Antifouling Surface Coatings for the Next Generation of Nanomedicine: Toward in Vivo Immune Evasion

Keywords::

• antifouling
• biomimetic
• immune evasion
• nanoparticles
• PEGylated
• zwitterionic

Nanoparticles extend the possibility of improving therapeutics and diagnostics like no other means. Originally, the goal of first-generation therapeutic nanoparticles was to improve circulatory times and facilitate a reduction in cardiotoxicity for chemotherapeutics. However, since these early attempts, the nanoparticle field has expanded greatly to include a broad range of previously unimaginable therapeutic and imaging modalities. The most iconic examples of this are the COVID-19 vaccines, which collectively have saved the lives of millions of people [1]. The successes observed for the COVID-19 vaccines have since catapulted second-generation nanoparticle-led therapeutic approaches heavily toward vaccines promoting antitumor immunity. This newer approach has the potential to surpass traditional treatment regimens, effectively preventing metastasis and minimum residual disease, through encouraging immune memory toward cancer cells [2]. Second-generation cancer vaccines are extremely promising, as are attempts toward harnessing the effects of trained immunity, but what if the aim, for example, is to drive gene-based therapeutics toward more distal sites such as the brain, without promoting inflammation?

For nanoparticles designed toward immune evasion, there remain fundamental design faults that have, in general, stunted the far-reaching potential of a range of possibly game-changing nanoparticle-led therapeutics and diagnostics [3,4]. Negative effects are particularly prevalent when attempting intravenous delivery. Often, intravenously (IV) administered nanomedicines show exceptional promise during evaluations in preclinical mouse models; however, fail to reach phase III human trials because they prove to be immunogenic in humans [5]. The overarching problems that befall these trials are routinely the result of the innate immune system and its vigilant response to the presence of foreign material in the blood.

The immune system is an elaborate maze of complex interactions and synchronized crosstalk between the innate first responders and the adaptive effector cells. Collectively, the innate immune system acts indiscriminately to try to eliminate a threat as quickly as possible. While a timely response is invaluable in controlling pathogenic invasion, for IV-administered nanoparticles it presents as one of the most significant hurdles to overcome. Complement system activation is justifiably the most problematic occurrence because activation results in a barrage of proinflammatory cytokines being released into the blood which communicates a signal for overactivation of all immune cells. However, the most damaging effect of complement is arguably the programmed proteolytic cleavage of C3 and C5 proteins that produce anaphylatoxins (C3a, C4a and C5a), as well as complement-activated basophils which release large doses of histamine into the blood. In reality, complement activation-related pseudo allergy (CARPA) has often been reported for various nanoparticle formulas and infusion hypersensitivity reactions in general remain one of the most common reasons for halting trials.

Surface fouling and the subsequent enveloping of a protein corona is recognized as the main reason why IV-delivered nanoparticles activate the innate immune system. Spontaneous and non-specific protein absorption occurs because of the various surface properties that nanoparticles possess, for example, the charge, hydrophobicity and unique functionalization properties [6]. Poly(ethylene glycol) (PEG) remains the gold standard antifouling approach for nanoparticles in the clinic. While PEG is praised for its stability-enhancing attributes, low cytotoxicity and superior antiopsonization properties, PEG is not the perfect biological material. The most well-established and commonly detailed flaw of PEGylated nanoparticles is the propensity for the development of anti-PEG antibodies and the potential to elicit potent B cell activation. More recently, PEG has also been identified as a trigger for hypersensitivity reactions through complement anaphylatoxins and cytokines released from activated basophils [7]. Within a real-world scenario, this effect is best evidenced by the wave of statistics emerging from the COVID-19 vaccination rollout. For example, of the ∼2 billion Moderna and Pfizer COVID-19 vaccines administered up until 2022, ∼123 people per million suffered vaccine-induced anaphylaxis [8].

