An overview on the investigation of nanomaterials' effect on plasma components: immunoglobulins and coagulation factor VIII, 2010-2020 review.

FVIII and immunoglobulins (Igs) are the most prominent plasma proteins, which play a vital role in plasma hemostasis. These proteins have been implemented frequently in protein therapy. Therefore, their maintenance, durability, and stability are highly essential. Herein, various approaches to improve protein functions have been investigated, such as using recombinant protein replacement. In comparison, advances in nanotechnology have provided adequate context to boost biomaterial utilization. In this regard, the applications of various nanoparticles such as polymeric nanomaterials (PEG and PLGA), metal nanoparticles, dendrimers, and lipid based nanomaterials (liposomes and lipid nanoparticles) in stability and the functional improvement of antibodies and coagulation factor VIII (FVIII) have been reviewed from 2010 to 2020. Reviewing related articles has shown that not only can nanomaterials adequately protect the structure of proteins, but have also improved proteins' functions in some cases. For example, the high rate of FVIII instability has been successfully enhanced by bio-PEGylation. Also, utilizing PEGylated liposomes, using the PEG-lip technique for coating nanostructures, leads to FIIIV half-life prolongation. Hence, PEGylation had most impact on the stability of FVIII. Likewise, PEG-coated liposome nano-carriers also presented such a good effect on stability improvements for FVIII due to their ability to tune the immune system by reducing FVIII immunogenicity. Similarly, Ig PEGylation and conjugation to magnetic nanoparticles resulted in increased half-life and better purification of Igs, respectively, without any loss in structural or functional features. Consequently, metal-organic frameworks and recent hybrid systems have been introduced as promising nanomaterials in biomedical applications. As far as we know, this is the first study in this field, which considers the applications of nanoparticles for improving the storage and stability of antibodies and coagulation FVIII. This journal is © The Royal Society of Chemistry.

Introduction

Coagulation factors and immunoglobulins (Igs) are two main orderly plasma proteins that play crucial role in blood hemostasis, which stop bleeding through the coagulation cascade, and develop immunogenicity against foreign external components. They are also significant components that are frequently used in protein therapy. In this regard, maintaining the efficiency and improving the stability of these two essential proteins is strongly being followed. However, the objective has been under-researched by scientists.

Advances in nanotechnology in therapeutic and medical applications could open new horizons to employ nanomaterials in protein therapy. Using nanocarriers has improved the efficacy of various therapeutics[1-5] (Fig. 1). Here we are going to review the applications of nanoparticles in the improvement of storage of antibodies and coagulation factor VIII from 2010 to 2020. As far as we know, this is this first study in this field, which considers the applications of nanoparticles for improving the storage of antibodies and coagulation factor VIII in order to introduce the most efficient strategies for sustainability and consequently improving the quality of the life of patients.

Fig. 1

Overview of nanoparticle effects on two essential plasma proteins.

Results and discussion

Here, the results of studies focusing on nanomaterials which were conjugated with protein factors such as FVIII and immunoglobulins will be highlighted.

Coagulation factor VIII

Coagulation factor VIII (FVIII) is a non-enzymatic glycoprotein composition containing six domains, NH2–A1–A2–B–A3–C1–C2–COOH, which serves as a critical component of the intrinsic coagulation pathway.[6-9] Coagulation pathways are illustrated in Fig. 2.

Fig. 2

Schematic image of extrinsic coagulation pathways and the role of FVIII.

The coagulopathy resulting from a deficiency in synthesis or activity of FVIII leads to type A hemophilia.[10-13] This bleeding disorder is characterized by spontaneous hemorrhage, particularly in joints and muscles due to the congenital x-linked disorder that occurs in 1 of 5000 male births.[14,15] Less prevalent hemophilia (type B) can also occur in 1 of 30 000 male births, resulting from FIX deficiency. FVIII is carried in plasma, associated with a protein factor, called von Willebrand factor (vWF), which plays a chaperone-like role in plasma to stabilize FVIII against dendritic cells' clearance and degradation.[16,17] Free FVIII is a labile component that just remains less than 2 hours in circulation. However, formation of the vWF-FVIII complex increases the FVIII circulation time to approximately 8–12 h.[18] Fresh frozen plasma (FFP) is the best and most accessible resource of FVIII. The temperature susceptibility of FVIII storage could restrict getting access to the high amount of FFP resource in a difficult situation.[19-22] Therefore, improving the half-life of the FVIII factor while trying to maintain its activity and efficacy has a crucial impact on life-threatening disorders such as hemophilia.[23] Various types of therapy for extending protein maintenance have been established for more than seven decades for hemophilia disorders including albumin-fusion FVIII and PEGylated factors, and some of them were commercialized and entered the market.[24-29] In recent studies, nanomaterial application in extending protein maintenance attracted several interests. Therefore, we will review related articles focusing on nano-based content. Prophylactic treatments, including standard FVIII infusion, basically could prevent frequent bleeding episodes. However, harmful consequences and disadvantages like pain, irreversible joint damage, and difficulties due to short interval infusion could significantly impact patients' life quality since prophylaxis for hemophilia A typically needs at least three times per week to provide a sufficient circulating level of FVIII. There is a critical point related to the immunogenicity of FVIII infusion and producing anti-drug antibodies that are called inhibitors, which neutralize the FVIII activity.[30] Evidence shows that plasma-derived FVIIIs are less immunogenic but have more exposure to contamination than recombinant FVIIIs.[31] However, recent studies have established the third generation of (recombinant FVIII) rFVIII that is both more efficient and less immunogenic than previous generations. Also, neutralization can be controlled by bypassing the inhibitory activity with activated prothrombin complex concentrates (APCC).[32-35]

