–100 nm size range were found to alter signalling processes essential for basic cell functions (including cell death)7, 40- and 50-nm nanoparticles demonstrated the greatest effect. 
Endocytosis and diffusion have been proposed as mechanisms for the uptake into cells of NPs with similar sizes as viruses. Endocytosis is the uptake of particulate matter, such as proteins and other nutrients, into eukaryotic cells via enclosure by the cell membrane. It is also exploited by these cells for the clearance of cell debris and foreign cells from the body.
Endocytosis pathways can be subdivided into four categories: namely, receptor-mediated endocytosis, caveolae, macropinocytosis, and phagocytosis.[
각 endocytosis pathway를 통해 형성되는 소포(vesicles)의 최대 크기 (dimension)는 정해져있지 않지만, 그 크기는 세포 내로 들어갈 수 있는 cargo의 양으로 제한 되어있을 가능성이 크다.
Whether RME or another type of endocytosis takes place is determined, among other factors, by the particle size. For particles, the rate of RME is determined by interplay of the decrease in free energy required to drive the cargo into the cell, the amount of available binding sites, and the wrapping time. Nanoparticle uptake by endocytosis is a complex interplay of the energy release by the number of bound receptors, the number of vesicles that can be formed from the membrane, receptor diffusion toward the attached particle, and time to complete this process. For large NPs, allowing only 1 particle to be taken up per vesicle, attachment occurs to numerous receptors at a time, resulting in a major change in free energy. A larger contact area with the cell membrane for elongated particles will also lead to a larger decrease in the free energy, but at the same time to less available binding sites for other particles. Large particles will require more time to complete wrapping. For small particles, several NPs can be taken up in 1 vacuole, but energy release is weaker because only single attachment sites exist. Because a local decrease in the Gibbs free energy is required to induce membrane wrapping, a minimum size for a single particle at a given ligand density must exist; otherwise endocytosis is energetically impossible. Uptake is only initiated after a certain number of receptors have been triggered. If the change in free energy is too small, membrane wrapping will not be induced. 
Endocytosis가 일어나 세포 내로 나노입자를 uptake 하기 위해서는 복잡한 상호작용이 있다 많은 세포 표면에 bounding 된 수용체들에 의한 에너지 발생의 복잡한 상호작용이 있다.
RME 의 속도는 세포 내로 cargo를 운반하기 위해 요구되는 자유에너지, 세포 표면에 binding 할 수 있는 site의 수, 입자를 감싸는 시간과 같은 여러 상호작용 감소에 의해 결정된다.
큰 입자크기에 대해 1개의 소포 당 uptake 할 수 있는 입자의 수가 1개라면, 나노 입자의 세포 막 표면으로의 부착은 동시에 많은 수용체들에게서 일어난다. 자유에너지의 큰 변화를 일으킨다.
Phagocytosis is a specific form of endocytosis involving the vascular internalization of solids such as bacteria by an organism, and is therefore distinct from other forms of endocytosis such as the vesicular internalization of various liquids (pinocytosis). In an organism's immune system, phagocytosis is a major mechanism used to remove pathogens and cell debris. 
Opsonized particulate substances and small solute volumes are internalized by a mechanism called phagocytosis. Opsonins are small molecules that enhance binding in phagocytosis (e.g., antibodies)
[Phagocytosis 3-STEP ]
Step 1. Unbound phagocyte surface receptors do not trigger phagocytosis.
Step 2. Binding of receptors causes them to cluster.
Step 3. Phagocytosis is triggered and the particle is taken up by the phagocyte.
동물세포에서 일어나는 phagocytosis의 경우는 면역 방어시 일어나는 endocytosis 로, 주로 외부 세포 (감염성있는, 박테리아), 세포 파편 (debris) 과 apoptotic 세포를 분해하는 역할의 메카니즘이다. 특정 세포에서도 endocytosis 메카니즘으로 작용한다.
(예: macrophage 대식세포, monocyte 단핵구, neutrophil 호중구)
Receptor-mediated endocytosis (RME)
세포 막 표면에 있는 수용체 (receptor)에 의해 물질의 endocytosis가 진행되는 메카니즘으로 Clathrin-, Caveolae 가 있다.
Clathrin-mediated endocytosis (~120 nm)
Clathrin-mediated endocytosis mediated by small (approx. 100 nm in diameter) vesicles that have a morphologically characteristic coat made up of a complex of proteins that are mainly associated with the cytosolic protein clathrin. Clathrin-coated vesicles (CCVs) are found in virtually all cells and form domains of the plasma membrane termed clathrin-coated pits. Coated pits can concentrate large extracellular molecules that have different receptors responsible for the receptor-mediated endocytosis of ligands, e.g. low density lipoprotein, transferrin, growth factors, antibodies and many others.
