Keywords

7.1 Introduction

Recent developments in the biomedical sciences such as hyperthermia cancer therapy (Hainfeld et al. 2004; El-Sayed et al. 2005; El-sayed et al. 2006), targeted gene, and drug delivery (Sandhu et al. 2002; Koo et al. 2005) involve the interaction of nanoparticles (NPs) with living organisms, at the cellular level (Alivisatos et al. 2005; Parak et al. 2005; Luccardini et al. 2007). All these applications of NPs involve administration of NPs by different routes. After administration by various routes, NPs come in contact with cells. NPs may not only enhance the pharmacological effects of drugs, but also cause unwanted effects in the target cells (Nishikawa et al. 2009). Many factors affect the biological response of NPs with cells.

Cellular response of NPs can be varied by changing the type of NPs. Changing of even one variable can induce a very different outcome for NPs exposed to cells (Oh et al. 2011). The biological responses of NPs are dependent on size, charge, chemical composition, shape and surface chemistry (Verma and Stellacci 2010). In addition, targeting moieties such as tripeptide glutathione, cell penetrating peptides, nuclear localisation signal peptides, and proteins can also affect the cellular response (Oh et al. 2011). These parameters can also affect the cellular uptake, and the biological response of NPs (Oberdorster et al. 2005; Xia et al. 2008a, b). NPs in the blood stream are known to encounter with plasma proteins and immune cells. Processes such as adsorption of proteins create unfavourable condition for cellular uptake. Uptake of NPs by immune cells may occur by various pathways and can be enhanced by adsorption of plasma proteins. NPs coated with targeting moieties activate their receptor-mediated internalisation. The activation of membrane receptors strongly depends on the size of nanomaterials. NPs coated with targeting moieties are also found to be activating cell signalling processes essential for basic cell functions including cell death (Jiang et al. 2008).

Cellular uptake of NPs also has an influence on response of NPs towards cells. They are involved in manipulation of signal transductions leading to the expression of cell functions (Nishikawa et al. 2009).

Owing to currently arising opportunities and concerns associated with NPs in living systems, it is of immense importance to develop an understanding of the complex processes that govern their cellular response and intracellular fate of nanomaterials (NMs). Interestingly, the interaction between NPs and molecules of the biological milieu results in the formation of a biological coating on the surface of NPs (Lynch and Dawson 2008). Therefore, biological entity which interacts with cells, tissues and organs, is completely different from the original surface of the NPs. Formation of biomolecule corona is a dynamic process and biomolecules like proteins and lipids present in the biological fluids compete for NPs surface to form biomolecule corona (Mahmoudi et al. 2011a, b). The biomolecule corona is usually enriched with about 10–50 proteins, that have the maximum affinity for the NPs surface, out of several thousand proteins of the biological milieu. New and advanced techniques are required for deep understanding of both the thermodynamics and kinetics of the biomolecule corona evolution and its subsequent biological impacts (Mahmoudi et al. 2011a, b).

The biological responses to NPs are greatly affected by the key forces at the bio-nano interface and also by the inherent characteristics of the NPs like size, shape, charge, coatings, surface modifications with targeting ligands, crystallinity, electronic states, surface wrapping in the biological medium, hydrophobicity, and wettability. Therefore, a thoughtful investigation of the NP biomolecule complex is essential for the development of therapeutically safe NPs (Nel et al. 2009). In this chapter, we have covered pathways for cellular uptake of NPs, monitoring of endocytic pathways, cellular response of therapeutic NPs, and formation, characterisation, and factors affecting biomolecule corona of NPs.

7.2 Pathways for Cellular Uptake of Nanoparticles

The invasion of NPs in human body occurs via inhalation, ingestion or through the skin. Once these tiny particles enter a biological milieu, they will inevitably come into contact with a huge variety of biomolecules including proteins, sugars and lipids which are dissolved in body fluids. Thus, the NPs have to be viewed as evolving systems which adapt to varying concentrations of the biomolecules present in the fluid. NPs enter the cells mainly through the passive diffusion or active transport, while nanomedicines got entry into cells via endocytosis which helps the drug to penetrate the specific cells and get accumulated (Fig. 7.1). The endocytosis pathway has been classified according to the proteins which play a major role in the process. Correspondingly, the mechanism of interaction of NPs with cytomembrane which governs the entrance and travel of NPs inside cells has also been extensively studied.

Fig. 7.1
figure 1

Different uptake pathways followed by NPs for internalisation inside the cells. NPs follow phagocytosis, clathrin mediated endocytosis, caveolin mediated endocytosis, clathrin and caveolin-independent endocytosis and macropinocytosis pathways