Current hemophilia research is focusing on the emergence of new or developed factor replacement therapies to extend proteins' half-lives. Among these recent studies, covalent binding of FVIII to polyethylene glycol (PEG; PEGylation) had the most citation, and then liposomes and rarely PLGA have been used in related experiments which have been brought out in order.

Poly ethylene glycol (PEG)

Basically, the first reaction of the body against nanoparticle entrance, as a foreign component, is forming a protein complex in a way that NPs are totally surrounded by proteins which is called “protein corona”. Opsonins are proteins that are responsible for opsonization and consequently evoke the antigen presenting cells (APCs) like macrophages for phagocytosis and plasma clearance.[36] Poly Ethylene Glycol (PEG) is a hydrophilic and biocompatible polymer that has been successfully established in biotech pharma to modify therapeutic proteins for inducing biocompatibility by anti-opsonization activity and prolonging the circulatory half-life of proteins. Herein, native FVIIIs have been investigated, using enzymatically new (PEG) conjugates (ranging from 10 to 80 kDa), to specifically attach a unique O-linked glycan to the FVIII B-domain. Therefore, the cleaving process, which is done by thrombin, leads to B-domain associated O-glycan PEG release. The activated FVIII (FVIIIa) can generally enter into the coagulation cascade. In vivo and in vitro potency of 10, 20, 40, and 80 kDa PEGylated FVIII have been tested. The results revealed a positive correlation between the half-life of FVIII factor and size of PEG. The study also illustrated that FVIII conjugated with a single-chain 40 kDa turoctocog alfa (N8-GP) had the most efficiency on the prolongation of FVIII in mice and rabbits and less in rats. The biochemical functionality of N8-GP was unchanged while the cellular uptake and in vivo clearance reduced.[37]

A patent was then investigated, using a B-domain of the factor VIII molecule which has been truncated and particularly coated with a hydrophilic polymer, such as a PEG group.[38]

Furthermore, similar research studies have been conducted on N8GP-rfVIII in order to prolong the half-life of the protein factor that showed similar results. However, there were no statistical reports in these studies.[39,40] Also, in another research, a glyco-PEGylated recombinant FVIII (N8GP) was first tested in a human trial to evaluate the safety pharmacokinetic profiles of N8-GP in comparison with previous FVIII products. Assessments illustrated that the terminal half-life of N8-GP averagely extended to 19.0 h (range: 11.6–27.3 h) or a 1.6-fold increase in plasma survival occurred. It could also be well tolerated without any noticeable safety concern.[41]

According to the nearly similar studies in 2016 and 2017, in addition to the native FVIII, rFVIII has also been examined to improve the biocompatibility and long-lasting circulation, employing novel PEG derivatives. The recombinant FVIII PEGylated by rurioctacog alfa pegol (BAX 855) in which approximately 60% of the 20 kDa PEG chains were localized to the B-domain showed a development in reducing the injection frequency per week without receiving FVIII inhibitory antibodies or unexpected adverse events. Moreover, turoctocog alfa pegol (N8-GP) was used, pursuing the trial phase III of the previous study. Two groups of patients including typical rFVIII injection and N8-GP treated rFVIII have been assessed, and prophylactic medication was also compared in two groups. According to the research, a single dose of up to 75 IU per kg N8-GP and two intravenous injections were well tolerated by 83.6% and 95.5% of the patients. In conclusion N8-GP could extend the half-life of FVIII (mean, 18.4 h) and prolong the factor plasma activity compared to the patients of previous study and favorably showed safety. No clinically severe concern was reported as well.[42-47] Herein, BAY rFVIII has been also implemented.[48]

Also, PEG-FVIII innovation has been patented by an American assignee with similar developing results.[49]

Liposomes

Liposomes are nano-to micro-sized lipid vesicles (approximately 100 nm to upper μm in diameter) composed of two spaces where a phospholipid bilayer has encapsulated the hydrophilic interior.[50] Although liposomes have different applications in therapeutic agent delivery, they are rapidly detected by dendritic cells in plasma circulation. In this regard, PEGylation is frequently being used in recent progress to improve the biocompatibility and extend the circulation half-life in bloodstream. Phosphatidyl derivatives are decent candidates used to increase the plasma circulation time in interaction with or without vWF in order to improve pharmacokinetics. For example, synthetic phosphocholine based liposomes (POPC) have been investigated in some research studies, using PEGLip, a new technology for PEGylation. The results revealed that PEGLip specifically bound to plasma-derived FVIII (pd-FVIII) that strongly depended on the presence of PEG polymer.[51,52] Besides, it has been proven in two other related investigations that PEGLip pd-FVIII significantly induced faster thrombin generation in preclinical studies and also shown that using PEGLip FVIII led to reducing the prophylactic infusion dose to once-weekly administration (Fig. 4), and it was well tolerated, which resulted in lower bleeding frequency as well.[53,54]

Fig. 4

Schematic image of IgG adsorption and purification from rabbit serum using hybrid magnetic nanoparticles.

