Artificial engineering of the protein corona at bio-nano interfaces for improved cancer-targeted nanotherapy

NP surface, displays an interface between biomacromolecules and NPs, governing their pharmacokinetics and pharmacodynamics. Upon interaction of proteins with the NPs, their surface features are modified and they can easily be removed from the circulation by the mononuclear phagocytic system (MPS). PC properties heavily depend on the biological microenvironment and NP physicochemical parameters. Based on this context, we have surveyed different approaches that have been used for artificial engineering of the PC composition on NP surfaces. We discussed the effects of NP size, shape, surface modifications (PEGylation, self-peptide, other polymers), and protein pre-coating on the PC properties. Additionally, other factors including protein source and structure, intravenous injection and the subsequent shear flow, plasma protein gradients, temperature and local heat transfer, and washing media were considered in the context of their effects on the PC properties and overall target cellular effects. Moreover, the effects of NP – PC complexes on cancer cells based on cellular interactions, organization of intracellular PC (IPC), targeted drug delivery (TDD) and regulation of burst drug release profile of nanoplat-forms, enhanced biocompatibility, and clinical applications were discussed followed by challenges and future perspective of the field. In conclusion, this paper can provide useful information to manipulate PC properties on the NP surface, thus trying to provide a literature survey to shorten their shipping from preclinical to clinical trials and to lay the basis for a personalized PC.


Introduction
Nanoparticles (NPs) have a chemically active surface due to their high surface-to-volume ratio. For this reason, in biological applications, NPs strongly interact with the environment resulting in the NP surface being covered with a complex layer of biological molecules [1]. The surface-associated proteins are called the protein corona (PC). The nature or structure of a protein may change after binding to the NP surface, thereby disrupting the functions of that particular protein and interfering with the intended function of the NP. The PC comprises the hard inner protein layer (hard PC), which has a very low exchange rate with the medium, and the soft outer protein layer (soft PC) has a high exchange rate with free proteins in the medium. Analysis of adsorbed proteins and their lifespan on the NP surface is essential for designing safe and productive nanoproducts [2].
The PC changes the size, dispersion, stability, and features of NPs, resulting in variations in their intended biological response with biological molecules, membranes, and physical barriers [3,4]. The biological characteristics of NPs are a function of the physicochemical characteristics (dimension, morphology, and functional group), the physiological environment, and the exposure time within the environment. Blood is often the first physiological environment that NPs interact with after intravenous injection, and plasma contains numerous proteins capable of interacting with NPs and forming a PC [5]. Proteins can be adsorbed to the NP surface in native or denatured forms depending on the inherent stability and hydrophobicity [6][7][8][9]. Some studies have demonstrated that NP-PC interactions regulated cellular targeting and uptake of therapeutic NPs while reducing the effects of unwanted NP cytotoxicity [4,10,11]. Yet NP-PC interactions can also lead to the rapid removal of NPs from the body and upregulation of inflammatory pathways. Indeed, NPs themselves can influence the selforganizing reactions of proteins and promote the induction of amyloidosis [9]. As a result, the design of NPs must factor in the PC for successful applications to be achieved that limit unwanted responses.
The PC layer mainly consists of opsonins which enhance the identification and clearance of NPs in the circulation by the mononuclear phagocyte system (MPS). Opsonization leads to NP clearance and low therapeutic efficacy of intravenously administered NPs [12]. As such, a central strategy to reduce MPS-mediated clearance is to engineer the PC for improved cancer-targeted nanotherapies. Several well-written reviews approach aspects of this important topic, including the characteristics of NPs and adsorbed proteins and alterations of the nano-plasma interface [13], how the PC is formed on the NP surface [14], characterization of the PC [15][16][17][18], the biological impacts of NP-PC complexes on nanomedicine implementations [17] and strategies for regulating the PC for biomedical applications [19][20][21][22][23][24]. In this comprehensive review, we discuss advances in NP-PC complexes for drug delivery and cancer treatment applications. We delineate how physicochemical NP characteristics alter the PC and discuss methods to modulate and quantify the PC. Further, we discuss how the PC modulates functional outcomes of nanoparticle drug delivery and the advancements in understanding the role of the PC in the area of cancer-targeted nanotherapies made to date.

Protein corona characterization
Numerous methods (Table 1) have been developed to perform PC analysis (Fig. 1); however, each approach has strengths and weaknesses, and as such, multiple methods should be combined to assess the functional effects of the PC adsorbed on nanocarriers fully. Although some of the analytical outputs overlap, the inconsistency of the analytical methods has resulted in conflicting information on the role of the PC on the biological activity of NPs. Indeed, it should be noted that several factors, including the stability of NPs, source collection storage, temperature, time, protein contamination and impurities, and purification processes, influence the accuracy of analyzing PC properties [25][26][27][28][29]. All the methods mentioned in Fig. 1, including quantitative, structural, and size analyses are performed in two forms, ex-situ or in-situ. For exsitu quantification, NPs are isolated and the PC is analyzed, whereas insitu measurements assess the effect of the PC on the biological behaviors of NPs.
Commonly, ex-situ analyses use chromatography, magnetization, and centrifugation (by semi-quantitative separation of the PC in the presence of complex biomolecule mixtures) to separate the PC from NP-PC complex for further analysis. Based on the PC formation, the dynamic protein exchange and the PC fingerprints (PCFs), extraction methods including multiple washes, sedimentation processes, or chromatography can interfere with the composition of the PC and bias analyses. After separation of the PC to analyze their quantitative and qualitative characteristics, electrophoresis and mass spectrometry methods are used to assess the composition of the PC. One of the main complications of these techniques is the lack of sufficient information about the homogeneity/heterogeneity of the PC, which may contribute to some contradictory outcomes found in the literature. In-situ techniques are used to explore the effect of physicochemical properties of NPs on the formation of the PC. Commonly, electron microscopy is used to study the morphology of PC-NPs and isothermal titration calorimetry (ITC) is used to assess the strength of the PC interaction with NPs. However, the need to dry the samples coupled with the heterogeneous composition of the PC can lead to significant errors in the quantification of the PC diameter and binding affinity, respectively. Finally, although zeta potential and DLS analyses can assess the surface charge and size of NPs in colloidal solutions, complex solutions such as blood make quantitative measurements challenging with these approaches and can lead to inaccuracies.

Table 1
A summary of the benefits and drawbacks of PC-NPs analysis methods [25][26][27][28][29]. The measurement method is not affected by the size of the ligand and the optical properties of the proteins

Physicochemical properties of NPs
The interaction of NPs in biological systems occurs at different timescales. Tightly adsorbed proteins with a low exchange rate in the hard PC can adhere to the NPs when they are endocytosed. In contrast, intracellular proteins can exchange proteins with a fast replacement rate in the soft PC during endocytosis. Therefore, NPs may display different biological behaviors depending on the mechanism of cellular entry and physicochemical characteristics. Various approaches relying on the synthesis of NPs, including changing their dimension, morphology, and functional groups are being widely used to attempt to modulate the characteristics of the PC layer.