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Various aspects such as pathways of entrance, factors affecting the pathways, functions of some proteins involved in endocytosis are still uncertain and are not absolutely proven. Such study is necessary for better understanding of the novel field of multifunction nanomedicines. NPs uptake into cells occurs through endocytosis (Jones et al. 2003), a process by which cells absorb NPs from outside by engulfing them within their cell membrane (Conner and Schmid 2003). This process of cellular uptake is further categorised into two phenomena, namely phagocytosis and pinocytosis (Fig. 7.1). Phagocytosis was originally discovered in macrophages. Phagocytes such as macrophages, neutrophils and monocytes destroy foreign particles such as NPs in blood through the phagocytosis process (Watson et al. 2005). Relatively, large NPs prefer this mechanism which initially involves recognition by opsonin viz., immunoglobulin (IgG and IgM), complement component (C3, C4, and C5) and blood serum proteins. Thereafter, the NPs bind to the cell surface receptors inducing the cup-shaped membrane extension formation. Such membrane extensions encircle the NPs and then internalise them, forming the phagosomes of diameter 0.5–10 µm which finally move to fuse with lysosomes (Aderem and Underhill 1999). Pinocytosis has been reported to occur by four different mechanisms: macropinocytosis, caveolin-dependent endocytosis, clathrin-dependent endocytosis and clathrin/caveolin-independent endocytosis (Fig. 7.1) (Swanson and Watts 1995; Patel et al. 2007; Xu et al. 2009). Macropinocytosis is a growth factor-induced, actin-driven endocytosis and a non-selective process for uptake of solute molecules or cargo. Macropinocytosis involves the formation of lamellipodia-like plasma membrane extensions. Interestingly, macropinosomes can uptake NPs of >200 nm in size (Walsh et al. 2006). Cellular uptake by macropinocytosis has been reported for PEGylated-poly-L-lysine NPs (Walsh et al. 2006). Receptor-mediated endocytosis process is assisted by specific proteins, either clathrin or caveolae (Bao and Bao 2005). Caveolin, a protein exists in most cells plays a dominant role in caveolae-dependent endocytosis process. This pathway bypasses lysosomes (Benmerah and Lamaze 2007), thus many pathogens including viruses and bacteria select this pathway to avoid lysosomal degradation (Medina-Kauwe 2007). Transferrin-coated PLGA NPs are highly absorbed by brain endothelial cells via caveolae pathway (Chang et al. 2009). Similarly, clathrin-coated pits have the ability to accumulate NPs only up to 100 nm (Pelkmans and Helenius 2002) and targeted (receptor) NPs are internalised by clathrin-mediated endocytosis (Walsh et al. 2006). The internalisation is more efficient for NPs smaller than the caveolae. Clathrin/caveolae-independent endocytosis is a distinct pathway which relies on cholesterol and requires specific lipid compositions. NPs endocytosis by cells not only depends on the size, but also on surface properties of the NPs. Carboxy dextran-coated superparamagnetic iron oxide NPs (SPION) have been internalised by human mesenchymal stem cells and the efficiency of internalisation was correlated with the amount of carboxyl groups on the NPs surface (Mailänder et al. 2008). Cationic NPs enter HeLa cells in greater amounts than anionic NPs (Harush-Frenkel et al. 2007; Dausend et al. 2008). NPs uptake may also depends on the length of the molecules on the surface coating (Chang et al. 2009), or the type of cells (Xia et al. 2008a, b).

7.3 Monitoring Endocytic Pathways

Researchers have been interested in identifying the different pathways that NPs use during their internalisation to cells. Either endocytic markers or inhibitors have been used for long time to locate NPs and confirm whether the corresponding pathway plays an important role in the uptake of the NPs. More recently, a combined approach has been postulated for better understanding of such mechanisms. Low-density lipoprotein (Duit et al. 2010) and transferrin (Liu et al. 2010a) enter into cells through clathrin-dependent endocytosis, and hence these are commonly used markers for clathrin-dependent endocytosis process. Similarly, caveolin-1 is mostly used as a marker of caveolae-dependent endocytosis, and dextran is the marker of macropinocytosis (Petrescu et al. 2009). Inhibitors can be used to block certain endocytic pathways to verify whether this route has been used by NPs to enter cells. It has been reported that amiloride, cytochalasin D and rottlerin can block macropinocytosis (Diken et al. 2011), whereas chlorpromazine (50–100 µm), hypertonic sucrose (0.4–0.5 m) and potassium depletion can be used to inhibit the clathrin-dependent endocytosis (Ivanov 2008). Similarly, methyl-β-cyclodextrin, filipin, nystatin and cholesterol oxidase can be used as the inhibitors for caveolae-dependent endocytosis (Diken et al. 2011).

7.4 Factors Affecting Cellular Response of Nanoparticles

NPs possess intricate structures and surface chemistry. In biological systems, they may play an undecided role (Xie et al. 2009). Response of NPs with cells depends on the type of cells, cell culture media, size, shape, surface charge, surface moieties, temperature and route of administration of NPs (Fig. 7.2). These are the key parameters for particle binding and wrapping by the surface membrane as well as defining the path of cellular uptake (Xia et al. 2008a, b). Type of cells and cell lines also affect the cellular response. Cell lines grown in different culture media showed different cellular response to NPs (Maiorano et al. 2010). In addition to this, type of NPs also has an effect on its fate inside the cells. NPs are synthesised from materials of different compositions. Biodegradable NPs are rapidly cleared from the cells (Chellat et al. 2005), while non-biodegradable particles are retained inside the body for longer duration (Niidome et al. 2006). NPs uptake by cells is also affected by their shape. Shape of NPs has not only affected the cellular uptake but also the internalisation. Spherical particles are internalised at a higher rate in endothelial cells (Jun et al. 2005).