Phosphatidyl derivatives like phosphatidylserine and phosphatidylinositol are two anionic phospholipids that can convert the immunogenic epitope to immune-tolerogen epitope that prolongs protein circulation against inhibitor development. The promising result of using these materials to reduce the immunostimulatory activity of inhibitors could extend the half-life of FVIII. However, the results indicate that liposomes are being exposed to rapid uptake by the reticuloendothelial system (RES). Hence, PEGylation is a strategy that has been investigated to make longer-acting FVIII.[55] PEGylation has beneficial effects on making a barrier against opsonin protein by surface hydration and neutralization of the surface to inhibit opsonin and RES cell attraction. Despite all benefits observed in subcutaneous administration, no positive therapeutic effects have been demonstrated in an intravenous infusion due to liposome PEGylation, using passive PEG transfer.[56-58]

Moreover, joint research studies presented the efficacy of using B-domain deleted (BDD)-rFVIII conjugated soy phosphatidylinositol lipid nanoparticles in reducing inhibitor development and 1.5-fold and 1.4-fold increases in the terminal half-life of BDD-rFVIII to 18 hours in mouse clinical trials and dogs, respectively. Simultaneously, the immunogenicity assay has been evaluated in the presence of labeled anti IgG as well.[59-61] Furthermore, in similar research in 2018, it has been demonstrated that subcutaneous administration of Lys-phosphatidylserine could extend the half-life to 1.6 fold by reducing the immunogenicity.[62]

There are also several reports on employing different types of lipid nanoparticles (LNP), like lipid nanotubes (LNT) and lipid nanodiscs (LND), to investigate and screen the influence of modifications on the protein structure and function. For example, in empirical research, LNTs, helical organization of FVIII on lipid nanotubes, were used to characterize how a difference in sequence can correlate with structural and functional changes. As a result, the methods described in this article can identify the sequences, which will improve the recombinant FVIII function when modified. Therefore, this can lead to significant clinical implications for drug discovery in both thrombosis and hemostasis fields.[63] Also, similar studies were conducted using lipid nanodiscs as delivery systems for FVIII, in which rFVIII was assembled on lipid nanodiscs. The results indicated that this complex had a synergistic effect on hemophilic mice's clotting process.[64,65]

PLGA

Poly lactic-co-glycolic acid (PLGA) is a biocompatible copolymer that has been classified as a safe component and licensed by FDA. PLGA has been used in the same PEG related applications in therapeutic procedures. Herein, a research article was published in 2015. Tolerogenic nanoparticles (NPs) were introduced, using developed tolerogenic synthetic vaccine particles (SVP) by incorporating rapamycin in the self-assembling of PLGA to induce immunologic tolerance and avoid the inhibitor development process in protein therapy. Rapamycin is commonly used as an immunosuppressant drug to prevent transplant rejection and immune response, as well as induce tolerance to FIX and FVIII in mouse models. The nanoparticle delivery of rapamycin and antigens to the same APC may facilitate tolerogenic antigen presentation, leading to induction of antigen-specific response of regulatory T cells. Since viral vesicles are quickly taken up by dendritic cells, biocompatible and biodegradable PLGA has been used to prolong the circulation half-life. Tolerogenic nanoparticles instead of FVIII replacement therapy showed potential to improve the efficacy and safety profile of FVIII in the treatment of hemophilia patients, either for managing or prevention of bleeding.[66] The results are illustrated in Table 1.

