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Author: Evan Scott
As understanding improves about how the immune system functions, engineers can begin employing principles of rational design to modulate immune responses for therapeutic applications. Key tools in this frontier of immunoengineering have emerged from biomaterials and nanoscale science, such as theranostics: the combined delivery of therapeutic and diagnostic agents. By providing a means of tracking and quantifying cells that are targeted and modulated during vaccination and immunotherapy, -theranostics makes it possible to approach the immune system less as a mysterious “black box” and more as an interlinked network of cells and signaling molecules that can be mapped for improved reproducibility and understanding. Immune -theranostics holds promise for realizing technologies that harness the full potential of immunotherapy in the treatment of a wide range of inflammatory disorders.
The immune system is a dynamic and highly responsive network of bio-active molecules, cells, and tissues. It must continuously maintain the homeo-stasis of its host body within a strict set of physicochemical boundaries while being ever ready to address an equally complex and evolving repertoire of invading pathogens and heterogeneous cancers. Adding to this complexity is the uniqueness of each patient’s immune system—women, men, children, neonates, the elderly, and the diabetic can each have distinct immune responses to the same stimuli. Furthermore, prior exposure to particular inflammatory molecules and conditions, such as certain foods or regional infections, can have significant impacts, even preventing allergic reactions or making some vaccines ineffective in specific parts of the world.
While the protective abilities of the immune system have long been tapped for the generation of vaccines, its potential to be directed toward the treatment of cancer and inflammatory disorders has been explored only relatively recently in the form of immunotherapy. But what methods are available to controllably and reproducibly modulate this system, which varies from person to person and based on sex, age, and disease state? To address this need, immunoengineers apply principles of rational design, biomaterials science, nanoscale science, systems analysis, and numerous other engineering disciplines to better assess, control, and customize immune responses for safe and reproducible therapeutic applications.
A New Frontier for Engineering: Rational Immunomodulation
Immunoengineering is a relatively new field, but its concepts have always been a core component of biomaterials science. Materials development for biological implants and in vivo controlled delivery have historically focused on minimizing inflammation. Biomaterials are therefore usually optimized to inhibit the activation of inflammatory immune cell populations in tissues and biological fluids, to decrease toxicity, increase the therapeutic efficacy of delivered agents, and extend the lifetime of implanted devices.
Now, instead of a focus on preventing inflammation, advances in the development of nanoscale bio-materials (nanobiomaterials, NBMs) permit the design of mate-rials to directly elicit therapeutically beneficial -responses from the immune system (Allen et al. 2016; Scott et al. 2017). The immune system interacts with NBMs based on a never-ending battle with viruses. Nanoscale lipid vesicles released by immune cells are essential components of cell-cell communication and signaling, and biomimicry of these nanostructures -presents a pathway for probing, modulating, and monitoring immune responses.
With these developments theranostics—the combined delivery of therapeutic and diagnostic agents—has emerged as a vital tool for identifying and tracking immune cells that are modulated by delivered drugs and immunostimulants (Allen et al. 2018; Karabin et al. 2018). “Immunotheranostic” strategies are significantly enhancing the ability of engineers to reproducibly generate immune responses by monitoring which components are modulated at the organ and cellular level during immunotherapy and vaccination (Du et al. 2017, 2018).
Previous Methods of Vaccine Development: Treating the Immune System as a Black Box
Vaccination is fundamentally the process of training the immune system to recognize and eliminate pathogens either prophylactically or therapeutically and can thus be considered one of the first forms of immunotherapy. Although it may seem obvious that immunology should be a key component of vaccine design, this has not always been the case.
Rational vaccine design requires an understanding of the immune system that has not yet been achieved, but the urgency to aid the sick and prevent the spread of infection has presented no alternative other than the use of trial-and-error methods. As a result, most immunization strategies were developed by treating the immune system as a black box. Antigens (molecular components of pathogens) and adjuvants (“danger signals” that stimulate inflammatory cells) are randomly combined in formulations that serve as the input into the system. The output from the black box is the (hopefully) lasting and protective immune response. With little understanding of the mechanism by which antigens and adjuvants achieve this output, formulations are selected that generate the safest and most effective prevention or removal of infection, with lasting immunological memory to respond quickly to future pathogen exposure.
But complex cell-cell interactions occur and dozens of signaling molecules known as cytokines are released by inflammatory cells during an immunization. It is critical to know which cells contribute to these responses and whether the same cells can be reproducibly stimulated across different human populations. Importantly, different immune cells express different combinations of cytokines, often in amounts proportional to the extent of their exposure to the adjuvant, and this network of activated inflammatory cells and released cytokines forms an emergent system that can be tailored for specific therapeutic applications.
By employing targeted NBMs to control and monitor which immune cells are modulated during vaccination, theranostics provides a means to explore this black box to better correlate the input vaccine or immuno-modulatory formulation with the output immune response.
Engineering Nanobiomaterials for Targeted Immunomodulation
NBMs are key tools in immunoengineering and have attracted much attention for their ability to deliver therapeutics and imaging agents to specific cells and tissues (Allen et al. 2016; Scott et al. 2017). This versatility has demonstrated improved efficacy and deployment of vaccine formulations by providing triggered or bioresponsive mechanisms for controlled release, transporting combinations of bioactives with diverse solubility, and allowing control over reproducibility, speed, and cost of production (Scott et al. 2017).
Among the range of available NBMs, self-assembled NBMs composed of synthetic amphiphilic polymers are especially advantageous for vaccination and immunotherapy because of their versatility in chemistry and structure (Allen et al. 2016). These traits allow better mimicry of viruses, which possess physicochemical and structural characteristics that dictate their interactions and processing by critical immune cells known as professional antigen presenting cells (APCs).