The effect of NP size
The surface chemistry of the NPs defines the essential nature of the proteins in the PC. However, for NPs, the size and the surface features are dominant factors that play a key role in altering the nature of the bioactive proteins in the PC and thereby the biological interactions. The data on the effect of NP size on the PC is contradictory and independent of the NP material. Therefore, it is not easy to distinguish the influence of size from other controlling parameters on PC formation. Indeed, the variation in the ratio of the accessible surface area to protein concentration for the different NPs can influence the effect of NP particle size.
It has been shown that NP dimension and surface properties regulate the PC properties. For example, it was reported that six different polystyrene (PS) NPs with multiple functional groups and two different sizes displayed various effects on the composition of the PC around NPs. Indeed, it was seen that the highest number of identified proteins in 100 nm and 50 nm plain PS NPs with 49 identified proteins were followed by 50-nm amine-modified and 50-nm carboxyl-modified PS NPs with 42 identified proteins [30]. However, it was reported that PC formation on the surface of PLGA-and PLGA-PEG-NP with a size of more than 100 nm was not dependent on the size of the NPs [31]. Several proteins were recognized in the PC of PLGA-NP, revealing that this system is prone to opsonization. In contrast, PEGylation generated a significantly lower amount of adsorbed PC with potential long-circulating properties, which provide an enhanced permeability and retention effect (EPR).
Size of NPs can play a key role in the kinetics of the PC formation, PC composition, and amount of PC adsorption. For example, based on the Small-angle X-ray scattering, MS: mass spectrometry, XPS: X-ray photoelectron spectroscopy, LC-MS: liquid chromatography-mass spectrometry, FCS: fluorescence correlation spectroscopy, ICP-MS: inductively coupled plasma mass spectrometry, FTIR: Fourier-transform infrared spectroscopy, NMR: nuclear magnetic resonance, ITC: isothermal titration calorimetry, XRD: X-ray diffraction, CD: circular dichroism kinetics of the PC formation, Piella et al. [32] found that the temporal process in the evolution of the PC is faster for small NPs than that of the larger counterparts, further suggesting the remarkable role of size in the kinetics of the PC formation. In another study, it was seen that gold nanostars (AuNSs) and Au nanorods (AuNRs) with average sizes of 40 and 70 nm, respectively, had different PC compositions. Larger AuNSs were determined to adsorb a larger amount of proteins relative to other AuNPs [33]. Therefore, the authors ascribed the higher amount of proteins to the higher surface area of larger AuNSs. Furthermore, Chellathurai et al. [34] reported a direct correlation between NP size and protein adsorption, as they determined that the small NP size and the hydrophilic surfaces inhibit the formation of a large amount of protein adsorption.
It should be noted that colloidal stability and homogenous size distribution are key factors for elucidating the effects of different sizes on the PC properties. For spherical NPs, particle dimension is determined with the surface curvature as the increase of size reduces the ratio of S/V and corresponding free energy. This is consistent with several studies that reported a dimension-dependent impact on NP-protein interaction [35,36]. In another study, it was also shown that amorphous silica (SiO 2 ) NP with a size of around 1000 nm show more protein adsorption than those with 50 nm [37]. In contrast, it has been demonstrated recently that PC composition around SiO 2 NPs are not affected by the size of the NPs [38]. Also, it was seen that an increase in AuNP size from 5 to 80 nm reduces the protein binding affinity [39]. Therefore, it should be noted that the size difference sometimes is not large enough to influence PC properties remarkably [40]. When NPs are smaller than 10 nm, they are not prone to form a PC and result in protein denaturation [32]; conversely, when NP size enhances up to about 80 nm, the interacted proteins display structural alterations, which result in further protein-protein interaction and adsorption. The multilayer PC consists of about two-or three layers enhances the thickness of the PC [32]. In general, the difference in homology between the PC [30] and protein abundance [41] in NPs with similar sizes can be associated with the NP material, the surface charge of NPs, and the analytical limitations of the methods applied.

Shape of NPs
NP shape is considered an important factor that has an apparent effect on the formation of the PC and the corresponding therapeutic potential of NPs. For example, a larger PC is found on rod-like mesoporous SiO 2 NPs compared to spheres that had less adsorbed protein, including albumin and fibrinogen [42]. Additionally, studies have shown a differential affinity of fibrinogen and complement C3 adsorption on Au/Fe 3 O 4 NPs with a flower-like morphology (FLNPs) relative to Au/Fe 3 O 4 NPs with a spherical core-shell morphology (CSNPs) [43]. Importantly, this difference in PC composition triggers inflammatory cytokine release and significant macrophage mortality. It can be inferred that the roughness of NP surfaces could serve as a steric blockade against the interaction of fibrinogen [44]. Contrary to another study, superparamagnetic iron oxide NPs (SPIONs) were found to enhance inflammation in macrophages [45]. It was found that SPIONs conjugated with Au particles for FLNPs could not increase the pro-inflammatory behaviors of macrophages [43]. This data shows that the Fe 3 O 4 with a petallike morphology of FLNPs can avoid the attachment of some proteins like fibrinogen. It was also found that nanocage AuNPs had the lowest affinity to plasma proteins and enhanced biocompatibility relative to other morphologies (spheres, rods, stars) due to areas of high curvature and maximum ligation on its flat surfaces, which could avoid opsonization [46]. Interestingly, mesoporous silica NPs with a spherical shape generated both homogeneous a soft and hard PC with a 10 nm thickness and higher adsorption of albumin, whereas rod-and faceted-shaped mesoporous SiO 2 NPs, primarily possess a soft corona [47]. These data were consistent with the approximate dimensions anticipated for albumin interacting with metal oxide NPs [6,48,49].

Surface chemistry
Different functional groups specify multiple chemical microenvironments (charge, hydrophobicity, etc.) on the NPs, influencing their interaction with the plasma proteins. The PC is known as a biomolecular interface consisting of a "soft" and a "hard" corona specified with "short (several seconds)" and "long (many hours)" representative exchange rates, respectively. In a study, SPIONs were functionalized with three different groups: citric acid (CA), poly(acrylic acid) (PAA), and double layer of oleic acid (OAOA) [50]. It was found that the PC formation promotes over time. Although CA and PAA-functionalized SPIONs showed the equilibrium in 120 min, it was observed with a slower rate for OAOA-functionalized SPIONs covered with dysopsonins, perhaps due to the noncovalent basis of the second OA. Thus, it was found that a double layer of OA can be used as a potential functional group to cover SPIONs to be used as contrast agents for imaging. Furthermore, binding patterns of proteins on the NP surface are not just associated with the relative protein concentrations in plasma. The interaction of opsonins with hydrophobic functionalized NP and negatively charged NPs (proteins with pI > 5.5) thermodynamically is more favorable. For example, it has been shown that biomaterial surfaces with hydrophobic properties mediate further attachment of IgG protein relative to albumin [51]. It was shown that the positive charge of polyethylenimine (PEI, branched, 750 kDa) -coated SPIONs is changed toward a negative charge when NPs exposed to cell culture medium associated with an additional content of proteins [52]. The formation of the PC decreased the interaction of NPs with human brain microvascular endothelial cells (HBMEC). It should be noted that the type of NP affects the identity of the NP-associated PC. For example, it has been shown that while the charge and high hydrophobicity of synthetic NPs increase the formation of the PC, their properties seem to show different behaviors [53]. The high population of dysopsonins shows how the protein-based NPs can help in the cellular uptake of different drugs. Pustulka et al. [54] indicated that specific proteins in the PC could affect NP recognition by the cells and internalization by macrophages. In general, its surface features are very prominent factors of early interactions of NPs with immune cells and likely a more significant influence than size when NPs are exposed to the bloodstream [55].