Fig. 7.2
figure 2

Factors affecting cellular response of NPs a Size Positively charged NPs are well taken up by cells. b Shape Spherical NPs are efficiently taken up by cells c Temperature High temperature allows both cellular uptake and interaction, whereas at low temperature only interaction of NPs takes place. d Cell culture media NPs incubated in RPMI media showed more cell uptake than NPs incubated in DMEM media

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Surface chemistry of NPs has immense role to play in the cellular response of NPs (Bartneck et al. 2010) Polyethylene oxide (PEO)-coated gold nanorods with amine end groups have exhibited anti-inflammatory properties, whereas carboxy terminated led to proinflammatory effects. NMs surface chemistry has influence on the expression of inflammatory genes and phenotype of macrophages (Bartneck et al. 2010). Surface charge of NPs is also reported to affect the response of NPs with cells. NPs with cationicsurface charge show positive response with negatively charged cell membrane. The internalisation of negatively charged NPs is believed to occur through non-specific binding and clustering of NPs on cationic sites of the plasma membrane and their subsequent endocytosis. Negatively charged NPs have displayed a less efficient rate of endocytosis. NPs with positive surface charge depolarize the plasma membrane leading to increased Ca2+ influx (Yue et al. 2011). Positively charged NPs normally escape from lysosome and reside in perinuclear region, whereas the negatively charged NPs prefer to co-localise with lysosome (Yue et al. 2011).

Cellular uptake of positively charged particles into lysosomal compartments could lead to cytotoxicity by acidifying proton pump (Boussif et al. 1995). In addition, the positively charged NPs (Fig. 7.2) have been shown to target cell membranes through strong binding to phospholipid components, which can lead to membrane disruption (Asokan and Cho 2002).

Charged NPs can also activate complement system, hemolysis and thrombogenicity. Charged NPs are more aggressively invoked complement system than neutral ones (Bartlett and Davis 2007; Nagayama et al. 2007). Gold NPs activate the immune system (Bastus et al. 2009). Bare lipid NMs show high cellular uptake due to non-specific internalisation through charge interaction of the positively charged NPs with the negatively charged cell surface. The PEGylation of the NPs limited the non-specific charge interaction and resulted in reduced cellular uptake of NPs (Wang et al. 2009).

Targeting moieties attached to the surface of NPs decide their fate inside the cells. NPs with specific recognition moieties bound to the surface have a good potential for site-selective uptake as well as improved specificity for drug targeting (Dinauer et al. 2005). This strategy is used to direct NPs to cell surface carbohydrates, receptors and antigens (Sinha et al. 2006). Moieties attached to the surface can include any molecule that selectively recognises and binds molecules on target cells (Sapra and Allen 2003). Antibodies, oligopeptides, carbohydrates, glycolipids and folic acid are the most widely used moieties for targeting different cells and tissues. Targeted NPs can bind more specifically to cancer cells than normal cells. NPs without specific moieties can attack normal cells as well. Peptide-conjugated gold NPs activate the macrophage system. Macrophage activation by gold NPs depend on the peptide pattern at the NPs surface (Bastus et al. 2009). Targeted NPs are internalised by cancer cells through receptor-mediated endocytosis enhancing cancer cell killing (Hu et al. 2010). NPs are internalised by different uptake pathways which also affect the cellular response (Xia et al. 2008a, b; Kumari and Yadav 2011). Multiwalled carbon nanotubes (MWCNTs) and quantum dots delivered by nano-channel electroporation undergo nonendocytic uptake in BEAS-2B cells and result in higher cell death (Zhao et al. 2015). Cellular uptake of NPs is also affected by the type of cells. Different types of cells internalise NPs to a different extent. Gold NPs with positive surface charge have shown more cell internalisation ability than those with negative surface charge in non-phagocytic cells. However, the internalised amount of negatively charged gold NPs was similar with that of the positively charged gold NPs in phagocytic cells (Liu et al. 2013).

Polystyrene NPs have been efficiently internalised by human ATI cell line (TT1) cells, while uptake of NPs by primary human ATII cells was negligible (Thorley et al. 2014). It has been shown that extent of NPs uptake, kinetics of NPs uptake and NPs internalisation mechanisms differ between primary cells and phenotypically linked cells (Lunov et al. 2011). Pericellular matrix (PCM) of cell has been documented for a role in accumulation and enhancing uptake of NPs (Zhou et al. 2012). Cellular uptake of NPs is also modulated by cell surface area and membrane tension. It has been reported that cellular internalisation is enhanced linearly with cell surface area and decreased exponentially with increasing membrane tension (Huang et al. 2013). Cell culture media also has an influence on NPs uptake and internalisation. TiO2 NPs incubated in three different media showed different extent of uptake and internalisation. Uptake of TiO2 NPs was maximum in media with highest amount of protein (Strickland et al. 2013). Gold NPs incubated in RPMI media showed more cellular uptake than NPs incubated in DMEM media (Maiorano et al. 2010).

7.5 Cellular Response of Therapeutic Nanoparticles

NPs are increasingly used in the field of drug delivery. NPs show various responses with cells. These include interactions with cellular membrane and cellular uptake, signalling pathways, ROS production, cell cycle dysregulation and necrosis or apoptosis (Jones and Grainger 2009). Many in vitro models are used to study the response of NPs with cells (Kumar et al. 2012). Metallic and polymeric NPs are extensively used in the field of nanomedicine due to their ease of preparation and surface modification. Understanding the cellular response of metallic, polymeric and silica NPs has recently fascinated the attention of scientific community.

