Impact of protein coronas on nanoparticle interactions with tissues and targeted delivery

Abstract

A major challenge in advancing nanoparticle (NP)-based delivery systems stems from the intricate interactions between NPs and biological systems. These interactions are largely determined by the formation of the NP-protein corona (PC), in which proteins spontaneously adsorb to the surface of NPs. The PC endows the NPs a new biological identity, capable of altering the interactions of NPs with targeting organs and subsequent biological fate. This review discusses mechanisms behind PC-mediated effects on tissue distribution of NPs, aiming to provide insights into the role of PC and its potential applications in NP-based drug delivery.

Introduction

Significant advancement in nanomedicine, driven by the extensive usage of nanotechnology, has received substantial attention due to its potential in precise diagnosis, tumor targeting and effective treatments for a range of diseases [1–3]. Recently, there has been a substantial increase in the development of nano-sized delivery systems. This upsurge is attributed to their capacity to selectively deliver the payloads to the targeted tissues, which is capable of enhancing the effectiveness of nanomedicine and minimizing the side-effects [4,5]. Despite these substantial and promising advantages offered by their applications, nanomedicine often fails in later stages of development or clinical trials [6,7]. These issues arise from either excessive off-target or inadequate on-target accumulation of nanomedicine [8,9]. A critical factor has been attributed to the generation of protein corona (PC) and its impacts on the interactions between nanoparticles (NPs) and targeted organs. It has been well documented that when NPs are introduced into biological fluids, biomolecules (e.g., nucleic acids, proteins, lipids, and sugar moieties) spontaneously adsorb, interact, and evolve on the surface of NPs [10–12]. This process gives rise to a structure reassembling a crown called the biomolecular corona. The biomolecular corona’s composition may determine the ultimate fate of NPs [13–15]. Among all biomolecules, the predominant biomolecules that form the biomolecular corona are proteins, known as NP-protein corona (PC) and most studies that investigate the impact of biomolecular corona formation have focused on PC [16]. The emergence of PC endows NPs with “biological identity” distinguishing it from its original physiochemical properties, leading to the modification in the pharmacokinetics and biodistribution characteristics, and subsequently cellular uptake of NPs [17,18]. In this review, we first discuss the impacts of PCs on the tissue-targeting ability and subsequently summarize targeting strategies based on various tissues. Recent advances and challenges in tissue-specific targeting strategies of NPs will then be discussed in depth followed by future perspectives. This review only focuses on targeting strategies for intravascularly administrated nanomedicines.

Impact of protein coronas on the tissue-specific targeting distribution

The targeting abilities of NPs are determined by functionalizing their surface with the targeting moieties (e.g., antibodies, small molecules, and nucleic acids), which can guide NPs to specific organs/tissues for drug delivery. However, the proteins absorbed on the surface of NPs can conceal the inherent or engineered reactivity of NPs, by blocking cell membrane receptors to inhibit internalization and lose the protection of NPs from opsonization (i.e., the immunological response that targets NPs for removal), causing rapid clearance and off-target accumulation [15,16]. Here, we discuss the mechanism of interactions of NPs with different target tissues with a particular focus on the brain, lungs, liver, and spleen.