FVIII and studied nanomaterials

R	Types of NPs	Size (nm)	Electrostatic charge	Effects on plasma derivatives' viability	Effects on plasma derivatives' function	Ref.
(1) Polymeric nanomaterial: PEGylation
1	O-Glycan PEG-FVIII	40 kDa		Unfolding effect on vWF, reduced activation in presence of vWF	Reduce degradation and increase circulation time	37
2	B-domain truncated FVIII-PEG	PEG variants			(O-Glycan) BDD-FVIII-280 13 to 19 hours, 1.9–2.1 fold increased	38
					2 × 40 kDa PEG BDD-FVIII 40 kDa PEG 280 16 to 20–22 hours, 1.7 fold
					(O-Glycan) BDD-FVIII 80 kDa PEG 280 15 to 21 hours, 1.9 fold
3	PEGylated FVIII variants	Domain variant PEG		Improved pharmacokinetics and activity, attenuated immune-response	Extended half-life with 95% efficiency, 2 fold increased	40
4	Glyco-PEGylated recombinant FVIII (N8GP)			Noticeable reduction in cell clearance without any safety concerns	Half-life of N8-GP extended to an averagely of 19.0 h (range: 11.6–27.3 h) or a 1.6-fold increase in plasma survival	41
5	Rurioctocog alfa pegol (BAX 855, Adynovate®)	Full-length factor VIII, 20 kDa PEG			95.9% bleeds controlled with one or two doses and extended to 1.4–1.5 fold increased	42
6	Turoctocog alfa pegol (N8-PG)	B-domain truncated, 40 kDa PEG			Extended the half-life of rFVIII to 18.4 hours	43
7	BAX 855 (PEGylated ADVATE™ (rFVIII))	20 kDa PEG reagent to FVIII-octocog alfa		No adverse safety concern, no subject developed antibodies to CHO proteins	1.3–1.5 fold half-life extension, twice-weekly prophylaxis with BAX 855 was safe and efficacious in paediatric with severe haemophilia A	45
8	B-domain replaced by sialic acid FVIII conjugated (PEG)	Branched 40 kDa N8-GP (turoctocog alfa pegol)			Subcutaneous administration, prolonged 19 h stability in patients with severe hemophilia A, corresponding to a 1.6-fold prolongation versus standard FVIII	47
9	BAY 94-9027 BDDFVIII-PEG	60 kDa (PEG) molecule site specifically attached via a maleimide linker to a cysteine amino acid		No relevant changes in PK characteristics after multiple doses	1.4 fold half-life increased	48
(2) Liposomes
1	PEGLip/pd-FVIII	DSPE-PEG 2000 (2 kDa)		Reduce the affinity of inhibitor	Extended circulation and improve survival rate in mice	51
2	Conserved peptide associated PEGLip FVIII	80–100 nm			Reduced endothelial uptake	52
3	PEGLip pdFVIII	80–100		Faster and more efficient thrombin generation	Enhanced FVIIa haemostatic efficacy 2 h extended half-life	53 and 54
4	FVIII–PI		Neg. (−)	The C2 domain of FVIII; similar binding region with von Willebrand factor within immunodominant epitope	Protected FVIII from degradation	56
					Reduce immunogenicity
5	(FVIII–PI/PEG)	DMPE-PEG2000			Adverse effect on immunogenicity in intravenous administration due to PEG passive transfer	57
6	Soy-PI linked BDD-rFVIII		Negative	Decrease the electrorepulsion due to B-domain deletion, decrease the inhibitor affinity to attach	Extended plasma survival and hemostatic efficacy prolong circulation and decrease the immunogenicity	59–61
7	Lyso-phosphatidylserine particle	193.9 ± 69.2 nm	−27.64 ± 4.00 mV (negative)	Converting an immunogen to a tolerogen, induction of T cell regulators by	Reduced anti-FVIII antibody responses, by means of inducing tolerance towards therapeutic proteins. It is not reported quantitatively	62
(3) Lipid nanoparticle (LNP)
1	FVIII-nanodisc complex	15 nm	Negatively charged ND rich in phosphatidylserine (PS)	Improving procoagulant activity due to shielding effect of ND on FVIII binding site to lip-protein receptors	Increased the stability and reduce the amount of required FVIII, thus lower the treatment cost	64 and 65
(4) Polymeric nanomaterial: PLGA
1	PLGA coated SVP-rapamycin encapsulated			Inhibited expansion of antigen-specific T cells and induced antigen-specific regulatory T cells or, antigen-specific immune tolerance	Prevent the immunogenic response and reverse inhibitor formation	66

To sum up, comparing reviewed nanostructures showed that nanosystem-conjugated recombinant FVIII which was covered by derivatives of biocompatible PEG polymers like N8-GP has showed the most effective structure in increasing the circulation time in blood. For example, an international patent (WO) of the o-glycoPEG system and some other research studies have shown the increase of stability up to 19 hours and successful reduction of infusion frequency to just twice a week.[40,42,43,49]

Generally, improving the stability is related to two mechanisms: first, increasing the circulation time by limiting cell uptake by dendritic cells and hiding from the reticuloendothelial system can be possibly accompanied by PEGylation. Next, the implementation of phosphatidylserine and inositol derivatives such as Lys-PS throughout the liposome PEGylated systems can attenuate immune response by converting immunogenic compounds into tolerogenic and increasing the durability of protein factors.[62]

Regardless of the current protein function improvement approaches, the state of the art method using mRNA-based gene delivery has been currently investigated. However, probable mutations, high costs, and unknown adverse drawbacks are intensely challenging that restricts its utilization in clinical therapies, and improving the current prophylactic treatment is preferable.[67,68]

Immunoglobulins

In 1890, von Behring and Kitasato discovered antibodies in the serum of a rabbit infected by tetanus, claiming that the antibodies had specific functions.[17,69-71] Human blood plasma contains an excessive number of globular proteins, specifically Igs known as gamma globulins.[72,73] This protein family consists of a wide range of autologous molecules that constitute immune responses in diverse physiological and pathological processes in vertebrate animals.[74] Igs or antibodies are glycoprotein molecules with a Y form-like structure that includes two similar domains for recognizing antigens and two similar domains with effector functions (Fig. 3).[75] This family is divided into five classes: immunoglobulin G (IgG), IgA, IgM, IgD and IgE, considering their concentration in blood.[17,76]

Fig. 3

Image of PEG-linked liposome conjugated rFVIII structure and function.