7.5.1 Metallic Nanoparticles

Toxicity of silver nanoparticles (Ag NPs) to human hepatoma cells is the result of oxidative stress and is independent of the toxicity of Ag+ ions (Jun et al. 2005). Polysaccharide-coated and uncoated Ag NPs are distributed differently and cause different levels of DNA damage in mouse embryonic stem cells (MES) and mouse embryonic fibroblast cells (MEF). Both types of Ag NPs induce p53 protein expression, DNA double-strand breakage and apoptosis responses in MES and MEF cells (Ahamed et al. 2008). Upon exposure to 6.25 μg/mL Ag NPs, morphology of both HT-1080 and A431 types of cells remain unaltered. However, at higher concentrations (6.25–50 μg/mL) of Ag NPs, cells became less polyhedra, more fusiform and shrunken. Changes in the levels of catalase and glutathione peroxidase in A431 and A431 types of cells are statistically insignificant. Ag NP exposure caused the DNA fragmentation in cells (Arora et al. 2008). Citrate-stabilised NPs show significant cellular response, while cellular treatment with nucleic acid or BSA functionalised NPs caused no detectable changes in gene expression, cell cycle progression or apoptosis induction (Massich et al. 2010). Nucleic acid-modified gold NPs have been reported for less immune response (Massich et al. 2009). The L929 cells become round and even shrunken on exposure to TiO2 NPs (Jin et al. 2008). Moreover, TiO2 NPs-treated cells either show condensation of fragmented chromatin or directly necrosed. Cells cultured in a medium containing 300 μg/mL TiO2 have increased the number of lysosomes and damaged some of the cytoplasmic organelles. Gold NPs with weakly bound ligands have shown significant cellular responses, while gold NPs with strongly bound ligands have shown weak cellular responses (Massich et al. 2010). Gold NPs have also been observed to induce cell death in human carcinoma lung cell line A549. In contrast, BHK21 (baby hamster kidney) and HepG2 (human hepatocellular liver carcinoma) cell lines remain unaltered by gold NPs treatment (Patra et al. 2007). ZnO NPs showed dose-dependent toxicity in RAW 264.7 cells, higher cellular uptake and elevated intracellular ROS level (Hong et al. 2013).

7.5.2 Silica Nanoparticles

SiO2 NPs are extensively used in the field of cancer therapy (Hirsch et al. 2003), DNA delivery (Bharali et al. 2005), drug delivery (Venkatesan et al. 2005) and enzyme immobilisation (Qhobosheane et al. 2001; Chen and von Mikecz 2005). SiO2 NMs have caused decrease in number of molecules released per mast cell granule (Maurer-Jones et al. 2010). Exposure to 25–500 μg/mL of mesoporus SiO2 NPs have been reported to inhibit cellular respiration in a concentration and time-dependent manner (Jin et al. 2007). Dye-doped silica NPs have shown low level of genotoxicity and cytotoxicity against the tested A549 cells (Jin et al. 2007). Silica nanotubes have exhibited growth inhibition in epithelial breast cancer cell line (MDA-MB-231) and primary umbilical vein endothelial cell line (HUVEC) (Nan et al. 2008) in a concentration-dependent manner.

7.5.3 Polymeric Nanoparticles

PLA NPs have elicited a strong cytotoxic T lymphocyte (CTL) response and a strong T helper cells-biased cytokine release in mice (Ataman-Önal et al. 2006). HIV envelope glycoproteins 140 carrying carnuba wax NPs have been reported to induce strong cellular/humoral response without inflammation (Arias et al. 2011). Polysiloxane NPs were endocytosed via caveolae in human aortic endothelial cells and enhanced nitric oxide release (Nishikawa et al. 2009). The incorporation of a bombesin peptide or RGD peptide via a PEG spacer in polymeric NMs was resulted in receptor-mediated cellular uptake and high gene silencing efficiency in U87 cells (Wang et al. 2009). Ligand-receptor recognition between cRGD and αvβ3 integrin has mediated the surface binding of RGD-targeted NMs to HUVECs and probably induced cRGD- targeted NPs to enter cells through caveolae and localised in the perinuclear regions (Liu et al. 2010b). cRGD functionalization of PLGA NPs has appreciably improved NP accumulation in tumour cells in vitro and resulted in improved tumour accumulation of NPs in a mouse model (Toti et al. 2010). Cationic polystyrene NPs have caused necrotic and apoptotic cell deaths in BEAS-2B and RAW264.7 cells, respectively (Xia et al. 2008a, b).

7.5.4 Quantum Dots

Quantum dots (QDs) with unique photochemical properties such as high photoluminescence and photo stability have shown great potential as a bimodal imaging agent, cancer diagnostics and drug delivery (Lee et al. 2010). Cetuximab-coated quantum dots have shown enhanced uptake in EGFR overexpressing A549 cells (Lee et al. 2010). Quantum dots have also undergone transformation in biological systems (Mahendra et al. 2008; Pettibone et al. 2013). Speciation of four types of CdSe/ZnS QDs in HepG2 cells has been studied in a recent report and it was observed that two chemical forms, named as QD-1 and QD-2, have been detected in HepG2 cells. QD-1 and QD-2 has been confirmed as QD-like NPs and a kind of cadmium metallothioneins complex, respectively (Peng et al. 2015). Matrix metalloproteases (MMP-2 and MMP-7) decorated quantum dots also have shown enhanced uptake in HT-1080 cells (Zhang et al. 2006). Ligand-conjugated QDs were not accumulated in vesicles of the early sorting pathways and were also able to reach the lysosomes of dendritic cells (Cambi et al. 2007). Tat peptide-conjugated quantum dots (Tat-QDs) has been internalised by macropinocytosis. The internalised Tat-QDs have sticked to the inner vesicle surfaces and trapped in cytoplasmic organelles and actively transported by molecular machines such as dyneins along microtubule tracks (Ruan et al. 2007).