The impact of protein coronas on brain-targeting capability

The blood-brain barrier (BBB), which serves as the endothelial barrier between the circulating bloodstream and the brain, is notably restrictive due to a relatively higher density of endothelial cells and tight junctions [19]. Thus, the BBB is able to prevent the delivery of NPs from extravasating into brain. Targeted strategy for the intravenously administered NPs across the BBB could potentially harness endocytosis process (Figure 1a). The process involves the recognition of specific receptors on the luminal endothelial cell membrane, followed by endocytosis into the endosome, intracellular transport, and ultimately exocytosis from the endothelium (Figure 1a) [20,21]. The use of transferrin (Tf)-conjugated NPs is the most commonly used targeting strategy to deliver drugs into the brain via receptor-mediated endocytosis pathway [22]. However, PC can act as a shield, preventing Tf from binding to Tf receptors and other receptors on the cell surface [23,24]. Dawson et al. [23] reported that Tf-conjugated NPs is unable to interact with Tf receptor on the cell membrane after incubation in serum, resulting from the shield effects of PC on the Tf protein. Xiao et al. [24] reported that the PCs impact the transcytosis of Tf-functionalized NPs through BBB and attenuate their ability to target brain tumor cells. Similarly, polymeric NPs coated with the HIV-1 trans-activating trans-activator peptide (known for being able to cross the BBB) and/or alpha neural/glial antigen 2 (known to be able to target oligodendrocyte precursor cells) are unable to cross the BBB, neither specifically target oligodendrocyte precursor cells, likely because of the formation of PCs [25]. In addition to Tf-based delivery systems, the apolipoprotein (e.g., ApoE)-based strategies have also been reported as promising tools for brain-targeted delivery [26]. Certain surfactant-modified NPs, such as poly(ethylene glycol)-/polysorbate-modified NPs, have the capability to absorb apolipoproteins such as ApoE or ApoB, and thus forming the apolipoprotein-rich PCs after systemic administration. By interaction with lipoprotein receptors on the BBB, these proteins absorbed on the surface of NPs can facilitate the entry of NPs into the brain [27,28].

Figure 1.

A schematic illustration of organ-specific targeting mechanisms of nanoparticles (NPs). (a) NPs cross the blood–brain barrier via receptor-mediated endocytosis. (b) Lung-selective targeting via caveolae-medicated endocytosis. (c) Opsonization mediate Kupffer cell interactions. (d) Opsonization mediate splenic macrophage interactions. Figure created with BioRender.com.

The impact of protein coronas on lung-targeting capability

When foreign NPs are within the bloodstream and tissues, they typically undergo clearance by the reticuloendothelial system (RES) [5,29]. RES, a part of the immune system, comprises a system of phagocytes such as macrophages and monocytes. These cells are primarily located on the vascular wall of the liver (i.e., Kupffer cells), spleen (i.e., splenic macrophages), kidneys (i.e., mesangial cells), and lung (i.e., lung macrophages). However, most NPs are typically taken up by liver Kupffer cells after systematic administration [30]. This sequestration presents a significant obstacle to achieving tissue-specific targeting of NPs. The PC consisting of apolipoprotein E (i.e., ApoE) absorbed to the surface of NPs might promote the phagocytosis of lung macrophages [31–34]. Scheffler et al. [33] reported that gold nanoparticles (AuNPs) bound with human serum albumin and ApoE can significantly accumulate in the lungs when compared to control NPs (i.e., citrate stabilized AuNPs). Kim et al. [32] synthesized a liposome consisting of 1,2-dioleoyl-3-trimethylammoniumpropane/ dioleoylphosphatidylethanolamine, which can specifically absorb apolipoprotein A-I to increase the accumulation in lung tumors and reduce the hepatic accumulation. Although there was an increased accumulation of NPs in the lungs, it did not have any benefit as the NPs were also rapidly eliminated by the RES. Recent studies have developed lipid NPs functionalized with specific protein antibodies (e.g., plasmalemma vesicle-associated protein) for lung-targeted mRNA delivery in vivo [31,35]. These lipid NPs can internalize into the lungs via the mechanism of caveolae-mediated endocytosis (Figure 1b) to achieve lung-selective targeting.

The impact of protein coronas on liver-targeting capability

Being the largest RES organ, the liver presents a heightened capability to eliminate NPs in comparison to other RES-rich organs (e.g., spleen, kidney, and lung). Based on the postulated mechanisms of hepatic sequestration of NPs in a previous study [36], the process of delivering NPs to liver is through the following steps: (1) interact with Kupffer cells and liver sinusoid endothelial cells, (2) if they escape from these cells, they would transport to the space of Disse, (3) interact with hepatocytes, and (4) if they escape from all of these cells, they will be eventually eliminated from the body (Figure 1c). While the exact molecular mechanisms governing interaction between Kupffer cells and PC is still not fully elucidated, several studies have identified several specific receptors that can bind to the PC on NPs and be recognized by Kupffer cells [37–40]. The phagocytosis of NPs by Kupffer cells is primarily mediated by opsonin proteins [e.g., immunoglobulins and complement (C3)] [41,42], leading to the NP accumulation in the liver and rapidly elimination from circulation [43–45]. Recent studies have identified the PC composition using analysis of mass spectrometry, revealing that the highly-bound proteins involve opsonin proteins, especially complement C3 absorbed onto the surface of NPs [46,47]. On the other hand, to prevent the NPs interaction with Kupffer cells, one strategy is to coat NPs with neutral-charged polymers such as polyethylene glycol (PEG) to deter the opsonin proteins absorbed to the PC on the surface of NPs [48,49]. This method can prolong the circulation lifetime of NPs but it varies by the degree of PEGylation on the surface of the NPs [50].

