Biodegradable Polymers for Application as Robust Immunomodulatory Biomaterial Carrier Systems.

Chitosan, a natural biopolymer derived from the chitin in shellfish, is considered more environmentally friendly than synthetic alternatives due to its renewable and sustainable origin. When formed into microparticles through standard techniques, chitosan exhibits varying degradation times depending on its chemical modifications and environmental conditions.[ 119 ] For instance, unmodified chitosan can degrade within a few weeks to several months, while cross-linked chitosan may take significantly longer, potentially up to several years.[ 120 ] Chemical modifications, such as carboxylation, alkylation, and quaternization, enhance its solubility and biological activity.[ 121 ] These modifications allow chitosan to be tailored for specific uses, such as immunomodulatory cancer or infectious disease vaccines, where its degradation rate can be controlled to optimize efficacy. Chitosan’s properties as a mucoadhesive polymer make it particularly valuable in the formulation of mucosal immunomodulatory formulations. Its ability to form hydrogen and covalent bonds with mucosal surfaces enhances the retention time of chitosan at mucosal membranes, allowing for a longer sustained release of encapsulated cargo at that site. Additionally, chitosan exhibits immunomodulatory properties by inducing the release of a wide range of cytokines, chemokines, and growth factors from innate immune cells.[ 119 ] This activity can be harnessed to develop therapies that modulate immune responses, making chitosan a promising candidate for immunomodulatory applications. Overall, the unique combination of biodegradability, chemical versatility, mucoadhesive strength, and immunomodulatory effects makes chitosan a powerful immunomodulatory biomaterial Chitosan particles have been used to deliver a wide range of immunomodulatory therapies including checkpoint antibodies for cancer therapy ( Figure 1 ).[ 122 , 123 ] The tumor microenvironment is a significantly tolerogenic one wherein multiple agents are at work to suppress immune surveillance and clearance of the tumor. One pathway that has been identified as a large contributor to this environment is the program cell death 1 (PD1) and PD ligand 1 (PDL1) checkpoint cascade ( Figure 7 ). Similar to the PD1 pathway, the CTLA-4 pathway is also considered a checkpoint mechanism, which can block co-stimulation during antigen-presentation. Therapeutic antibodies are clinically available to block PD1, PDL1, and CTLA-4 (e.g. Keytruda) that can be given IV to patients to reduce these checkpoint responses. Checkpoint inhibition works best at the local tumor site and in immunologically ‘hot’ tumors which have a high amount of T cells, tumor antigens, and antigen-presenting cells present.[ 124 ] Two of these ‘hot’ tumors are melanoma and lung cancer. Song et al. used chitosan nanoparticles to deliver both immune checkpoint peptides (anti-PD-1) and ICD inducing chemotherapeutic doxorubicin ( Figure 1 ) for the treatment of lung cancer in a mouse model.[ 125 ] The inclusion of ICD can help generate antigens within the tumor for presentation to T cells, however, delivery within the cancer is preferred to limit off target effects. They formulated these agents into glycol chitosan nanoparticles that were approximately 250nm in size. An anti-PDL1 antibody was functionalized to the polymer at approximately 5-10 weight percent using click chemistry and modifications to perform the conjugation in water, which is an efficient and environmentally friendly synthesis method. In both a subcutaneous and metastasis lung cancer model with CT26 cells, survival time was significantly increased and tumor burden lessened with co-therapy chitosan particles, in comparison to doxorubicin particles alone, soluble doxorubicin, and PBS controls. In vivo toxicity studies illustrated that the chitosan nanoparticles carrying doxorubicin and anti-PDL1 antibodies were significantly less toxic than doxorubicin alone. When evaluating in vitro the effects of the doxorubicin and checkpoint-loaded chitosan nanoparticles, the greatest amount of IFNγ, and DC and CD8 + T cell activation was observed with the co-therapy, compared to the individual therapies. Their studies showed that in CT26 cells, the co-treatment chitosan nanoparticles induced damage-associated molecular patterns (DAMPs) at an increased rate as measured by calreticulin-positive cells. Overall, the incorporation of both agents into biodegradable chitosan nanoparticles significantly modulated the tumor microenvironment to help clear the tumor. Alginate is a natural polysaccharide polymer derived from brown seaweed with broad biomaterial uses because of its biocompatibility and biodegradability.[ 126 ] Micro and nanoparticles of alginate can be formed through ionic gelling, as well as standard methods like emulsion and spray drying. In nanoparticle form, alginate’s degradation time can vary significantly based on factors such as the degree of oxidation and the environmental conditions. For instance, partially oxidized alginate particles can degrade within a few days to weeks, while more stable forms may take several months.[ 127 ] This variability in degradation time allows for tailored applications in drug delivery and tissue engineering, where controlled release and scaffold stability are crucial. One of the most notable features of alginate is its ability to form hydrogels ( Figure 8 ). These hydrogels are most often formed through ionic cross-linking with divalent cations like calcium.