Igs play a prominent role in medical conditions. They are essential in the diagnostic field and have considerable importance in diverse therapeutic approaches like replacement therapy and immunomodulatory therapy.[17,77-79] Moreover, because of their useful characteristics such as high specificity, selectivity, good stability, and solubility, they have been adopted to treat cancer, autoimmune disease, and targeted drug delivery (extracellular or intracellular delivery) in rheumatic, infectious, inflammatory, and neurological conditions.[80-83] Human Ig can be used in various modes, for instance, subcutaneous (SCIG), intravenous (IVIG), and intramuscular (IMIG) administration.[84-87] However, all administrations mentioned above have their advantages and disadvantages.[72,78,88,89] Although there are wide Ig therapeutic approaches for clinical use, applying them in clinical trials is not quite as simple as it might seem. Difficulties are due to challenges in Ig purification and preserving them against aggregation, having a cytotoxic effect in some cases, short half-life, and structural and functional modifications.[81,82,90,91] Consequently, since a diverse range of nanostructures have been implemented to optimize the use of Igs, like improving the maintenance and functionality of Ig, protein stabilization, and preventing aggregation, it is intended to gather some reports whereby conjugation and interactions between different Igs and nanostructures were experimented with obtaining a better view of this administration. As we know, any interaction or adsorption might lead to conformational and functional changes in proteins.[81,82,92]

PEGylation

In 1977, the first adopted PEG was in conjugation with bovine serum albumin and its immunoprotective effect was confirmed. Since then, applying PEGylation in therapeutic and pharmaceutical approaches has been boosted. It can be used to block enzymatic degradation, increase the safety of therapeutics, and improve the half-life of biomolecules.[93,94] It has also been implemented to improve the solubility and reduce the mAb fragments' immunogenicity.[94,95] Below, some of these research studies have been reviewed.

PEGylation was applied to improve the stability of antibodies by Roque and coworkers in 2015. The antibody fragment (Fab) was conjugated to 30 kDa PEG and was tested within the aggregating condition which was induced by temperature and mechanical stress. They also considered the structural change of Fab upon its interaction with PEG. The result indicated that PEGylation was an effective method to preserve antibodies in stressed conditions. Although the secondary structure of the protein did not change in the presence of PEG, tertiary structure stability experienced moderate disturbance.[95]

In addition, an empirical research study has been conducted by Reichard and coworkers in 2016 to enhance the circulation time of antibodies in targeted organs. In order to achieve this purpose, a single chain variable fragment (scFv) has been developed with a terminal cysteine (ScFv7F9Cys) which showed higher affinity for conjugation. In another research ScFv7F9Cys was conjugated to three deferent sizes of PEG: PEG5K, PEG20K, and PEG40K. Also, according to analysis via SDS-PAGE, only one PEG was conjugated to each antibody fragment. Although regarding past research studies all three PEGs could increase the half-life of ScFv, in the mentioned research only 20 kDa PEG was used for conjugation due to the highest binding activity to the experimented drug. In conclusion, PEGylation successfully increased the half-life of ScFv7F9Cys. Similar to previous research, any conformational or functional change of immunoglobulin was not reported. The colloidal stability of the solution illustrated the stability of Igs upon conjugation.[93]

In 2017, PEG conjugated to an antibody fragment via a site-selective thiol approach was studied to increase the remaining time within the lungs. In this research, the effect of the two-armed 40 kDa PEG conjugate to a murine anti-IL-17A Fab's monomer on the antibody's local half-life in mouse lungs was measured and compared with that in rats and rabbits. As a result, conjugation dramatically increased the residence time of anti-IL-17A Fab' monomer in all three species' lungs and did not affect Ab's binding activity. However, conjugation markedly raised Ab's inhibitory ability in cell culture. Since no noticeable changes in Ig usability have been observed, we can conclude that no considerable alteration happened in the antibody fragment structure.[96]

Metal NPs

Metal NPs have been applied in various therapeutic and pharmaceutical approaches, such as anti-bacterial agents, drug-delivery vectors, and biosensors. Due to their particular characteristics like the ease of synthesis, chemical stability, and good conductivity, gold NPs and silver NPs have been applied in biochemistry, medicine, and analytical chemistry. Biological implementation of metal NPs due to their large surface area and free surface energy faces a challenge in maintaining nano-systems' colloidal stability. NPs, as meta-stable systems, excessively tend to aggregate upon physical or chemical stresses. NPs tend to decrease their high surface energy via interaction with other molecules to form protein corona shells or even aggregation. In this respect, a wide range of materials including surfactants, polymers, and proteins have been used to boost particles' colloidal stability in various conditions.[97-100]