7.5.5 Liposomes

Liposomes have long been considered good candidates for drug delivery. Effect of surface charge on the binding and endocytosis of liposomes has been investigated on human ovarian carcinoma cell line (HeLa) and a murine-derived mononuclear macrophage cell line J774. HeLa cells have been found to endocytose positively charged liposomes to a greater extent than either neutral or negatively charged liposomes (Miller et al. 1998). In contrast, the extent of liposome interaction with J774 cells was greater for both cationic and anionic liposomes than for neutral liposomes (Miller et al. 1998). Tumour penetrating peptides (TPP) targeting liposomes have exhibited remarkably increased cellular accumulation by PC-3 tumour cells than bare liposomes (Yan et al. 2014). Liposomes modified with octaarginine have enhanced the efficiency of cross-presentation of ovalbumin in mouse bone marrow-derived dendritic cells (Nakamura et al. 2014). Hyaluronan (HA) coated liposomes have shown cellular uptake via lipid raft-mediated endocytosis in A549 cells. Once within cells, HA-liposomes have localised primarily to endosomes and lysosomes (Qhattal and Liu, 2011). Aptamer targeted liposomes have shown enhanced binding specificity and selectivity to CD44 expressing A549 and MDA-MB-231 cells (Alshaer et al. 2015). Annexin A5 functionalised liposomes bind to phosphatidylserine exposing apoptotic K562 cells with high specificity (Garnier et al. 2009).

7.6 Protein Corona Formation on Therapeutic Nanoparticles

Upon entering in biofluids, NPs surfaces were rapidly covered by selective sets of blood plasma proteins forming the protein corona (Cedervall et al. 2007a, b; Lindman et al. 2007; Mahmoudi et al. 2009; Mahmoudi et al. 2010; Walczyk et al. 2010). Upon entry of NPs into biological millieu, they were initially surrounded by high concentrations of free protein. Proteins moved towards the NP surface either by diffusion, or by travelling down a potential energy gradient. Protein adsorption in the neighbourhood of the NPs surface has occurred spontaneously only if it is thermodynamically favourable (Walkey and Chan 2012). In other words, if:

$$\Delta {\text{G}}_{\text{ads}} =\Delta {\text{H}}_{\text{ads}} - {\text{T}}\Delta {\text{S}}_{\text{ads}} < 0$$

where ΔGads, ΔHads and ΔSads are the changes in Gibbs free energy, enthalpy and entropy, respectively, during adsorption, and T is the temperature.

The formation of covalent and non-covalent bonds between NPs and protein, rearrangement of interfacial water molecules or conformational changes in either the protein or the NPs surface contribute to favourable changes in enthalpy (ΔHads < 0), or entropy (ΔSads > 0) (Walkey and Chan 2012). Type of protein (Lindman et al. 2007) and physiochemical properties of NPs determine the mechanism involved during adsorption and protein corona formation (De et al. 2007). Proteins interact with NPs surface through a portion known as domain during adsorption and protein corona formation. Adsorption of high-molecular weight protein kininogen to iron oxide NPs has occurred through its domain 5 (Simberg et al. 2009). Adsorption of proteins to NPs surface may involve interactions through many domains of the proteins (Walkey and Chan 2012). ΔGads determines the stability of the protein NPs corona. Proteins with large ΔGads stay with NPs surface, while proteins with small ΔGads desorb and return to solution (Norde 1994). Hydrophobic NPs interact strongly with proteins than those of hydrophilic (Walkey and Chan 2012). Hydrophobic or charged NPs cause more conformational changes in protein than their hydrophilic counterparts. Conformational changes of proteins are either reversible or irreversible and depend on the structure and chemistry of protein and NPs (Walkey and Chan 2012).

Protein corona is further divided into two types, viz. hard corona and soft corona (Fig. 7.3). Hard corona consists of an inner layer of selected proteins with a lifetime of several hours in slow exchange with the environment (Fig. 7.3). Soft corona comprises of an outer layer of weakly bound proteins, which are characterised by a faster exchange rate with the free proteins (Mahmoudi et al. 2010). It is now believed that the hard corona interacts with cellular receptors and defines the fate of NPs in a biofluid due to the long lifetime of the hard protein corona (Mahmoudi et al. 2011a). It has been documented in a study that negatively charged NPs did not show the formation of hard protein corona while positively charged NPs show the formation of hard protein corona (Casals et al. 2010). First monolayer (hard corona) of transferrin binds irreversibly to polystyrene NPs and second monolayer (soft corona) is capable of exchanging proteins with solution (Milani et al. 2012).