PEGylation
NPs with sharp interfaces and low steric repulsion are potential platforms for protein adsorption. The use of hydrophilic materials, like PEG [56][57][58][59][60], poly(2-oxazoline) [61,62], or polysarcosine [63,64] can reduce the interaction of proteins with NPs by creating a lower active surface area, hydrophilic interface, and high steric hindrance [65]. A PEGylation strategy has been chiefly applied for improving the pharmacokinetic properties of several NPs [66]. PEG-functionalized NPs are usually referred to as "stealth" NPs because PEGylation enables the NPs to escape the RES. Along this line, Assali et al. [67] showed that AuNRgraphene oxide hybrid (NR@GO) had a significantly higher affinity to generate a PC than a modified NR@GO-PEG. Although the unfunctionalized NR@GO platform showed high affinity to opsonin proteins, the modification of the NP surface with PEG attracted dysopsonins which resulted in more circulation time and cellular uptake. Moreover, they found that the PEG-functionalized NR@GO platform can be combined with low-power therapy to increase gene delivery in the cells. These data may accentuate the role of surface features in PC patterning and designate the effect of functionalization in regulating the PC composition for potential gene/drug delivery.
Earlier, Gref et al. [68] showed that PEG-functionalized polymeric NPs could have different PC patterns than unfunctionalized NPs. They showed that PEG-functionalized PLGA NPs enhance blood circulation time while decreasing liver uptake relative to their unfunctionalized counterparts. Also, PEGylation of NPs results in lower interaction of opsonin with NPs and improved circulation time. It should be noted that PEGylation leads to reduced cellular uptake once they extravasate and increased cytotoxicity to off-target tissue due to longer circulation time. Also, the grafted PEG does not sufficiently extend over the NP surface.
It has been also demonstrated that stealth carriers can trigger the immune system to produce PEG-based IgM [69], which noticeably mitigated the half-life of injected PEG-modified liposomes [70]. PEG configuration and the length of the PEG lipid chain on the NP surface can play a crucial role in addressing the challenges mentioned above. It has been reported that PEG can cover the NP surface in three conformations, including mushroom, transition, and brush modes ( Fig. 2A), the latter of which is the most favorable mode (>8 mol%) for optimal covering of the NP surface followed by transition (4-8 mol%) and mushroom configurations (<4 mol%). Indeed, steric hindrance and entropy are the main factors that inhibit the interaction of proteins with brush mode PEGylated NPs. Additionally, it has been shown that a M W of 2 kDa has the highest potential for mitigating the recognition by immune cells [71]. However, developing a stable platform such as micelles, liposomes, and nanogels with widespread application in nanomedicine with a brush configuration will be challenging. Photos et al. [72] proposed that a nanocarrier can be developed by introducing brushed PEG through hydrating a polymer monomer that had increased hydrophobic forces in a self-congregated mode. Also, it has been shown that post-inserted PEG could be arrayed in an optimal density (>10 mol%) for the brush mode onto the LPD (liposome-polycation-DNA) NPs. In this system, the authors indicated that the lipid bilayer could be highly stable through charge-charge repulsion, decreased liver uptake, and enhanced siRNA delivery into the cancer cells [73].
Indeed, it can be claimed that using alternative interaction to alienate the repulsion among the PEG molecules; e.g., charge-charge interaction derived from the nanocarrier or hydrophobic force among the monomers of the nanocarrier (micelles, liposome), play a key role in providing a high mol% of PEG arrayed in the brush configuration [74].
Recently, it has also been shown that the polymer topology designates the formation of the PC on core-shell NPs [62]. Indeed, it was displayed that the cyclic poly(2-ethyl-2-oxazoline) (PEOXA) brushes result in denser NP shells than their linear forms, complexly inhibiting the formation of the PC as well as reducing the colloidal stability of NPs.
Also, coating NPs with polymers and ligands through regulation of PC can alter the circulation time, cellular uptake, colloidal stability, systemic targeting of NPs.
In this context, it was found that coating NPs with poloxamine-908 mitigates opsonization but results in the function of dysopsonins in the serum via a reduction in the surface hydrophobicity of particles [78]. Another study demonstrated that poloxamers and poloxamines, due to their amphiphilic properties, can interact with hydrophobic moieties. For example, poloxamer 407-modified PS NPs had an extended circulation time [79] and a reduced number of attached proteins, including clusterin and Apo C-III [80]. Indeed, it should be emphasized that the size and molecular weight of multiple ligands and targeting groups along with their corresponding short-or long-range interactions potentially trigger the cellular uptake [81] and formation of the PC [82]. Another study showed that functionalization of single-walled carbon nanotubes (CNT) and SiO 2 NPs with Pluronic F127 enhanced their colloidal stability but significantly reduced attachment of serum proteins [37]. It has also been shown that PEG and PF127 can be applied to enhance the colloidal stability of SiO 2 NPs by preserving the interaction of molecules to the NP surface, whereas they provide a minimum impact on the PC composition [83].
Another study showed that polysorbate-modified solid lipid NPs as stable nanocarriers could be applied for systemic targeting of drugs to cross the BBB [84]. Another report found that polysorbates can be used to develop a PC pattern that aims to drive the niosome targeting in vivo [85]. In some other studies, it has been shown that functionalization of CNT [86] and AuNPs [87] with polycarbodiimide and (3-mercaptopropyl) trimethoxysilane (MPTMS), respectively, can modulate the PC properties and delivery into cancer cells.

Self-peptides
CD47, an integrin-related protein found on the surface of multiple cells [88], results in the low interaction of cells with macrophages through receptor CD172a [89,90]. Therefore, adsorption of CD47 on NPs can prolong NP blood half-life and increase tumor targeting (Fig. 2B). Also, it has been reported that blockage of macrophage uptake by CD47 was not associated with the NPs in the range of 100 nm to 10 μm [89]. Therefore, coating NPs with CD47 as a self-peptide can inhibit phagocytic function [91] and promote cancer therapy. Indeed, improving the circulation time of NPs is emerging for cancer therapy. For this reason, Rodriguez's group designed and produced a 21-amino acid self-peptide based on human CD47 that could block phagocytic uptake and the corresponding clearance [89]. It was shown that "self" peptide-functionalized PS NPs provided a notable prolonged circulation time relative to scrambled peptide or PEG functionalized NPs in NSG mice. Also, self-peptide significantly (16-fold) increased the ability to deliver paclitaxel (PTX) to human lung cancer cells. Therefore, selfpeptides can be potentially used for nano-based cancer therapy following injection with remarkably prolonged circulation and promoted cancer cell accumulation relative to PEGylated NPs.