Magnetic particles (MN) are among the metal NPs with a metallic core and various shells. Polymer shells preserve naked magnetic particles against air and aggregation.[101] Ig conjugation to MNs due to its specificity has been used to improve Ig purification and recovery or cell detection and isolation.[90,102-105] In 2020 a hybrid magnetic NP with an Fe3O4 core coating with a siliceous shell, which was enriched in the polysaccharide κ-carrageenan (HMNPs), has been implemented to achieve the high volume of Ig purification from rabbit serum (Fig. 5). Fortunately, the hybrid achieved its goal and did not alter the secondary structure of immunoglobulin neither upon adsorption on HMNPs nor after the desorption step.[106]

Fig. 5

PAMAM-dox-trastuzumab conjugate, attached to HER-2 receptor.

As for other metallic NPs, in 2015 monodispersed IgG-folinate functionalized silver NPs were obtained as bio-nano composites for biological experiments. IgG was selected for protecting silver NPs from aggregation or macrophage recognition in the body. The results showed that AgNP-IgG-CF (AgNP-IgG-calcium folinate) conjugation was successfully functionalized, and no structural/functional changes have been reported.[99]

Another research on metal nanoparticle-Ab conjugation was published in 2020 in which stable conjugates of antibodies with Au-NPs (GNPs) have been proposed. Mab 2H3C6 is a monoclonal antibody against the protein MPT64 of Mycobacterium tuberculosis, and Mab2H2F6, a monoclonal antibody 2H2F6, against the structural protein of the Sharkey plum virus has been conjugated to GNPs. The results showed that GNPs might aggregate upon interaction with antibodies because of their light chain decay during preparation, storage or conjugation. Although conjugation of GNPs to the fragment antibody which was purified from unstable fragments introduced a stable compound, there is no evidence to support that antibody fragment conjugation with GNPs has negative consequences on Ig stability.[97]

Dendrimers

Dendrimers are a relatively new class of highly branched polymers, which have been firstly introduced in the middle of 1970. Their high monodispersity, 3D poly-branched, core–shell like structure, internal cavities to entrap molecules like drugs, and different functional groups on the surface are the main properties that allow them to be employed in various applications as drug delivery, imaging, antiviral and transfecting agents.[107-111]

PAMAM (polyamidoamine) or PPI (polypropyleneimine) dendrimers are the most prominent dendrimers. Dendrimers are categorized into cationic (e.g., PAMAM-NH2), anionic (e.g., PAMAM-COOH), and neutral (e.g., PAMAM-OH) dendrimers based on their surface functional groups. Surface charge modifications can significantly diminish polymers' toxicity and improve their biocompatibility: the molecule size and number of terminal groups are directly related to dendrimers' generation. Moreover, different protonation patterns will mainly make different structures. PAMAM G4 (4th generation) with a diameter of 45 Å has been demonstrated to have lower cytotoxicity among other larger generations of PAMAM-NH2. So, it is the first candidate as a drug delivery system in comparison with other PAMAM generations.[108]

In empirical studies, dendrimers have been investigated in pharmacy and medicine. The results illustrated their potential for enhancing the solubility of hydrophobic components in drug-delivery systems. Besides, cationic dendrimers can be decent non-viral vehicles for gene delivery in therapeutic applications. However, toxicity is the main factor that should be considered. Cationic dendrimers have more toxic activity among others. Positive surface charges or groups (such as amine groups) can interact with negatively charged lipid membranes and lead to hole formation. To reduce the mentioned toxic effects, modifications of dendrimers like surface coating with biocompatible polymers like PEG, an acetyl group, or saccharide moiety can significantly reduce the toxicity. In PAMAM dendrimers, amine group substitution with hydroxyl groups seems to reduce the toxicity. Also, they can interact via possible hydrogen bond formation, which mainly occurs in biological reactions.[108,109]

Several studies have investigated the effect of PAMAM derivatives on different blood components.

In 2018, a PAMAM based delivery system has been developed, using PAMAM dendrimer linked trastuzumab, delivering doxorubicin (dox) to breast cancer cells in which human epidermal growth receptor 2 (HER-2) had been overexpressed on the cellular surface. Trastuzumab is a humanized monoclonal antibody against HER-2 and provides specificity for conjugated structures. The results showed that PAMAM-dox-trastuzumab conjugates were more effective and had less toxicity than PAMAM-trastuzumab conjugates or free doxorubicin (Fig. 6).[112] In 2018 PAMAM dendrimer-trastuzumab conjugates have been tested with different drugs, docetaxel (doc) or paclitaxel (ptx), against HER-2-positive SKBR-3 and HER-2-negative MCF-7 by the same team. The results showed the high selectivity of PAMAM-doc-trastuzumab and PAMAM-ptx-trastuzumab conjugates for HER-2-positive cells and confirmed the unity of trastuzumab.[113] PAMAM-dendrimer protected the whole conjugate during the process in both types of research, and binding between the dendrimer and trastuzumab did not change the IgG's characteristics.[112,113] In 2016, a PAMAM-dendrimer and humanized anti-HER-2 antibody (herceptin) conjugate have been implemented in a nanohybrid structure, which successfully bound specifically to HER-2 overexpressing cells as a targeted nano-imaging agent. The experiments indicate that the herceptin structure and function remained after covalent binding to dendrimers.[114] As a result, the PAMAM-dendrimer-immunoglobulin conjugate had satisfactory usage in the targeted compound delivery.