Fig. 7.3
figure 3

Nanoparticles inside the biological systems. NPs form soft and hard corona after interaction with plasma proteins. Fate of NPs is decided by the protein corona of NPs

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7.7 Characterisation of Protein Corona on Nanoparticles

Upon contact with biological fluids, NPs interact strongly with proteins and other biomolecules, which drastically alter their surface characteristics. NPs with improved and changed biological activity will have the influence on NPs bio-distribution and their in vivo fate. Therefore, it is essential to apply and develop analytical tools and techniques to investigate the interactions of NPs with proteins in order to understand the protein corona composition and their possible biological activity. When NPs are meant for biological applications, it is necessary to investigate their physicochemical characteristics in the biological milieu (Mahmoudi et al. 2011a, b). As reported, many studies have been performed to shed light on the protein NPs association/dissociation processes in serum and plasma. As more than 3700 proteins coexist and compete for binding to the NPs surface, the determination of binding rates, affinities, and stoichiometries of protein association, and dissociation with NPs in biological fluids is particularly tedious and complicated process (Mahmoudi et al. 2011a, b). Protein corona is characterised by five parameters like thickness and density, identity and quantity, arrangement and orientation, conformation, and affinity of proteins on NPs. Together, these parameters describe the interaction of NPs with a biological environment (Walkey and Chan 2012). The composition and structure of the protein corona has been studied using either in situ or ex situ techniques. In situ techniques measure the protein corona, while the NPs is dispersed in a biological milieu. These techniques are limited in number and typically provide the least amount of information. Ex situ measurements require isolation of the NPs with its bound protein from the biological environment (Walkey and Chan 2012). Methods used for the characterisation of protein corona are Fourier transform infrared spectroscopy (FTIR), Raman spectroscopy, fluorescence correlation spectroscopy, differential centrifugal sedimentation (DCS), isothermal titration calorimeter (ITC), LC-MS, electrophoresis, size exclusion chromatography, and dynamic light scattering.

7.7.1 Fourier Transform Infrared Spectroscopy (FTIR)

FTIR spectroscopy is known as an important technique for the examination of protein conformation in H2O-based solution, resulting in a greatly expanded use in studies of protein secondary structure and protein dynamics in the past decade (Kong and Yu 2007). FTIR spectroscopy has been used for characterising the interactions of NPs with proteins (Wang et al. 2012).

7.7.2 Raman Spectroscopy

Raman scattering technique is a vibrational molecular spectroscopy in which a laser photon is scattered by a sample molecule and during the process energy is either lost or gained (Kengne-Momo et al. 2012). The amount of energy lost is seen as a change in energy (wavelength) of the irradiating photon which is characteristic for a particular bond in the molecule. The Raman signal produces a precise spectral sample fingerprint, unique to each atom, group of atoms or individual molecule (Kengne-Momo et al. 2012). A recent study has used surface enhanced Raman scattering spectroscopy to study changes in the proteins secondary structure as well as the effect on integrity and conformations of disulfide bonds immediately on the NP surface (Grass and Treuel 2014).

7.7.3 Fluorescence Correlation Spectroscopy

Fluorescence correlation spectroscopy (FCS) is a technique used to study the molecular movements and interactions. This technique monitors the fluctuation in the fluorescence signal from fluorescently labelled molecules inside the small confocal volume (Röcker et al. 2009; Maffre et al. 2011). In FCS, one usually considers the dynamics of number fluctuations in an open sampling volume of a macroscopic system that fluctuates about average equilibrium concentrations which are determined by the surrounding medium and its thermodynamics (Röcker et al. 2009; Maffre et al. 2011). The concentration fluctuations of each species can occur by in situ chemical reactions and by diffusion of each species in and out of the sampling volume. FCS is a highly accurate method to simultaneously determine binding affinities and the thickness of the protein corona on NPs (Röcker et al. 2009; Maffre et al. 2011). This method permits the quantitative observation of protein adsorption in situ on NPs (Treuel et al. 2014). Recent study has used FCS to quantitatively monitor HSA adsorption onto dihydrolipoic acid quantum dots (Treuel et al. 2014).

7.7.4 Differential Centrifugal Sedimentation

Differential centrifugal sedimentation (DCS) is based on the ability to separate NPs of the same density by mass, i.e. size (Cölfen 2004). DCS is a fast, accurate and relatively inexpensive, resolves multimodal size distributions, and uses relatively small sample volumes (Krpetic et al. 2012). DCS have been extensively used to measure size distribution, and hydrodynamic radii of NPs (Machtle 1999; Müller 2004, 2006). In addition to particle size, DCS has been used to determine changes in surface structure, study of binding isotherms of NPs and showed the attachment of DNA binding protein to NPs (Salvati et al. 2013). DCS can also be used for measuring thickness of protein corona.

7.7.5 Isothermal Titration Calorimetry

Isothermal titration calorimetry (ITC) is the only technique that can simultaneously determine all binding parameters in a single experiment. ITC measures heat transfer during binding which enables accurate determination of binding constants (KD), reaction stoichiometry (n), enthalpy (∆H) and entropy (ΔS). This provides a complete thermodynamic profile of the molecular interaction. ITC also helps in elucidating the mechanisms underlying molecular interactions. The strength of protein interactions can be assessed using ITC and it also provides additional information on the thermodynamics of protein adsorption (Lindman et al. 2007).