In attempts to reduce immune recognition, efforts have been placed toward modifying basic PEG architecture. Switching linear PEG-lipids to branched for instance is proposed to ensure better coverage and resist C3 protein absorption [6,9]. Additionally, substituting the long ethylene glycol sequence of PEG for short oligoethylene glycol oligomers is potentially appealing as a way of reducing overall immunoreactivity. Chemical additions such as hyaluronic acid, sulfoxide and, in particular, fluorine show excellent antifouling potential, as does PEG substitution with polyglycerol [10,11]. While the antifouling nature of PEG is controlled through the hydration layer formed by hydrogen bonding, zwitterionic polymers (ZPs) display antifouling properties through a hydration layer formed through electrostatic interactions. ZPs contain opposing charge groups within the repeating units, making them neutrally charged, which also helps prevent protein adsorption and ensures good stability in biologically relevant solutions. Polycarboxybetaine and polysulfobetaine are both promising examples of ZPs being heavily explored for their future potential to circumnavigate immune responses [12].

Drawing inspiration from nature, biomimetics is a way of imitating living organisms to help aid in the development of more biologically tuned materials. ZPs inspired by phosphatidylserine (PS) present an interesting approach in this respect. PS is a naturally occurring lipid containing an immune-responsive phosphoserine head group capable of initiating immune tolerance toward immunogenic proteins. Cell membrane cloaking is another exciting alternative biomimetic approach aimed eloquently toward immune evasion. Red blood cells (RBCs) [13], platelets [14], macrophages [15], mesenchymal stem cells and cancer cells [16] have all been explored as a way of reducing immune stimulatory responses because of the potential to utilize naturally occurring receptors on the cell membranes. For nanoparticles dressed in RBCs, platelets and cancer cells, CD47 on the cell membranes presents as an ingenious way of saying ‘Don’t eat me!’ to monocytes and neutrophils in the blood, thereby increasing circulatory times and reducing the toxic toll on the liver and spleen. Meanwhile, alternative homing receptors help drive nanoparticles toward the intended sites.

Biomimetics approaches also extend to the attachment of endogenous proteins which may help improve immune evasion and simultaneously address challenges relating to effective passage across physiological barriers – most notably the blood–brain barrier. Transferrin is particularly appealing in this respect due to the abundance of iron transporter receptors along the length of the blood–brain barrier and on brain cancer cells. Additional methods being aimed toward better brain targeting include the grafting of neurotrophic endogenous peptides, viral proteins, phage receptors and venom neurotoxins – collectively determined non-toxic to neural cells [17]. It is well-established that the protein corona effect defines the biological fingerprint of all nanoparticle-led therapeutics and diagnostics - controlling the actual, rather than intended outcome. Finding a way of advantageously manipulating protein corona formation presents an exceptional opportunity to effectively reengineer the nano-surface for targeted drug delivery [18]. Protein precoating and the artificial recreation of coronas ex vivo have already been extensively employed in attempts to achieve a stealth effect and reduce immune clearance [19]. Importantly, this is an up-and-coming area of brain disease research, with studies such as those reported by Zhang et al., eloquently demonstrating the future potential to better target the brain without eliciting unwanted innate immune responses [20].

Over the years, more studies have begun to look at small numbers of proinflammatory cytokines as markers for evading innate responses, as well as macrophage behavior – answering the question of whether nanoparticles are being phagocytosed. While these experiments may indeed be an important predictor of in vivo success in regard to increased circulatory times, they fail dismally in painting the whole picture of the innate immune response. In general, fewer studies describe the impact of neutrophils or properly take into account the harmful effects of complement when deciding the immune evasion potential of preclinical nanoparticle formulations. Moving forward, the importance of understanding the complexity of nanoparticle-innate immune interactions should be fully realized. In this regard, proteomics approaches may be best placed to study how complement proteins, neutrophils and various other proinflammatory precursors interact with the membrane coatings or many building blocks that make up the third-generation synthetic nanoparticle materials. All in all, this is an extremely exciting era for nanoparticle therapeutics and diagnostics – driven forward by inspired ideas and new methodology that guarantees nanotechnology remains at the forefront of life-changing medical breakthroughs well after the eventual fall of COVID-19.

Writing disclosure

No writing assistance was utilized in the production of this manuscript.

Financial disclosure

The authors have no financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Competing interests disclosure

The authors have no competing interests or relevant affiliations with any organization or entity with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.