Fig. 6

Co-based MOF system and surface functionalization regulation of the system for specific protein enrichment.

As explained before, PLGA has a similar mechanism of action as a coating agent to avoid opsonization and phagocytic attack of macrophages. So, this polymer can be used as a PLGA-PEG dual coating which is capable of enhancing features as well.

Targeted drug delivery in cancer treatment has shown promising future for the next evolution of liposomal drug carriers. Liposomes have been used in different forms such as antibody-targeted PEGylated liposomal drugs that have showed preferable therapeutic effects over their non-targeted correlates. Interacting with antibodies, by covalent or non-covalent bonds, antibody fragments, peptides or small ligands can enhance the selectivity of liposomes for different diseased cells. Antibody-targeted liposomes usually called immunoliposomes (IL) increase specific binding to tumor-associated internalizing antigens on the surface of cancer cells.[115]

In 2011, IgG antibody was conjugated to the surface of hepatitis B surface antigen (HBsAg)-loaded liposomes. This in vitro study was focusing on the targeting potential, localization pattern, and immunogenicity of the complex. This conjugation was formed by a glutaraldehyde coupling method. The positive zeta potential of optimized liposomes has not been altered after antibody coupling onto the surface. The coupling of antibodies on the surface of liposomes reduced the release rate of encapsulated antigens. In fact, antibody coupling led to the steric barrier on the surface of liposomes. According to the results and practical structure of the hybrid, we can conclude that the structure and function of IgG were maintained as well.[116]

Metal–organic frameworks (MOFs)

Metal–organic frameworks (MOFs) are the most recent nanomaterials that are widely employed in research due to their special benefits such as tunable porous and crystalline structures and high surface areas. This nano-scale porous structure is mostly used in separation, catalysis, storage, sensing and gas adsorption, as well as drug delivery. Generally, MOFs are generated by chemical coordination of metal ions and organic linkers to form a hybrid structure. They also have been implemented as a host for biomolecule encapsulation in recent years.[117-120]

In 2019, a hydrophilic MOF-based nanomaterial UiO@GO@PEG was produced from a zirconium-based MOF UiO-66-NH2 coated with a graphene oxide (GO) layer and hydrophilic polyethylene glycol (PEG) to enhance the stability and hydrophilicity of nano-structures. This hybrid was prepared for adsorbing glycoproteins from complex matrices. Three glycoproteins (IgG, γ-Glo and b-Lf) and five non-glycoproteins (α-La, BSA, Mb, Lys and cyt-c) have been used to prove UiO@GO@PEG functionality in protein adsorption under pH 7. The result showed that the adsorption efficiency of the nanostructure is 96.9% for IgG, 75.9% for γ-Glo, 76.8% for b-Lf that were much higher than those of non-glycoproteins which illustrate selective adsorption for glycoproteins. IgG was recovered with 0.5% (m/v) NH4OH solution with 82.6% efficiency and it was also isolated from human serum as a real biological system to investigate adsorption selectivity. No functional or structural change of IgG has been reported.[121]

In 2020, regulation of the surface properties like hydrophilicity and charge characteristics of Co-based MOF nanostructures by operating various organic building units was reported. Co-MOF-OH, a MOF coated with abundant hydroxyl groups, showed selective adsorption and enriched efficiency toward IgG at physiological pH (7.4). The enrichment factor for IgG from the IgG/human serum albumin mixture with a mass ratio of 1 : 50 was obtained up to 97.7. The result indicated that the structure of IgG did not experience any changes during the enrichment process and practicability of Co-MOF-OH in a complex biological solution was confirmed by the selective enrichment of IgG from human serum samples.[122]

The results are listed in Table 2.