The impact of protein coronas on spleen-targeting capability

In addition to the Kupffer cells in the liver, splenic macrophages also play an very important role in the uptake of NPs [51]. Tavares et al. [51] reported that spleen can sequester more NPs after removing liver Kupffer cells. In a mouse model where Kupffer cells were removed, the authors administered AuNPs and estimated the amounts of NPs in various tissues using both fluorescent-labeling optical imaging and element-based analysis through inductively coupled plasma–mass spectrometry (ICP-MS). They found significantly increased amounts of 50–200 nm NPs in spleen and concluded that the NPs might be removed by other RES organs when the major one (i.e., liver) is not functioning. Notably, the main mechanism for the removal of NPs from blood circulation in spleen is the internalization of opsonized NPs [52]. The formation of PCs on the NPs can promote the effects of opsonization of NPs by splenic macrophages. For example, if the composition of PCs consists of opsonized proteins such as complement, immunoglobulins and apolipoprotein [46,53,54] that can interact with the receptors on the surface of RES cells, NPs can be recognized by splenic macrophages and subsequently removed from circulation [55]. In addition to opsonized proteins, other proteins absorbed onto the surface of PCs have the capacity to be recognized by scavenger receptors [56]. The additional proteins introduce an additional mechanism to the clearance of NPs by RES [57]. The uptake of NPs by splenic macrophages usually takes place in the marginal zone (MZ) in spleen (Figure 1d). An earlier study [57] demonstrated that MZ macrophages can uptake polystyrene NPs. Notably, this uptake process does not rely on the lectin-like receptors but instead involves the scavenger receptors as well as the albumin-coating NPs. Additionally, it was observed that small liposomes (100–200 nm) exhibit a tendency for selectively internalized by MZ macrophages.

Role of protein coronas in the recent advances of organ-specific targeting strategies

In recent years, the growing understanding of PCs has paved the way for techniques to manipulate the adsorbed PCs on the surface of NPs [14,58]. This approach enables the utilization of these PCs for active targeting receptors on certain cells and thus harnessing the endogenous system for the purpose of organ-specific drug delivery [59]. There are two proposed strategies (Figure 2) to address the shielding role of the PC on targeted NPs (Tables 1–2).

Figure 2.

Illustration of advanced nanoparticles (NPs) delivery systems for organ-specific targeting. Two advanced NPs delivery strategies have been developed for organ-specific targeting: artificial and biomimetic approaches. In the artificial approach, NPs are pre-incubated with specific proteins like apolipoprotein, lipoprotein, transferrin, and peptides before introduction into biological fluids. The biomimetic approach involves decorating NPs with cell membranes, endogenous proteins, or biomolecules to confer targeting capabilities to the NPs. Figure created with BioRender.com.

Table 1.

Artificial protein corona-based organ-targeting strategies.

Target organs	Type of nanoparticles	Proteins	Mechanisms	Ref.
Brain
	LNPs	Apo E; Apo E4	Lipoprotein-medicated endocytosis	[27,61]
	AuNPs	Apo B100	Lipoprotein-medicated endocytosis	[62]
	HSA-NPs	Apo I	Lipoprotein-medicated endocytosis	[28,65]
	PNP	Tf	TfR-medicated endocytosis	[64]
Lung
	PEG-NPs	CD47	Unknown	[73]
Liver
	SNAs	C1q A, C1q B, and C1qC	Opsonin-mediated phagocytosis	[63]
	PNP	Fetal globulin	SR-medicated phagocytosis	[66]
	AuNPs	Albumin	Opsonin-mediated phagocytosis	[67]
	LNPs; HAS-NPs	Apo E	Opsonin-mediated phagocytosis	[68–70]
Spleen
	V-NPs	Opsonin	Opsonin-mediated phagocytosis	[71]
	QDs	Apo B-100	Opsonin-mediated phagocytosis	[72]

Note: Apo, apolipoprotein; Tf, transferrin; LNPs, lipid nanoparticles; AuNPs, gold nanoparticles; HSA-NPs, human serum albumin nanoparticles; PNPs, polymer nanoparticles; PEG-NPs, polyethylene glycol nanoparticles; SR, Scavenger receptors; SNA, Spherical nucleic acids; V-NPs, virus nanoparticles; QDs, Quantum dots; Vtn, vitronectin; RES, reticuloendothelial system.