Perspectives on the modulation of PC based on physiochemical properties of NPs
In general, it was found that the size (Fig. 3A) [32], charge ( Fig. 3B) [92], shape ( Fig. 3C) [33,42,93], surface roughness [94,95], and alternation in ligand structures and corresponding steric hindrance [96] can extensively influence several PC characteristics, including the amounts of adsorbed proteins in hard and soft PC, the extent of structural changes of protein, and overall formation of PC [33,42,[92][93][94][95][96]. Indeed, it was found that neutral/zwitterionic or negatively-charged NPs with a spherical shape, small sizes, and rough surfaces can be used to inhibit the formation of PC. However, positively-charged NPs with a nonspherical shape, large diameters, and smooth surfaces can form a thick PC layer. Therefore, the advancement of unique strategies to regulate the physiological properties of NPs will allow for precise manipulation of the characteristics of the PC and introduce new strategies to recruit the full capacity of the PC.
However, the modulation of PC based on these parameters still needs further investigation. Changing the physiochemical properties of NPs can trigger several unwanted effects, such as protein conformational changes, toxicity, and macrophage uptake, which cause rapid clearance of engineered NPs from the body. Also, the formation of PC is regulated through different adsorption dynamics on the surface of NP, including the direct adsorption of soluble proteins and protein aggregates and multistep adsorption. Furthermore, desorbed and reabsorbed protein species can play a key role in regulating the formed PC. Notably, diffusion of proteins to the surface followed by initial interaction with proper residues and the final attachment are three main stages in the formation of PC. The engineering of the physiological parameters of NPs could alter several parameters, including adsorption kinetics, heterogeneous protein interfacial dynamics, the protein structure, protein layer density, and distribution of adsorption orientations, leading to further PC changes. On the other side, the challenges related to the fabrication of NPs with well-defined physiological parameters still hinder the profound examination of these parameters. The few extensive studies involving a large diversity of NPs still hinder the development of a standard formula that can be applied to introduce a strategy for manipulating the PC at bio-nano interfaces for improved cancer-targeted nano-therapy. Therefore, exploiting the physiological properties of NPs and external stimuli to modulate the characteristics of PC and its effect on biological systems describes an important strategy to advance more promising NPs for cancer therapeutics.

Protein pre-coating
The specific attachment of targeting moieties to receptors on the targeted cell surfaces could increase NP uptake. However, targeting ligand-covered NPs often show low potency in vivo, and the NPs widely reach the RES rather than the targeted cells [97]. Indeed, the most prominent cause for the limited specific targeting is the formation of the PC, which acts as a barrier and introduces a new biointerface that complicates NP biodistribution, cellular internalization, intracellular targeting, and rate of drug release of the NPs. To address these drawbacks, another potential alternative approach is to pre-coat NPs before exposure to the blood system to enhance the adsorption of specific proteins that are biologically targeted to the target cells. Actually, upon interaction with the blood proteins, these NPs acquire a PC that results in the active targeting of the selective tissues (Fig. 2B). For example, it has been shown that manipulating the PC [98] using polysorbate precoatings [99] promotes apolipoprotein (Apo) adsorption to the PC and associated transport across the blood-brain barrier. Pre-coating can also lead to the formation of the PC, which resulted in the active targeting of NPs to some cancer cells [100] and hepatic stellate cells [101]. In another report, positively charged-functionalized liposomes pre-coated with an artificial PC were used as potential candidates for selective targeting of tumor-associated macrophages in the treatment of cancer [102]. Moreover, it was reported that ApoE pre-coating before intravenous delivery increases NP circulation time compared to their unfunctionalized and IgE-covered counterparts [103]. Indeed, precoating NPs enables the regulation of the PC properties and underlying cellular interactions [104].
The main challenge in pre-coating NPs is the feasibility of engineering NPs to control the properties of the PC and the associated active targeting of NPs. It should be explored whether the targeting potential of NPs pre-covered by a PC could be changed by the non-specific attachment of a high abundance of plasma components. It was found that precoating of NPs with γ-globulins could increase the feasible adsorption of opsonins [105,106], whereas pre-coating with HSA, could mitigate the attachment of opsonins onto NPs [107] and improve colloidal stability and probable cellular internalization [108,109]. It was then found that NPs pre-coated with γ-globulins were entirely covered with some opsonins (immunoglobulins and complement factors) [105]. Interestingly, it was seen that the PC with opsonin did not promote NP internalization by RAW 264.7 macrophages [105]. Therefore, the authors concluded that other PC components shielded the opsonins and inhibited their interaction with relevant receptors on the immune cells. Therefore, targeting potency of NPs can be affected by non-specific protein-protein interactions and spatial attachment of the recruited proteins.
It can be suggested that pre-coating of NPs with proteins probably regulate the PC composition, stability of NPs [110], their macrophage uptake [111,112], targeting specificity [113], dynamics of protein adsorption [114], cellular uptake [115], and their cytotoxicity [116]. Indeed, pre-coating of NPs can enable the scientists to design the PC which offers the probability to control cellular uptake. Opsonin depleted plasma can be used as a pre-coating system as it shows the ability to mitigate the interaction of NPs with phagocytic cells. However, an important challenge is to have a stable system that enable the stealth properties to be preserved even if pre-coated PC engineered NPs are reexposed to plasma [104], which heavily depends on the timeline of the PC formation (corona interactome) [117]. Table 2 summarizes more reports on the pre-coating of NPs by external proteins for cancer therapy which mitigated the attachment of plasma proteins, thereby blocking the recognition of NPs by the immune system and promoting their targeting capability.

Other factors
Additional factors (Table 3) influence the properties of nanocarrier PC, which consequently changes the interaction of NPs with cells. This occurs through changes in cellular uptake, cytotoxicity, and biodistribution. These reports aim to explore the PC formation to regulate the nanocarriers´interaction in vivo and ultimately develop their clinical translation. For example, it has been shown that a magnetothermal strategy can be used to quantitatively engineer the in vivo PC properties of IONPs mediated magnetic nanodrugs (Fig. 4A). Indeed, magnetothermal modulation is based on localized heat transfer by IONPs under an external alternating magnetic field (AMF) and can lead to the regulation of the PC identity [134]. Both in vitro and in vivo outcomes reveal that magnetothermal modulation can result in fewer opsonins, increase the pharmacokinetics of NPs, and improve cancer nanotherapy (Fig. 4B) [134]. Another earlier study has shown that the uptake of PEGylated AuNR (GNR)@BSA platforms by HeLa cells is independent of the surface charge ( Fig. 4C(i)) because of the stealth characteristics of the PEG modification [135]. However, the BSA pre-coating mitigated the cellular uptake of NPs relative to PEGylated counterparts without the BSA; this effect was charge-dependent ( Fig. 4C (ii)). Then, the analysis of the photothermal effect induced by laser irradiation (λ = 805 nm, 500 mW, 4 W/cm 2 , 3 min) on the GNR surface revealed a notable reduction in the amount of BSA attachment on the PEGylated GNR with a positive charge, both in completed and non-completed cell media (Fig. 4C (iii)). Nevertheless, no-apparent difference in the cellular internalization of the GNR@BSA platforms with and without laser irradiation was detected except for CTAB-modified GNRs (Fig. 4C (iv)) [135].    In another study, the effect of intravenous injection and the subsequent shear flow was investigated on the PC properties [136]. It was reported that several hard PC could be observed in SDS-PAGE (Fig. 4D (i) as plasminogen (88 kDa) and albumin (66 kDa) were identified as the primary and abundant proteins. Densitometric analysis via ImageJ software quantified the amount of plasminogen in the PC enhanced under flow (Fig. 4D (ii)), where increasing flow rate resulted in a significant change in the tertiary (Fig. 4D (iii)) and secondary (Fig. 4D (iv)) structures of plasminogen. This data was assessed by fluorescence and circular dichroism techniques, respectively [136].