Immunoglobulins and nanomaterials

R	Types of NPs	Size (nm)	Electrostatic charge	Effects on structure of Ig	Effects on function of Ig	Ref.
(1) Polymeric NPs: PEG
1	PEG	20 kDa	No charge	No change	Increased half-life	93
2	PEG	10.7 nm/40 kDa	No charge	No change	Increased half-life	96
3	PEG	30 kDa	No charge	Moderate disturbance in tertiary structure	Ig increased hybrid affinity	95
(2) Metal NPs
1	Gold NPs (AuNPs)	24.9 nm	Negative (citrate)	No change	No change	97
2	Silver NPs (AgNPs)	26 nm	Negative (citrate)	No change	No change	99
3	Iron NPs	Core: 48.9 (±5.9)	−25 mV	No change in secondary structure	High purification of Ig adsorption yield: 90%	106
		Shell thickness: 20.7 (±2.3)
4	Iron oxide	Hydrodynamic size: 33 nm	−51 mV	No changes	No changes	105
(3) Polymeric NPs: dendrimer
1	PAMAM dendrimers	PAMAM G4 dendrimer	Positive	No change	No change	112 and 113
2	PAMAM dendrimers	PAMAM G5 dendrimer	459.0 ± 28	No change	No change	114
(4) Polymeric NPs: PLGA
1	PLGA	221–252 nm	No charge	No change	Ig functionality maintained after released	81
2	PLGA	−<10 μm	No charge	No change	Ig functionality maintained after encapsulation	92
(5) Liposome and LNP
1	Liposome	360.23 ± 15.11	+8.23 ± 0.51 mV			116
(6) Metal–organic frameworks (MOFs)
1	MOF (UiO@GO@PEG hybrid)	500 to 600 nm	−29.6 mV	No change	No change	121
2	Co-based MOF	Hybrid system > 10 μ	−40 mV	No change	No change	122

Herein, we have mainly considered immunoglobulin usage in different therapeutic areas: treatment of cancer, as transporters of toxins or radiolabelled isotopes to cancerous cells, and also as anti-autoimmune agents for treatment of autoimmune diseases and neurological disorders, in which Ig has been conjugated to a nanostructure.[106] In this review NPs have been implemented on antibody or antibody fragments upon interaction.

Noticing the bio-conjugations as mentioned earlier, we can see moderate behavior for Igs upon interaction with NPs. Ig has mostly been used due to its higher specificity in drug delivery systems or cell isolation.[104] In conjugation to metal NPs, magnetic NPs had the most common implementation regarding antibodies' most important applications: bio-separation and purification purposes.[90]

Additionally, using other metal NPs, the well-functionalized structure and good colloidal stability were investigated. Encapsulation of metal nanoparticles via bio-compatible materials plays a critical role in both colloidal and Igs' structural and functional strength.[99,123]

MOF nanostructures have different applications in interaction with Igs. Purification and enrichment of immunoglobulin G have been explained before, which demonstrated that noncovalent interaction between Igs and MOFs does not change the protein stability and function of Igs during the enrichment process.[121,122]

In the field of targeted drug delivery, Igs conjugated with dendrimers, particularly PAMAM-dendrimers, were used in various studies. The results illustrated that the stability of antibodies was maintained within conjugation.[112-114]

Likewise, liposomes are mostly used in targeted drug delivery and their successful function for delivering molecules without affecting their stability has been confirmed. Similar to using dendrimers, Igs have been implemented to targeting liposomes.[115,116]

PEGylation has shown promising results in increasing the half-life of antibodies and antibody fragments during therapy in the polymeric nanostructure.[93,96] PEGylation causes prolonged systemic exposure via decreasing the amount of proteolysis and renal elimination. It also increases solubility and reduces immunogenicity.[80] Moreover, it successfully protects the Ig structure in unfavorable conditions.[95]

Conclusion

During the last few decades, bio-system applications in medical and pharmaceutical approaches have been developed. Nowadays, several types of research are conducted based on macromolecules containing antibodies, recombinant proteins, and nucleic-acid-based products after a long time focusing on small molecules as biopharmaceuticals.

According to the innovation aspect of the article, it is worth mentioning that this is the first time that reports from 2010 to 2020, dealing with the nanostructures' effect on plasma protein stability, have been reviewed and collected. To conclude, the results revealed that nano-based systems with PEG derivatives have the best improvement. Also, utilizing PEGylated liposomes, using the PEG-lip technique for coating nanostructures, leads to FIIIV half-life prolongation. This however is the only research that has been reported about PLGA coating.

Among different nanostructures reviewed in this study, including metal NPs (GNPs, AgNPs, and magnetic NPs), dendrimers, PEG, and PLGA, choosing the best nanoparticle for conjugation with diverse Igs depends on the impact of nanomaterials on the stability of the structure and consequently function of Igs indeed. We should select a suitable nanostructure according to the implementation of Ig conjugated to the nanoparticle. Reviewing the last decade's papers, iron oxide magnetic nanoparticles have demonstrated proper function in the separation and isolation of stem cells or other cells and Ig purification from plasma. Liposomes and dendrimers are suitable vehicles for targeted drug delivery. PLGA and PEG have been used for encapsulating Ig and protect it against aggregation, phagocytosis, and reduce toxicity or immunogenicity. These properties make PEG and PLGA suitable for coating other nanostructures and nano-hybrid structures regardless of their application. Moreover, PEG has been considered as a stabilizer for Ig in the stressed situation to enhance Ig's half-life in targeted tissues. Fortunately, no dramatic structural changes and loss of Ig functionality in conjugation with NPs have already been reported. MOFs are the recent applicable nanomaterials that have been frequently used in different studies. However, further investigations should be implemented for safety validation in biomedical applications. In conclusion, the results showed that MOFs are promising hybrid systems that can promote the usage of nanomaterials in the near future.

Conflicts of interest

There are no conflicts to declare.