Table 2.

Biomimetic protein corona-based organ-targeting strategies.

Target organs	Biomimetic Approach	Type of nanoparticles	Proteins	Mechanisms	Ref.
Brain
	Biomolecule modification	LipNPs	Apo A1, E, J	Lipoprotein-medicated endocytosis	[82]
	Biomolecule modification	LipNPs	Apo E	Lipoprotein-medicated endocytosis	[83]
	Biomolecule modification	PTX/Aβ-CN-PMs	Apo E	Lipoprotein-medicated endocytosis	[26]
Lung
	Endogenous protein coating	LNPs	Albumin, fibrinogen	Caveolae-medicated endocytosis	[31]
	Endogenous protein coating	LNPs	ApoE, β2-GPI, or Vtn	Caveolae-medicated endocytosis	[84]
Liver
	Biomolecule modification	RcP-NPs	RBP4	RBP receptor-medicated uptake	[89]
	Biomolecule modification	DNA tetrahedron	Lipoproteins	Lipoprotein-medicated endocytosis	[85]
Spleen
	Endogenous protein coating	LNP	Apo H	Opsonization/spleen-targeted delivery via phagocytosis	[84,86]
	Endogenous protein coating	LNP	Apo E, Fg, C3	Opsonization/spleen-targeted delivery via phagocytosis	[33,87]
	Cell membrane decoration	DEX-NPs	C3b	Opsonization/spleen-targeted delivery via phagocytosis	[88]

Note: Apo, apolipoprotein; Tf, Transferrin; LNPs, lipid nanoparticles; LipNPs, Liposomal nanoparticles; AuNPs, gold nanoparticles; RcP, Retinol-conjugated polyetherimine; RBP, Retinol binding protein; Fg, fibrinogen; C3, complement 3; DEX-NPs, dextran-coated ferrous nanoparticles.

Pre-coating strategy: Artificial engineering of protein coronas

This first strategy is the use of artificial PC on tissue-specific targeting delivery. The concept of the artificial PC involves pre-coating NPs before introducing them into biological fluids to enhance the absorption of specific proteins that is capable of targeting to specific cells [27,28,60–73]. When these NPs interact with the blood proteins, they acquire the artificial PC that is able to actively target to the selective tissues [60]. For example, it was demonstrated that modifying the PC through polysorbate pre-coating can enhance apolipoprotein absorption on NPs and thus increasing the transport across the BBB [27,61,62,74]. In addition, Dal Magro et al. [27,61] reported that lipid NPs are able to increase the transport to the brain parenchyma and higher accumulation in the brain when incubated with apolipoprotein E4 or E-derived peptides in vitro, compared to unmodified particles. For liver targeting delivery, the absorption of apolipoprotein and complement on NPs was used to further promote the accumulations of NPs in liver [63]. The retinol binding protein [75] and lipoproteins [76] can target hepatocytes and serve as targeted ligands on the NPs to promote the long-term accumulation in liver. In another study, liposomes with positive charge functionalization were pre-coated with artificial PC and explored as possible nano-carrier for targeted delivery to tumor-related macrophages in cancer therapy [77]. One of the main challenges of the artificial PC approach, however, is that the active site of PC should be positioned in the outer corona layer to facilitate interaction with specific cell receptors. For example, although pre-coating silica NPs with the serum proteins γ-blobulins could enrich the PC with immunoglobulins and opsonin which have high targeting efficacy and recognized by macrophages, there was no substantial enhancement of macrophage uptake of NPs compared to NPs without pre-coating [78]. The authors concluded that other protein binding onto artificial PCs by non-specific protein-protein interactions results in the shielded effects and inhibits the interactions between opsonin and relevant receptors on the immune cells [78]. Hence, these findings underscore the importance of exposing the functional binding sites to cell receptors, which is difficult to manipulate from the spontaneous absorption of such proteins onto the surface of NPs.