[ 128 ] This hydrogel nature makes alginate an excellent candidate for delivery of immunostimulatory proteins like cytokines ( Figure 8 ). Additionally, alginate exhibits immunomodulatory properties, which can be beneficial in treating inflammation and autoimmune diseases. It can modulate immune responses by interacting with immune cells and influencing cytokine production, going beyond simple drug release to manipulate immune cell behavior through tissue engineering accroaches. One immunomodulatory use that alginate hydrogels have been used extensively for is the transplant of cells, such as islet and stem cells.[ 126 , 129 ] Alginate encapsulation of cells can be used to help dampen immunomodulatory responses to foreign cells by the patient receiving the transplant. In 1949, Burnet and Fenner introduced the Self/Non-Self model of the immune system, which explained how the immune system distinguishes between the body’s own cells and foreign entities, triggering immune responses against the latter.[ 130 ] This model clarified how the immune system effectively responds to foreign cells and proteins, as they are deemed non-self. In 1989, Janeway expanded this concept with the Infectious Non-Self model , highlighting the role of the innate immune system and introducing Pathogen Associated Molecular Patterns (PAMPs). He proposed that receptors like TLRs recognize these patterns, initiating innate immune responses. In 1994, Dr. Polly Matzinger introduced the Danger model , suggesting the immune system responds to perceived dangers rather than non-self and can become activated to both foreign and self-molecules called Danger Associate Molecular Patterns (DAMPs).[ 131 ] By encapsulating foreign cells in an alginate hydrogel, the immune system is unable to easily access its PAMPs and DAMPs and allows the cell to work as a drug depot to modulate response or work therapeutically. As an example of using alginate hydrogels to evade the immune response Ghanta et al.[ 132 ] encapsulated mesenchymal stem cells (MSCs) ( Figure 1 ) for treatment of acute myocardial infarction (MI). Spherical alginate particles were made that encapsulated approximately 40,000 MSCs per particle using a co-axial needle with alginate and barium chloride. These core-shell structures helped to protect the cells inside the core of the particle. Cells remained viable within the capsule for at least 14 days and released paracrine factors during that time, such as the angiogenic protein vascular endothelial growth factor (VEGF). In an ischemic rat model, MSC alginate hydrogels were administered and monitored through an echocardiogram. The MSC alginate hydrogels reduced Left ventricular ejection fraction (a measurement of MI damage) significantly compared to MSC injection alone. All treatments were given intracardial in the left ventricle myocardium. Further, treatment with MSC loaded alginate particles reduced fibrosis formation, which is a measure of permanent MI damage. Overall, increased treatment efficacy was attributed to the increased survival of MSCs in the alginate hydrogel in vivo . Alginate carriers’ hydrogel nature makes it highly compatible and efficient in delivering immunostimulatory cell therapies as well as biologics. Hyaluronic acid (HA) is a natural polysaccharide polymer that is found in mammalian connective tissues and can be extracted from animal tissues or produced more sustainably by fermenting bacteria [ 133 ]. It is extensively used for dermal fillers [ 134 ]. HA has a variety of functional groups, which make it highly modifiable to tune properties of interest, and its anionic nature allows it to crosslink cationic polymers such as chitosan, introducing unique properties for delivery and biocompatibility. Like alginate, HA is highly hydrophilic, which makes it optimal for fabrication of hydrogel scaffolds and hydrogel-like particles. Electrospinning and chemical crosslinking are common methods for hydrogel and scaffold formation. HA is a component of the mammalian ECM, so it unsurprisingly has highly biocompatible and ECM-like properties when administered as a hydrogel. As previously mentioned, HA can have immunosuppressive properties, which can be desired in wound repair and tissue regeneration applications. HA hydrogels display soft, ‘ECM-like’ properties and are a ligand for CD44 receptor, which is expressed more extensively on M2-like macrophages than M1-like macrophages.[ 135 ] Consequently, HA-based hydrogels can recruit and promote M2-like macrophage polarization, reducing inflammation and increasing tissue repair. Kim et al. showed the mechanism of HA macrophage polarization using a THP-1 monocyte cell line.[ 136 ] Using a HA-collagen hydrogel formulation, they saw increased anti-inflammatory cytokine (IL-10) and decreased inflammatory cytokine (TNFα, IL-6) production in macrophages compared to a collagen hydrogel control – indicative of M1-like macrophage skewing. Similarly, HA’s affinity for cell adhesion ligand CD44 can be hijacked to preferentially attract M2-like macrophages in cancer to enhance delivery to these tumor-associated macrophages. Fernandez-Marino et al. employed this strategy to deliver hydrogel-like nanocapsules to M2-like tumor associated macrophages (TAMs) [ 137 ]. Using an orthotopic immunocompetent fibrosarcoma mouse model, fluorophore tagged HA nanocapsules were shown to significantly accumulated in tumors, as shown by IVIS and flow cytometry. Furthermore, in vitro M2-like macrophages experienced 3.1-fold greater accumulation of the HA particles than M1-like macrophages. Clearly, polymeric HA has biomaterial specific advantages in formulating cancer and tissue repair therapeutics.