PC effects on cellular interactions
In biological systems, the surfaces of NPs are generally covered by the PC resulting in the formation of a biointerface between NPs and the cell membrane. Hence, to further investigate the interaction of NPs and cells, the cell membrane composition and cell type should also be considered. For example, negatively charged cell membranes attract positively charged nano-based platforms with higher efficiency than negatively charged counterparts.
It has also been shown that the variations in cell type, secreted biomolecules, sex, cell stiffness, age, and mitochondrial and endoplasmic reticulum structures can play a potential role in the fate of the NPs/cell interaction [153]. For example, it was seen that the promoted accumulation of clathrin heavy chains leads to greater and faster quantum dot (QD) internalization in the male somatic fibroblasts and human amniotic mesenchymal stromal cells (hAMSCs) [153].
In general, as the cells produced different types of cytokines in the supernatant and minor variations in the protein source (serum and plasma) could remarkably change the profile of the PC, the type of the cell can influence the PC composition on the NP surface and therefore alter the biological behavior of NPs [154]. Therefore, the apparent changes found in the expression of different cells' paracrine signals could alter the NP-PC complexes' physicochemical properties and associated interactions with cells. Indeed, it has been shown that the origin of blood plasma (female and male) alters the biological interaction of NPs with cells [155]. Sexual dimorphism also occurs in the same cell types, including the endothelium, which leads to differences in protein expression and response to pharmacological compounds. One probable reason for different interactions of NPs in the biological systems with multiple cell types is referred to the distinct variation in zeta potential of NPs upon exposure to cell paracrine factors. NPs with low zeta potential do not potentially interact with the cells and show a lower success of cell uptake [54,156].
It has been reported that PC affects cellular uptake of NPs by phagocytic and nonphagocytic cells [4,157]. Indeed, the mechanism of NP uptake by cells depends on the size of NPs and the type of the cells [157]. The most regularly used pathway of NP uptake is through endocytosis, and more particularly, through phagocytosis (>250 nm) or pinocytosis (<100 nm). Also, the latter can be divided into four routes: clathrin-and caveolae-mediated endocytosis, clathrin-and caveolae-independent endocytosis, and macropinocytosis [158]. However, the presence of the PC can vary the capability of NPs to interact with cells  and the corresponding cell uptake [156]. Also, it has been shown that the size, shape, and PC can change the endocytosis pathways; however they do not alter the exocytosis mechanism [159].
As there are some changes in actin filament configuration and distribution between different cells with resultant influences on NP internalization, actin can play a crucial role in regulating endocytosisderived vesicles [160][161][162]. Therefore, actin filaments can regulate the function of endocytosis-derived vesicles through control of the shrinking, splitting, and trafficking [163]. It is logical then to suppose some notable variations in configuration and distribution of actin in multiple cell types could potentially influence the internalization and cellular trafficking of NPs. Bioinformatic analysis [164] and experimental data [153] also indicated that the integrity of the actin filament could influence the rate of NP uptake. Moreover, it has been reported that actin filament thickness can be directly related to the mechanical (stiffness) characteristics of cells [165]. Therefore, as different cells show multiple templates of actin filaments, one would anticipate that other cells may not show similar cellular stiffness, which influences membrane deformations needed for induction of endocytosis and thereby regulating cell type-specific uptake of NPs. The type of NPs upon interaction with cells could determine the mechanism of cellular uptake of NPs. For example, it was shown that although QDs can be brought into the female and male hAMSCs through clathrin-mediated endocytosis, AuNPs were detected extensively in non-coated vesicles [153]. Thus, it could not be confirmed that all NPs may be internalized by similar cellular uptake pathways.

Organization of the intracellular PC
It has been well reported that the hard PC regulates the interactions of NPs with cells. NPs normally interact with cells individually and are dealt with by active-mediated pathways [166][167][168]. When NPs are exposed to cells in a buffer alone (without PC), it usually causes leakage of the cell membrane [171,172]. However, NPs before contact with cells are covered by hard or soft PC, where the lifetime of the hard PC is usually several hours, typically longer than the lifetime recognition by receptors and consequent NP-PC complex uptake into cells [173,174], which is reflected in their biodistribution within tissues. After interactions with cell membranes, NPs are usually trafficked along the endolysosomal pathway depending on the cell type [169,170].
During internalization within the intracellular organelles, the assembly of the PC could be restructured, which may change the NP trafficking and cytokine release patterns [175,176]. Intracellular degradative systems degrade the NPs and reduce the proteins that  [136].
comprise the PC, which may cause extensive biological effects ranging from cytotoxicity to immune responses [175]. Therefore, exploring the fate of the PC intracellularly due to various possible degradation mechanisms is challenging. One possible approach is to expose NPs to fluorescently labeled serum to form NP-PC complexes. Subsequently, redispersion in normal serum would simultaneously track NP intracellular trafficking and the proteins comprising the PC [175]. It was reported that although serum fluorescent proteins were completely recycled/degraded during 8 h, when these proteins were taken into cells by NPs, even over 16 h, some degraded PC proteins could be found in the lysosomes [175]. The authors then claimed that the intracellular destiny of the PC regulated the NP internalization into the cells and resultant trafficking.
It was also shown that the PC could reduce the cytotoxicity induced by PS NPs and their capability to inhibit the autophagic pathway was restored after the degradation of the PC in lysosomes [177].
Indeed, as the PC is partially preserved on the NP surface, the adsorbed macromolecules in this form can play a crucial role in regulating subsequent cellular pathways. To understand the intracellular trafficking and cellular responses, employing organelle separation and fluorescence labeling of the PC, the intracellular evolution of NPs based on traveling within the cell can be determined. For sure, various bare surfaces influence the complexity of this evolution. As most of the proteins in the physiological pH have a negative charge, then the density of the PC on the negatively-charged NPs is higher than that of positively charged counterparts, resulting in significant accumulation of proteins in the lysosomes.
Another study indicated that Bi 2 S 3 nanorods@HSA-fluorescein isothiocyanate-FA corona (BN@HFFC) could be developed for selective targeting of lysozyme and photothermally increased platform for cancer treatment [178]. Also, it has been shown that the IPC-coated NPs have a greater internalization ability by HUVECs compared to bare AuNPs [176]. As it is challenging to investigate the original PC composition during uptake of NPs by cells due to the limited levels of extracted NPs and the low stability of the PC upon extraction, Wang et al. [170] designed a cross-linking approach by using paraformaldehyde (PFA) to cause the stabilization of the PC extracted from the cell lysis medium. Interestingly, it was seen that the original PC was extensively exchanged by intracellular proteins after internalization into cells, and the type of adsorbed proteins was dependent on the time. Lastly, it has been shown that 2D carbon-based NPs− graphdiyne oxide (GDYO) nanosheets can thermodynamically interact with an IPC composed of a signal transducer and activator of transcription 3 (STAT3), triggering the immunomodulation of macrophages by changing their phenotype. Indeed, the authors indicated that the recommended synthesized nanoplatforms could be used for local immunomodulation based on the structural assembly of the intracellular PC in macrophages [179].