Professional APCs—which include dendritic cells, macrophages, and B cells—are the most frequent targets of immunomodulatory NBMs because of their potency for cytokine release and T cell activation. T cells are the effector cells of the immune system that can directly kill virus-infected or cancerous cells (cytotoxic T cells) as well as direct or enhance functions of other immune cells (helper T cells).
Using a military hierarchy as an analogy, T cells can be considered both soldiers and noncombat support troops while APCs are the generals that direct their action. NBMs function as a direct line of communication to the generals by alerting them of imminent danger (adjuvant) and identifying targets (antigen) for elimination. After internalization by APCs, NBMs are degraded in intracellular compartments that contain a variety of enzymes and redox mediators (Owens and Peppas 2006), allowing transported payloads to modulate APC function for the activation of T cells.
Theranostics as a Tool to Improve Vaccine Design and Reproducibility
Continued progress in theranostics will allow early detection of disease, prevent unintended side effects of drugs, decrease the frequency and amount of administered drugs, and allow quantitative assessment of the accuracy of drug delivery in individual patients. Immuno-theranostic nanomedicine may thus revolutionize treatments for numerous inflammatory disorders, including cancer and heart disease, by providing power-ful new approaches not only for therapeutic delivery and diagnosis but also for personalized medicine and clinically relevant assessment of therapeutic efficacy.
Using viruses, bacteria, and other pathogens as inspiration, biomimetic NBMs can be engineered with physiochemical properties selected to stimulate or suppress specific APC populations while marking them for detection and quantification via multiple diagnostic modalities. As an example, theranostic delivery of a drug regimen to reduce vascular inflammation in patients with cardiovascular disease could allow a clinician to monitor the patient’s progress during treatment. Since not all patients will have the same response to anti-inflammatory drugs, the clinician could adjust the treatment as necessary by monitoring the levels of critical inflammatory cells in the patient’s arteries. NBMs targeting dendritic cells may serve such a function, as the level of these APCs in vascular lesions directly correlates with the risk of rupture and vascular occlusion (Bobryshev 2010). There is currently no noninvasive method to detect such unstable lesions in patients, many of whom could suffer heart attack or stroke without warning.
Engineering NBMs for Use with Diagnostic Imaging
NBMs can be engineered to be amenable to a variety of diagnostic methods depending on the specific need. Commonly employed imaging modalities include single-photon emission computed tomography (SPECT/CT), positron emission tomography (PET), magnetic resonance imaging (MRI), and fluorescence/-luminescence spectroscopy. MRI stands out for safety during repeated use, in contrast to techniques requiring high doses of radiation like SPECT/CT and PET. PET has superior spatial resolution (4–5 mm) to SPECT (10–15 mm) and high sensitivity that can detect picomolar tracer concentrations. Although lower resolution than PET, MRI enhanced with contrast agents (e.g., gadolinium-conjugated NBMs and superparamagnetic iron oxide nanoparticles) can be used to characterize various features of targeted tissues.
While fluorescence is impractical for clinical applications because of poor tissue penetration, it enables unprecedented quantitative analysis of cellular targeting in animal models, where organs and cells can be extracted for analysis by flow cytometry. This immunotheranostic strategy significantly enhances the ability to reproducibly elicit immune responses by monitoring which components are modulated at the cellular level during the development of vaccines and immuno-therapies (Dowling et al. 2017).
Conclusions and Future Directions
Theranostic NBMs hold great promise for diagnostic imaging and controlled delivery of therapeutics during immunotherapy, providing a much-needed method for mapping and understanding the complex network of inflammatory cells that contribute to elicited immune responses.
The immediate future directions of theranostics will likely focus on two critical issues. First, APCs will nonspecifically remove NBMs from circulation regardless of surface-conjugated targeting moieties like -antibodies and peptides, making selective APC targeting difficult to achieve. Avoiding uptake by off-target APC populations will require more advanced engineering of the nano/biointerface, such as precisely controlling the surface density and affinity of multiple targeting moieties (Nel et al. 2009), incorporating inhibitory signals like the CD47 (“don’t eat me”) peptide (Rodriguez et al. 2013), and optimizing NBM structure and size (Yi et al. 2016).
Second, the scalable self-assembly of monodisperse NBMs that mimic the complex nanoarchitectures of viruses remains a challenge. Current methods usually involve impractically complex polymers, low yield of the desired nanostructure, and difficulty with therapeutic loading, particularly dual loading of hydrophobic imaging agents and structurally sensitive water-soluble biologics. Recent advances in the commercially scalable technique of flash nanoprecipitation have demonstrated the scalable assembly of complex self-assembled NBMs from poly(ethylene glycol)-bl-poly(propylene sulfide) amphiphilic block copolymers (Allen et al. 2017). This method of impinging organic and aqueous phases in confined impingement jet mixers achieves highly reproducible and customizable nanoprecipitation conditions for the fabrication of polymersomes and bicontinuous nanospheres (Allen et al. 2018; Bobbala et al. 2018), which are unique NBMs capable of transporting lipophilic and water-soluble payloads simultaneously.
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Yi S, Allen SD, Liu YG, Ouyang BZ, Li X, Augsornworawat P, Thorp EB, Scott EA. 2016. Tailoring nanostructure morphology for enhanced targeting of dendritic cells in atherosclerosis. ACS Nano 10:11290–11303.
 NBMS are broadly defined as any biomaterial with at least one external dimension that is less than 1,000 nm.