7.7.6 Liquid Chromatography-Mass Spectrometry

Liquid chromatography-mass spectrometry (LC-MS) is a technique applied to a wide range of biological molecules. Mass spectrometers operate by converting the analyte molecules to a charged state, with subsequent analysis of fragment ions that are produced during the ionisation process, on the basis of their mass to charge ratio (m/z) (Pitt 2009). LC-MS plays an important role in several areas of clinical biochemistry and competes with conventional liquid chromatography and other techniques such as immunoassay. LC-MS has been used recently in a study to quantify the proteins in protein corona formed on SiO2 and polystyrene NPs. The composition of protein corona has been investigated by LC-MS on polystyrene NPs with average diameters slightly above 100 nm and resulted in identification of approximately 170 different adsorbed proteins (Ritz et al. 2015).

7.7.7 Matrix-Assisted Laser Desorption/Ionisation Time of Flight Mass Spectrometer (MALDI-TOF MS)

The sample for matrix-assisted laser desorption/ionisation (MALDI) is uniformly mixed in a large quantity of UV-absorbing matrix and then followed by time of flight mass spectrometry (Marvina et al. 2003).The matrix absorbs the UV light and converts it into heat energy. A small part of the matrix is heated and rapidly vaporised, together with the sample. Different matrices are used for different kind of samples. Derivatives of benzoic acid, cinnamic acid and other related aromatic compounds are usually approved as good quality MALDI matrices for protein analysis (Hillenkamp et al. 1991). Charged ions of various sizes are generated on the sample slide, a potential difference between the sample slide and ground attracts the ions in a direction. As the potential difference is constant with respect to all ions, ions with smaller m/z value and more highly charged ions move faster through the drift space until they reach the detector. Consequently, the time of ion flight differs according to m/z value of the ion (Marvina et al. 2003). MALDI-TOF-MS has recently been used in a study to identify the proteins of NPs protein corona of nanosized welding particles formed in vitro (Ali et al. 2015).

7.7.8 Electrophoresis

Electrophoresis is used for the identification of proteins after separation of the NPs protein complex that has been separated from excess plasma proteins. Most commonly used method for this purpose is two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) (Blunk et al. 1993; Gessner et al. 2002; Seehof et al. 2000). To identify individual proteins, it is common practice to compare the 2-D protein gels to a 2-D master map of human plasma proteins. However, differences in donor plasma and anticoagulants used during the blood collection process (EDTA, sodium citrate, lithium heparin), can result in variations in plasma protein maps, and hence may contribute to a misinterpretation of the 2D data when compared to a specific protein master map. The sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) is used for identifying proteins in hard protein corona (Winzen et al. 2015).

7.7.9 Size Exclusion Chromatography

Size exclusion chromatography (SEC) separates the NPs according to their size in dilute solutions (Sun et al. 2004). SEC is the second method investigated for its potential to reveal quantitative information on protein–NPs interactions. NPs and protein solution was passed through the chromatographic resin which allows protein and NPs to be resolved, but not different proteins. There has been a clear difference in the elution profile of HSA mixed with NPs, compared with free albumin, which implies an interaction between the protein and the NPs. Different proteins show different elution profiles when in protein NPs mixture than in free form. The rates of association/dissociation are very different from protein to protein, depending on the overall protein NP composition (Cedervall et al. 2007a, b). The strength of interactions of proteins with NPs can also be studied using SEC (Cedervall et al. 2007a, b).

7.7.10 Dynamic Light Scattering

Dynamic light scattering (DLS) measures the hydrodynamic diameter of NPs. DLS measures the size of NPs typically in the sub-micron region, also referred to as photon correlation spectroscopy or quasi-elastic light scattering. NPs suspended within a liquid undergo Brownian motion. DLS monitors the Brownian motion with light scattering. DLS measurements have been used to determine changes in the NP diameter before and after incubation with proteins. Proteins bind strongly to the NPs surface when the incubation time is increased, and they are stable against desorption in the serum free media. NPs hydrodynamic diameter is also increased as a result of the interactions of proteins with NPs. Adsorption of proteins onto the NPs surface has increased the overall size of NPs and not due to NPs aggregation (Casals et al. 2010). The thickness of the protein corona can be measured in situ using DLS (Walczyk et al. 2010).

7.7.11 Bioinformatic Tools

In silico simulation studies of protein NPs interactions is attracting attention as an alternative to experimental techniques (Walkey and Chan 2012). Simulation has been successfully adapted to study the adsorption of proteins on NPs. Simulation results have been used to study the interaction of a nanomaterial with individual amino acids to be observed over femto second timescales (Walkey and Chan 2012). Three strategies commonly used to simulate protein adsorption are quantum mechanical (QM), all atom empirical force field (AA) and coarse grained (CG) (Makarucha et al. 2011). Experimentally measured reference values are required to ensure the validity of simulation results. In silico simulations have been used to study the protein adsorption onto NPs as a function of surface ligand structure (Makarucha et al. 2011), surface curvature (Hung et al. 2011), and protein identity (Ge et al. 2011). However, computational power is presently unable to handle the complexity of competitive protein adsorption in a biological milieu, and there is a scarcity of availability of relevant force fields and descriptions of salvation (Walkey and Chan 2012).