Engineering the PC for targeted drug delivery
It should be noted that each NP shows a particular protein pattern governed by its physiological parameters. Indeed, a large number of reports have aimed to correlate the cellular internalization of NP-protein complexes to the PC composition by some simple pictures of NP-cell interaction; the more ample a PC, the more possible the interaction with cell receptors and, in turn, the more outstanding its function in enhancing uptake of NPs. Nevertheless, to explore the association between an NP-PC complex and selective internalization pathways, analyzing the specific site of protein interaction is a vital step [180][181][182]. Quantitative mapping of the links between NPs' material identity and biological behavior enables us to comprehensively reveal and regulate the nano-bio interactions in different biological fluids, which are pivotal for future perspectives in drug delivery systems. Therefore, based on the quantitative structure− activity relationship (QSAR) approach, it has been revealed that just a minor fraction of the several attached PC, PCFs, promote NP interaction with tumor cells and their potential uptake, which heavily depends on the interplay between physicochemical properties of NPs, PC, and charge variation [183]. In other words, for an in-depth and systematic analysis of the effect of PC on NP biological behavior, it is inevitable to advance the analytical assays and implement them with QSAR analysis to consider linear and nonlinear correlations between the defined descriptors and the resulting biological responses, rather than only exploring the influence of one factor over time. All successful QSAR models incorporated both liposome physicochemical properties and PCF descriptors to reliably obtain results from both physicochemical characteristics and NP− PC (Fig. 5A) [183]. The associations between the relative protein abundances (RPAs) of total proteins and cellular internalization could enable the scientists to regulate seven PCFs, including, Vitronectin, Apos (A1, A2, B, C2), vitamin K-dependent protein, and integrin β3) that increase the interaction of liposomes with tumor cells [130] as they are predominantly upregulated on several cancer cells and in tumor neovasculature.
Functional plasma proteins, including HSA [186], transferrin [187], and Apos A1, E and J [131], have shown to possess potential receptors on various types of cancer cells. Therefore, manipulating PC assembly with these proteins may increase the targeting efficacy of drug delivery systems. Although these proteins can mediate the cellular uptake of NPs through receptor-assisted transcytosis, it is rarely indicated that plain DDSs can enable the NPs to cross the cell membranes after attachment of such proteins in the blood [131]. One probable cause is that the orientation of these proteins in the PC layer is not uniform and their interaction with the receptor-binding pocket is not thermodynamically favored [181]. Therefore, it can be hypothesized that PC-mediated cancer cell targeting would be attainable by accurately manipulating the interaction of cancer cell-targeting plasma proteins on the NP surface [131]. For example, it has been shown that bio-inspired liposomes (sLip) labeled with a fluorescence probe (Dil) via conjugation with Aβ [25][26][27][28][29][30][31][32][33][34][35] (SP) can be exploited for precise orientation of Apos which results in the TDD of doxorubicin (DOX) to CNS (Fig. 5B) [131]. To develop targeted drug delivery systems, the functionalization of NPs for targeting and grafting by some polymers to reduce the macrophage uptake is necessary.
It has been shown that PEGylated NPs with short molecules and moderately functionalized with cyclic RGD had a superior targeting efficiency than those with a high density of peptide and long PEG molecules due to the reduced shield effect (Fig. 5C) [188]. While the PEGmodified nanoplatforms have been widely used in clinical medicine for several years [189], it has been shown that PEGylation cannot potentially shield the formation of PC and accordingly stimulates significant immune response [190]. Therefore, it has been demonstrated that the polyglycerol (PG) grafting can be used as an alternative based on its non-ionic and more hydrophilic nature than PEG to potentially shield NPs from PC formation and inhibit the subsequent macrophage uptake (Fig. 4D) [191]. Therefore, regulation of the PC for active TDD is an important factor that should be precisely optimized.
Another strategy for TDD is the application of zwitterionic coatings to avoid nonspecific interactions of targeting ligands and cell receptors [125]. Indeed, the application of zwitterionic modifications has been shown as a potential approach to prepare PC-free NPs [192,193]. This coating results in high biocompatibility, high safety, and longcirculating time [194,195]. Indeed, zwitterionic structures with the forms of low-molecular weight and polymeric agents can be used as coating compounds for multiple types of NPs [184,185]. Due to the zwitterionic feature of the cell membrane based on the presence of phospholipids, the modification of NPs with zwitterionic agents has been widely used as potential materials for drug carrier platforms [125,196,197].
Mapping the time evolution of the PC on the time scales of a NP's lifetime is crucial to predicting its behavior in vivo. Indeed, it has been shown that the RPA of opsonin proteins reduced over time, whereas that of dysopsonins was reported to increase in lipid-based vesicle NPs [118]. Therefore, these aspects of NP research require further research to realize the precise biological identity of NP− PC complexes in developing targeted drug delivery systems.
Based on the above reports, it can be concluded that the personalized PCFs concept affects the efficacy of targeted drug delivery as several factors, including gender, age, diseases, and pregnancy, all considerably influence the PC composition, which should be taken into account for the development of biocompatible and potential nanoplatform-based targeted drug delivery systems. Determination of specific proteins engaged in the associated biological response provide essential details and offer the prospect of being utilized for advanced nanoplatform design, controlled targeted drug delivery and other therapeutic targets in which regulation of nano-bio interactions is required.
PC-mediated targeted drug delivery can be achieved utilizing short peptides to bind exchangeable proteins with receptor binding domains with potential geometry. The reengineered NPs potentially control the binding of selective proteins onto the nano-surface for promising tumortargeting characteristics. Furthermore, their geometry and wettability can influence the PC composition and subsequent targeting efficacy of NPs grafted with different polymers such as PEG and PG. NPs modified with short PEG or PG molecules and loaded with moderate targeting ligand density show optimized targeted cell uptake and targeting potency. These details may guide the design of targeting nanoplatforms with optimized targeting potency. Moreover, zwitterionic coatings can be used as promising ligands of NPs, since they show both low-fouling features and capability to be utilized for targeting purposes. Zwitterionic ligands can provide the advantages of charged surfaces to establish potential interactions with cellular membranes and at the same time, through the formation of a strong hydration shell, can inhibit PC formation and relevant unwanted bio-responses.