Biomimetic approach to leverage protein coronas

The second method is so called the biomimetic approach [79–81]. The biomimetic method seeks to emulate natural biological environment and mechanisms by incorporating specific biological functionalities into synthetic NPs. This approach is aimed at achieving therapeutic effects or mitigating the adverse side effects associated with synthetic NPs. Following this concept, a wide array of biomimetic materials has been applied on the NPs, including coatings with endogenous proteins, cell membrane decoration, and biomolecule alterations [26,31,33,64,82–89]. Among these methods, the cell membrane decoration is one of the most useful and commonly used biomimetic approaches. For instance, the NPs decorated with cell membrane derived from a variety of cell types such as erythrocytes (i.e., red blood cells), which is capable of exhibiting the capability to diminish undesired immune reactions, circumvent elimination by macrophages and systemic clearance [90]. The red blood cells decorated NP delivery system had the lowest immunoglobulin absorption on the surface of PCs and prolonged circulation time. The red blood cell coatings reduce opsonization, leading to decreased interaction with macrophages [90]. Apart from the cell membrane decoration, the coating with endogenous proteins is another potent method. By pre-coated endogenous protein, the composition or abundant component of PCs can be manipulated to achieve stealth effect that extends circulation time and tissue-specific targetability. Dillard et al. [84] introduced a tissue-specific NP delivery system with endogenous protein coating strategy. Their approach endows NPs with stealth effect, preserving their targeting ability in non-liver tissues such as lung and spleen by reducing the binding of ApoE to NPs and escaping RES clearance [84]. Similar approach was also used in other non-liver targeting NPs [31,33,86]. To minimize the impact from PCs while ensuring stability, recent studies have explored the modifications in the generation or composition of PCs through biomolecule modifications while preserving the natural functionalities of PC components. For example, Zhang et al. [82] developed an innovative NP brain-targeted system with biomolecular modification. This system utilizes a short, non-toxic peptide derived from beta-amyloid peptide (Aβ1–42) to control the protein corona component, which is capable of interacting with the lipid-binding domain of the brain-targeting apolipoproteins (e.g., ApoA1, ApoE, and ApoJ) and thus facilitating the brain transport via lipoprotein-mediated endocytosis. This approach has been applied to other brain- and liver-targeted NPs [26,83,85,89].

Perspectives, challenges and conclusions

In this review article, we try to discuss mechanisms about targeting strategies of NPs to specific tissues and the dual impacts of PCs on those strategies. The targeting mechanisms of NPs were categorized based on specific tissues, ranging from ligand- or non-ligand-induced endocytosis, phosphatiosomes, and opsonization of RES. However, most NPs will inevitably adsorb serum proteins, altering the targeting ability of NPs. Recent studies have developed artificial and biomimetic PCs to address these limitations. Both the use of artificial and biomimetic PCs confers a pre-determined biological identity onto the NPs, empowering them with desired properties including prolonged circulation, tissue-specific targeting, and the ability to engage in cell-to-cell interactions, by precisely manipulating the interactions between functional plasma proteins and the surface of NPs or NP-PC interface. However, these approaches are still constrained by certain critical challenges that are yet to be thoroughly evaluated. For example, the use of biomimetic materials onto NPs can raise concerns regarding the potential to trigger the immune responses and overall safety, especially when non-autologous proteins or viral components are involved. Also, the intricate structure and composition are still significant challenges on the reproducibility and reliability of artificial PCs. There is an urgent need to establish an innovative and comprehensive paradigm for designing smart nanomedicine. This might be achieved by combining precisely tailored chemical synthesis with the versatility and biocompatibility of biologics. In conclusion, we anticipate that as nanomedicine and nanotechnology continue to advance, the role of the PC in the delivery strategy of NPs will become more clearly elucidated. The ability to manipulate the generation of the PC on the surface of NPs has the potential to overcome organ-specific targeting challenges, enabling precise, efficient, and safe delivery of nanomedicine.