7.8 Properties of NPs Affecting Protein Corona Formation

Interaction of NPs with biomolecules and composition of the resulting protein corona is affected by many factors (Foroozandeh and Aziz 2015). The physicochemical properties of NPs and the biological environment are vital parameters governing protein corona formation. Therefore, studying and understanding each of these parameters are essential for safe and smart designing of NPs for targeted drug delivery. The affinities and identities of proteins that bind to NPs are affected by the composition and surface chemistry of NPs. It has been reported that different proteins bind to different NPs with the same surface charge (Deng et al. 2009). Similar proteins can be adsorbed onto the TiO2 and SiO2 NPs, whereas significantly different proteins can compose the hard corona of ZnO NPs. The corona of TiO2 and SiO2 NPs were comprised of clusterin, apolipoprotein D and alpha-2-acid glycoprotein, while these were not observed in the corona of ZnO. Fascinatingly, transferrin, Ig heavy chain alpha and haptoglobin have been reported in the corona of ZnO NPs.

The size of NPs plays a vital role in the adsorption of proteins, conformational changes and composition of protein corona (Lundqvist et al. 2008; Lynch and Dawson 2008). It has been reported that the thickness of protein corona progressively increases as the size of NP increases. In addition, conformational change upon adsorption on the NPs surface showed enhancement with the size of NPs. Moreover, more proteins adsorbed on smaller sized NPs than on larger sized NPs (Dobrovolskaia et al. 2009). Interestingly, even 10 nm variations in NPs size have remarkably affected the protein corona composition (Tenzer et al. 2011). Shape of NPs has also affected the formation of protein corona on NPs surface (Deng et al. 2009). Surface charge of NPs also play vital role in the composition and formation of protein corona on NPs. Negatively charged NPs have been shown for enhancement in plasma protein absorption with an increase in the surface charge density of NPs (Gessner et al. 2002). In addition, proteins with isoelectric points (PI) of less than 5.5 adsorbed on positively charged NPs whereas proteins with isoelectric points of higher than 5.5 bound to negatively charged NPs (Foroozandeh and Aziz 2015). More proteins can adsorb onto the surface of hydrophobic NPs than their hydrophilic counterparts and lose their native structure (Roach et al. 2005). It has been observed that protein corona can drastically affect the cellular uptake and internalisations of NPs. Distinct corona signatures are indeed able to predict the cellular uptake of NPs. Hence, covering NPs surface with physiological proteins can indeed enhance or inhibit their cellular uptake, whereas the surface charge of the uncoated NPs appear to be less important (Monopoli et al. 2012; Lesniak et al. 2012; Zhang et al. 2014). Protein corona fingerprints can be modulated for increasing the cellular uptake of NPs across many biological barriers. It has been shown that apolipoproteins promote the transport of NPs across the blood brain barrier (Zhu et al. 2013) and different immunoglobulins and opsonins enable their uptake into monocytes (Riehemann et al. 2009), while dysopsonins inhibit the uptake of NPs (Cedervall et al. 2007a, b). NPs targeted with transferrin have been studied in foetal bovine serum albumin for their targeting efficiency (Salvati et al. 2013). It has been observed that serum decreased the overall uptake of NPs through transferrin receptors. These results have demonstrated that targeted NPs may lose their targeting abilities in biological media. This behaviour is attributed to the proteins in the serum forming a protein corona around the NPs, which masks the transferrin and stops it from binding to the targeted receptors on the cells. Hence, experiments carried out in vitro cannot be used to provide a conclusive analysis of targeting efficiency. Therefore, targeted NPs formulations should be smartly designed by taking into account the protein corona effect, and the problems faced due to the route of administration, organs, tissues and cellular uptake. This will help in developing targeted NPs with clinical applications and achieving the concept of personalised medicine (Gaspar 2013).

In addition to the properties of NPs, the composition of protein corona is also affected by the biological media in which NPs are incubated. It has been reported that formation of protein corona by utilising Dulbecco’s Modified Eagle’s Medium (DMEM) media is significantly time dependent, while using Roswell Park Memorial Institute (RPMI) media leads to different dynamics and reduction of protein corona (Maiorano et al. 2010). Besides NPs properties and biological milieu, other factors which affect protein corona at bio-nano interfaces are gradient plasma, plasma concentration, cell observer, temperature and cell membrane composition. Detailed investigations must be carried out to understand these ignored factors as to enable the development of better and effective nanomedicine (Foroozandeh and Aziz 2015).

Protein corona has been recently exploited for evading mononuclear phagocytic system. Albumins form tight binding to SiO2 NPs and may undergo rapid unfolding to form hard corona. Albumin has been documented to promote NPs uptake into cells that are expressing class A scavenger receptors and resulted in internalisation of the protein NP complex via receptor-mediated endocytosis (Mortimer et al. 2014).

7.9 Conclusions

NPs may not only promote the pharmacological effects of delivered therapeutic molecules, but also cause undesirable effects in the target cells. Factors like size, shape and surface chemistry of NPs can influence their cellular responses. NPs have been observed to induce cellular responses like ROS production, apoptosis, necrosis and expression of genes. Most of the studies involving the cellular responses of NPs have been carried out on in vitro cell lines, and therefore final validation is required with primary cells. Effect of various surface ligands and the use of targeting moieties should be investigated to better understand the cellular responses of NPs. NPs should no longer be viewed as simple carriers for biomedical applications, but can also play dynamic role in mediating biological responses. NPs-biomolecule corona should be more carefully characterised and quantified for safe and effective use of NPs in nanomedicine. The molecular design of basic molecules for NPs and the response between NPs and cells should be considered more carefully in terms of the activation of cell functions.