Engineering PC for the regulation of burst drug release profiles of nanoplatforms
A critical challenge in developing NP-based drug delivery systems is to alter the kinetics of drug release in a controllable manner [198,199]. The release profile data for different nanocarriers should be reevaluated by incorporating the impact of the PC on the rate of drug release to make it applicable for in vivo applications. It has been shown that the kinetics of NP drug release heavily depends on the presence of the PC (Fig. 6A) [200]. The type and amount of the PC and the relevant size and type of the nanomaterials have a profound effect on the sustained drug release of nanocarriers [200]. It was then demonstrated that the PC could cover the nanocarriers and, accordingly, varies the release profile of the anticancer agent from the NPs. The PC could mitigate the burst effect of either protein-modified NPs or platforms with surface adsorbed drugs (SPIONs).
Interestingly, the PC has been shown to slightly alter the drug release pattern of polymeric NPs [200]. In another study, it was seen that drug release from solid cationic Eudragit RS (EGRS) was further inhibited in comparison with anionic ethyl cellulose (EC) NPs due to different amounts of hard PC and formation of interparticle aggregates (Fig. 6B), which reflects the determining role of NP type along with their colloidal stability in controlled drug release in the presence of the PC [202]. Other studies have shown that the drug release profile was heavily associated with the composition of nanocarrier, stability, thermal sensitivity, and relevant changes in the PC profile [201]. For example, the rate of DOX release from traditional thermosensitive liposomes (TTSL) was slower than that of lysolipid TSL (LTSL) due to the presence of lysolipidsmediated long-lasting pores [201]. Indeed, it was found that after injection and separation of liposomes [ Fig. 6C(i)], the drug release profile of TTSL and LTSL ex vivo in buffer [ Fig. 6C(ii)] and full plasma [ Fig. 6C (iii)] was totally different, revealing that PC can hinder the burst drug release of nanocarriers [201]. However, the formation of the PC around and their PCFs to multiple quantified biological responses [183]. (B) Biodistribution of prepared liposomes in intracranial glioma. SP-sLip (SP-sLip/DiI) (red) were further accumulated than DiI-labeled sLip (sLip/DiI) in glioma region [131]. (C) Scheme illustration the interactions between cells and NPs with different surface modified groups [188]. (D) Exploring the macrophage uptake of NPs either with PEG or PG [191].
the lipid membrane was critical to regulating drug release from the liposomes during heating. Indeed, different types of NPs due to different compositions, thermosensitivity, and sol-gel properties along with different colloidal stabilities show different amounts of PC, affecting the payload release.
In addition to the influence of PC on drug release profiles, some reports have indicated that PC layering on the NP surface can serve as a reservoir with improved payload capability for different drugs or genetic materials [203]. The release of PC-loaded drugs can be manipulated in multiple approaches based on the characteristics of the NPs. An example of this is employing hyperthermia-stimulated release of DOX and DNA incorporated into the PC layer on the NIR-sensitive AuNRs (Fig. 6D) [203]. Upon laser irradiation-induced hyperthermia mediated by plasmonic properties of AuNRs, incorporated drugs, and genetic materials were released, likely based on the reorganization of the PC derived from slight protein conformational changes [203]. Therefore, in addition to drug release regulation, the PC can be exploited to incorporate drugs at a capacity much higher (~10 times) than that of covalent conjugation approaches [203]. As such, the PC can be used for dual loading and release of drugs [121] and DNA [203].
In oral delivery of drugs via NPs, the formation of the enzyme corona on the NP surface influences the delivery of drugs (Fig. 6E) [204]. The internalization of NPs into epithelial cells was reduced after enzyme corona formation. A significant amount of NPs could reach the colon and be degraded and metabolized, thereby releasing the payload [204]. Therefore, based on the tumor site, the type of NP-protein interaction and drug release profile can be varied. Furthermore, the PC showed no significant impact on the release profile of the drugs from oral nanocapsules (Fig. 6F) [200]. This data can be elucidated by the fact that the hard PC is significantly thinner (<10 nm) [205] than the nanocapsules shell with a dimeter of around 30 nm [203]. Additionally, the glass transition temperature of the polymers is significantly higher than that of the physiological temperature [203]. Both features cause the limited diffusion of the drug through the shell and through the hard PC. However, porous nanocapsules may likely show a drug release profile comparable to the one reported for the inorganic NPs [203].  [200]. (B) The effect of the PC on drug (dexamethasone) release by two NPs [202]. (C) The effect of PC on drug release by TTSL and LTSL with different phospholipid compositions. (i) Scheme illustration of the injection of and preparation of nanocarriers, (ii) drug release profile of nanocarriers in buffer, and (iii) drug release profile of nanocarriers in plasma [201]. (D) Exploiting the PC around AuNRs for loading and NIR-triggered drugs and genetic materials release [203]. (E) Scheme illustration of the formation of enzyme corona in the gastrointestinal tract (GIT) around the orally administered NPs [204]. (F) Formation of hard PC does not affect the drug release profile of nanocapsules [200].
In general, it can be concluded that the formation of hard PC remarkably mitigated the drug release from nanocarriers. In addition, the payload release can be affected by the additional shielding effect mediated by soft corona and the presence of excess proteins in the solution. It is also noticeable that the reduction in the drug release profile after a specific time can be associated with the drug stability and compatibility in buffer solution. Also, the composition of the hard PC, the type of polymer nanocarriers and their glass melting temperature, the interaction of proteins with core NPs, colloidal stability of NPs, drug stability, and hyperthermia can play key roles in the drug release profile of nanocarriers mediated by the PC.