Caveolae-mediated endocytosis (~80 nm)
Caveolae are the most common reported non-clathrin-coated plasma membrane buds, which exist on the surface of many, but not all cell types. They consist of the cholesterol-binding protein caveolin (Vip21) with a bilayer enriched in cholesterol and glycolipids. Caveolae are small (approx. 50 nm in diameter) flask-shape pits in the membrane that resemble the shape of a cave (hence the name caveolae). They can constitute up to a third of the plasma membrane area of the cells of some tissues, being especially abundant in smooth muscle, type I pneumocytes, fibroblasts, adipocytes, and endothelial cells. Uptake of extracellular molecules is also believed to be specifically mediated via receptors in caveolae.
Macropinocytosis (100 nm- 5 μm)
Macropinocytosis which usually occurs from highly ruffled regions of the plasma membrane, is the invagination of the cell membrane to form a pocket, which then pinches off into the cell to form a vesicle (0.5–5 µm in diameter) filled with a large volume of extracellular fluid and molecules within it (equivalent to ~100 CCVs). The filling of the pocket occurs in a non-specific manner. The vesicle then travels into the cytosol and fuses with other vesicles such as endosomes and lysosomes.
Several studies have shown that the cellular uptake of nanoparticles can depend on many factors, including the size and/or shape of the nanoparticle, sedimentation effects of large and dense particles, and the composition of the protein corona on the nanoparticle. 
Nanoparticle uptake by endocytosis is a complex interplay of the energy release by the number of bound receptors, the number of vesicles that can be formed from the membrane, receptor diffusion toward the attached particle, and time to complete this process. For large NPs, allowing only 1 particle to be taken up per vesicle, attachment occurs to numerous receptors at a time, resulting in a major change in free energy. A larger contact area with the cell membrane for elongated particles will also lead to a larger decrease in the free energy, but at the same time to less available binding sites for other particles. Large particles will require more time to complete wrapping. For small particles, several NPs can be taken up in 1 vacuole, but energy release is weaker because only single attachment sites exist. Because a local decrease in the Gibbs free energy is required to induce membrane wrapping, a minimum size for a single particle at a given ligand density must exist; otherwise endocytosis is energetically impossible. Uptake is only initiated after a certain number of receptors have been triggered. If the change in free energy is too small, membrane wrapping will not be induced. 
several nonphagocytic cells favored the uptake of smaller particles. Chithrani BD et al. demonstrated a clear relation between the size and number of gold particles being stabilized with citric acid ligands in each HeLa cell. There was an uptake optimum for spherical NP of 50 nm 
나노 입자의 uptake는 크게 두가지에 영향을 받는데, 그 요인은 다음과 같다. 
1) 나노 입자 자체의 성질 (Nanoparticle properties)
Previous research found a strong decrease in bead internalization and in the speed of the larger particles in comparison with 50-nm beads. This is in good agreement with the results of He et al. , who found that several nonphagocytic cells favored the uptake of smaller particles. They also found a clear relation between the size and number of gold particles being stabilized with citric acid ligands in each HeLa cell. There was an uptake optimum for spherical NP of 50 nm. An optimum based on the number of particles per cell does not necessarily lead to the same optimum in terms of mass. Most of studies have reported the number of particles per cell, expressed internalization of different-sized NPs by 3 different units (number of particles per cell, particle volume per cell, and particle surface area per cell). 
Overall, particle uptake by nonphagocytic cells shows a clear trend: uptake increases with particle size to an optimum of approximately 50 nm and decreases for larger particles [67,74,86,93–95]. For phagocytes, the uptake is not unequivocally related to particle size.
Surface charge (표면전하)
Surface functional groups (표면 작용기)
Zero surface charges, either by neutral surface groups (e.g., hydroxyl groups) or by zwitterionic ligands, have been shown to lead to low cellular uptake compared with charged particles [70,100–102,106]. This can be explained by the low NP affinity toward the overall negatively charged cell membrane. Zero surface charges cause the hindrance of nonspecific protein adsorption [123,124], and a strong hydration layer via electrostatic interaction may be formed.
2) 실험 조건 (Experimental condition)
Cell type (세포종류)
세포 종류 [ 암세포 vs 정상세포, 식세포(phagocytic) vs 비식세포 (nonphagocytic) ] 에 따른 차이가 세포 내 나노 입자의 uptake에 영향을 미친다. 암세포의 경우 정상세포보다 표면에 있는 수용체 (receptor)의 수가 다르다.
따라서 세포 표면에 결합(binding)할 수 있는 site의 수와 uptake에 영향을 미친다.
- Feng Zhao, Cellular uptake, Intracellular Trafficking, and Cytotoxicity of nanomaterials
- Katja Kettler et al. Cellular uptake of nanoparticles as determined by particle properties, experimental conditions, and cell type.
- Robby A. Petros et al. Strategies in the design of nanoparticles for therapeutic applications
- W.JIANG et al. Nanoparticle-mediated cellular response is size-dependent
- 위키피디아_Endocytosis https://en.wikipedia.org/wiki/Endocytosis
- 위키피디아_phagocytosis https://en.wikipedia.org/wiki/Phagocytosis
- Jong An Kim et al. Role of cell cycle on the cellular uptake and dilution of nanoparticles in a cell population
- Chithrani BD et al. Determining the size and shape dependence of gold nanoparticle uptake into mammalian cells.
- Figure2, Katja Kettler et al.