Bioinspired PC strategies for enhanced biocompatibility
It has been widely demonstrated that inorganic NPs can be used as potential materials for drug delivery [206] and combined cancer therapy [49,[207][208][209]. Hence, advancements in the control of the anticancer characteristics and biocompatibility can increase the applications of inorganic-based NPs for selective and potential image-guided combined therapies [210]. Therefore, an affordable and flexible bioinspired PC design approach can be used to advance NP-PC complex-based tumor detection and combined on-demand therapy. Bioinspired PC design can lead to colloidal stability and biocompatibility of nano-based platforms for promoting the efficacy of NPs for clinical treatments. And more exceptionally, the pre-coated PC as a nanoshell reduces the unwanted toxicity to the off-target cells and tissues by mitigating the release of inorganic metal ions and induction of oxidative stress after intravenous injection. Nanoshells can be used to generate inorganic materialsderived toxic ions that trigger oxidative stress and inflammatory responses in tumors under the external stimuli (radiation, magnetic field) [210]. For example, He et al. [210] applied BSA as a bioinspired PC to promote the biocompatibility of silver hybrid hollow AgAu-Raman tag DTTC-BSA SERS/photothermal bimodal platforms for imaging-guided photothermal tumor ablation.
It has also been shown that plasma PC can promote the biocompatibility of heteroparticles having individual parts of different materials. Although their unique morphology and structure can result in performing multimodal and versatile approaches in cancer management, including contrast and therapeutic agents and drug carriers, their heterogeneous nature can lead to profound cytotoxicity against normal cells. However, enhanced biocompatibility [ Fig. 7A(i)] and cellular uptake [ Fig. 7A(ii)] were found in Janus Au-thiol@Fe 3 O 4 -SiO 2 -PEG and Au-thiol@Fe 3 O 4 -SiO 2 -PEG-NH 2 NPs with the PC irrespective of amine groups [211]. Indeed, the authors discussed that the Au domain of fabricated Janus NP has potential antioxidant properties, whereas Fe 3 O 4 -based NPs triggered oxidative stress, which changed the biocompatibility of NPs evidenced by cellular ATP level [ Fig. 7A(i)] [211]. Additionally, the Janus structure increased the colloidal stability of NPs reduced by NH 2 functionalizatio,n and following exposure to serum proteins; all NPs showed a negative zeta potential value which prevents inter-NP interactions and associated aggregation mediated by van der Waal's forces resulted in improved cellular uptake [ Fig. 7A(ii)] [211].
Biocoronated Au@Pd nanodendrites (NDs) with different surface functionalization groups is another approach that significantly promoted the biocompatibility against breast (MCF-7), lung (A549), and kidney embryonic (HEK 293) cells [212]. Differently charged-Au@PdNDs had different affinities to blood proteins. Negativelycharged Au@PdNDs generated a significantly larger PC than positively-charged ones [ Fig. 7B(i)]. The adsorption affinity of proteins was also mediated by protein type, concentration, and incubation time [212]. Additionally, a stronger binding affinity of BSA and fibrinogen were found to both PEG-and protamine sulfate (PS)-functionalized Au@PdNDs. Whereas Au@PdNDs.PS exhibited a higher affinity towards γ-globulin than negatively-charged NPs. Furthermore, the secondary structural changes of proteins following exposure to NDs were associated with the NP concentration and surface properties predominant in fibrinogen. Based on the improved compatibility determined in different cell lines [ Fig. 7B(ii)], it was suggested that a similar internalization mechanism mediated the cellular uptake of PC-ND complexes, which needs further investigation in the future [212]. Generally, the PC is formed on all NPs in vivo, leading to decreased targeting efficiency, immune cell interactions, and rapid blood clearance with limited tumor accumulation. However, it is also feasible to exploit the PC for better biocompatibility and drug delivery by leveraging it to load and stimulate the NPs and release a drug/photosensitizer for simultaneous PPT, PDT, and chemotherapy. Indeed, NPs can be precoated with blood plasma followed by loading multiple drugs/agents. The intravenous delivery of NP-PC complexes can lead to significant biocompatibility and higher tumor accumulation, reaching a peak within a shorter period of time post-injection relative to non-treated samples. Additionally, localized laser irradiation (especially in the case of AuNP) of tumors can lead to a notable tumor temperature increase within a short period of time and the generation of reactive oxygen species (ROS) for PDT. Although significant NP accumulation can be found in the major RES organs, no acute cytotoxicity can be observed histologically post-treatment [128]. Therefore, it can be indicated that the PC on the NP surface can be constructive for the loading and delivery of drugs/agents, whereas the bioinspired PC could make the nano-based platform applicable for drug delivery and therapeutic implementations.

The importance of the PC in clinical trials
The PC formed on the surface of NPs stemming from different blood plasma proteins can affect the biological behavior of NPs differently [213]. Based on the type of NPs and the characteristics of the adsorbed protein, the biological identity of NPs and their corresponding clinical implications can be heavily influenced [214]. Indeed, the rapid formation of a plasma-derived PC with dynamic heterogeneities critically affects NP pathophysiology [215]. Therefore, manipulating the formation of PCs around nanocarriers through predictive patterns could improve therapeutic NPs against cancer. Indeed, by manipulating the formation of the PC around NPs, the gap between scientific data in this field and preclinical or clinical activities can be reduced. According to Scopus and PubMed sites, approximately 1,800 papers in medicine, 3,100 in pharmacology/toxicology, and 3,420 in biochemistry/biomolecules/genetics have been published about the design, synthesis, and biomedical applications of NPs. However, only a small number of NPs, approximately 283, have entered the clinical stages according to the data reported on the clinical trial site (http://www.ClinicalTrials.gov). Although a variety of reasons can be cited for the difference between scientific discoveries and clinical applications, one persistent challenge is the poor knowledge of the biological function of NPs and their interaction with proteins in vivo. Numerous papers have shown that the PC directly affects (negative or positive) on regulating NPs as therapeutics [28,29,216]. Therefore, these effects can be considered as friend or foe based on the type of PC and the objectives of the study. Generally, studies published to date have indicated unexpected effects of the PC on nanocarriers' translational or therapeutic performance in vivo. This is despite the in vitro studies used to predict the formation of the PC on nanocarriers. Because the PCFs shows different effects on the biological behavior of nanocarriers [139,217], the concept of a personalized PC and its specific biological responses must be well-considered in future studies (Fig. 8). Based on the development of predictive patterns, therapeutic applications of NPs can be improved by manipulating the composition of the PC. For this purpose, the first step is to examine the target tissue to determine that the PC adsorbed on the nanocarriers can be effective in greater permeability, higher stability, and targeting of the nanocarriers. The second step is to study the effect of PC on the therapeutic performance of nanocarriers such as drug release. This should be done by incorporating more physiological 3D in vitro models [218] and systems that incorporate flow [219,220], mechanical forces [221][222][223], and multicellular populations [224,225] for in vitro screening, in addition to larger animal models that more closely recapitulate human physiology.

Conclusions, challenges, and future perspective
When NPs interact with biological systems, multiple proteins are adsorbed/absorbed onto the NP surface because of the high surface free energy of NPs. This process forms the PC on NPs. The appearance of the PC results in alterations in the intrinsic physicochemical properties of NP and their corresponding therapeutic properties and cytotoxicity. Detailed information about NP-protein interactions in biological systems is necessary to artificially engineer the composition of the PC and advance potential nano-based platforms for cancer nanotherapy. Therefore, studies are required to understand multiple mechanisms that regulate the formation of the PC to develop effective NP therapeutic systems. The manipulation of the PC formation could be engineered by modulating the physicochemical properties of NPs or modifying NP surfaces by different selective ligands (peptides), stealth polymers (PEG), or pre-coating with proteins to develop potential cancer-targeted nanotherapies.
Therefore, promising DDSs with promoted targeting capability, selective cancer therapy, mitigated cytotoxicity, and low MPS uptake can be developed by regulating the PC properties. Indeed, for the potential development of NP-based therapeutic systems, they should have the ability to mitigate the adsorption of opsonins while promoting the binding of dysopsonins upon interaction with the biological fluids. Nevertheless, due to various unharmonious outcomes of PC and associated cellular responses, we should address several main challenges in the field as follows: (i) Developing more advanced and standardized methods to evaluate and quantify the PC, as studies have demonstrated that several errors can arise from the PC preparation, extraction, and analytical methods used for quantification. (ii) Based on several reports that the PC affects cellular interactions and trafficking, efforts should focus on understanding these mechanisms to enable clinical translation.
(iii) More efforts should be focused on understanding how different PC components regulate cellular interaction and targeting. NP physicochemical properties can be tuned to develop nanoplatforms engineered for higher efficacy. (iv) Efforts should focus on delineating the effects of the biological microenvironment on PC formation and stability, given observed differences in sex and cell and tissue type interactions. These studies should also aim to close the gap between in vitro and in vivo studies in the PC field to incorporate more advanced physiological models and standardized reagents. (v) Lastly, studies should also incorporate changes observed in the tumor microenvironment (TME), such as pH, temperature, and blood flow, to understand how the PC composition changes dynamically in tumor cells to develop effective nano-based drug delivery systems for cancer therapy.