These aforementioned examples provide an example of the breadth of which degradable polymers have been used to deliver cutting-edge immunotherapies. Still, judicious selection of biodegradable polymers is paramount to immunotherapy carrier system design for the payload and disease of interest. For example, hydrophobic polymers like PLGA or Ace-DEX may be preferred in the context of cancer or infectious disease particle vaccines because of their biocompatibility, tunable degradation, and ability to enhance MHCI and MHCII antigen-presentation. However, in the case of a nucleic acid cargo for a particle vaccine, materials like PBAEs or poly lysines may provide a better delivery platform due to electrostatic interactions from their cationic nature. As previously discussed, in addition to a polymer’s intrinsic immunomodulatory properties, particle size is another important factor in immunotherapy carrier design. The physical and chemical properties of a polymer can influence the constructs that it can form, as well as their size profile and distribution. Particularly, each polymer and its synthetic process will have different compatibility with fabrication methods as well as the homogeneity between polymer strand lengths. For instance, naturally derived polymers like alginate may have more heterogeneous polymer strand lengths than a synthetic PLGA, so the size distribution of nanoparticles could be broader. Furthermore, natural polymers like alginate are less soluble in organic solvents,[ 138 ] so fabrication processes like microfluidics or nanoprecipitation may not be viable options. Alternatively, spray drying microparticles for an infectious disease subunit vaccine may be desirable because of the scalability for large scale production.[ 139 ] However, certain proteins and peptides can be sensitive to the elevated heat exposure in the spray drying process and solvents, so an alternative particle formulation strategy is needed in those cases.[ 140 ] Fabrication methods and polymer molecular weight consistency can be critical factors to consider for particle size and morphology, and thereby immune cell trafficking and biodistribution. When using degradable polymers in preclinical applications, sustainability considerations should be prioritized. Natural versus synthetic-derived materials for polymer and immunomodulatory use can influence environmental outcomes. QS-21, has illustrated the environmental effects of limited natural products versus synthetic. QS-21 is a saponin adjuvant derived from the Chillian soapbark tree ( Quillaja saponaria ) [ 13 ]. It, or similar extracts from the tree, have been used in immune stimulating complexes (ISCOMs) in veterinary vaccines (e.g. Equilis Prequenza) but also recently in human FDA approved vaccines (i.e. Shingrix, Norvavax’s COVID vaccine, and preclinically for Mosquirix). However, QS-21 is an extremely complex molecule, and its isolation results in the destruction of the soap bark tree, which is limited in supply.[ 13 ] Access to the tree for isolation of the natural product has been a significant supply chain issue for QS-21 and only very recently, its synthesis has been reported in bioengineered yeast, however full evaluation of the product in humans has yet to be explored.[ 141 ] The same issue with the identification of the natural product QS-21 could also result in limitations around natural polymers used for immunomodulatory use, especially in consideration of the impact of climate change. Natural, semi-synthetic, and synthetic polymers each play distinct roles in sustainability in biomedical applications. Natural polymers like alginate and chitosan are derived from renewable sources such as seaweed and crustacean shells, respectively. These polymers are biodegradable and biocompatible, making them environmentally friendly options for immunomodulatory systems. Semi-synthetic polymers, such as acetalated dextran, are chemically modified natural polymers that combine the benefits of natural polymers with enhanced properties like increased stability and controlled degradation rates. Synthetic polymers like PLGA and PBAEs are engineered to have specific properties, such as precise degradation rates and mechanical strength, which are crucial for applications like controlled drug release and regenerative medicine. The environmental impact of using synthetic versus natural polymers is significant. If robustly available, natural polymers are generally more sustainable due to their biodegradability and lower environmental footprint. For instance, alginate and chitosan degrade naturally without leaving harmful residues, whereas synthetic polymers like PLGA and PBAE, although biodegradable, may degrade into acidic by-products. When synthesizing biodegradable polymers and fabricating carrier systems, the sustainability of the chemicals used is a critical consideration. Solvents and reagents used in the synthesis and formulation (e.g. emulsion, coacervation) of synthetic polymers can be hazardous and non-renewable, posing environmental and health risks. In contrast, the preparation of nanoparticles using natural polymers often employs water-based processes and benign solvents, which are more sustainable. For example, the use of alginate and chitosan in nanoparticle synthesis typically involves mild conditions and eco-friendly solvents, reducing the overall environmental impact. In comparison, synthetic formulations may involve more hazardous chemicals like dichloromethane. Therefore, while synthetic polymers offer tailored properties for specific applications, the use of natural and semi-synthetic polymers presents a more sustainable approach, particularly in the context of environmental impact and the sustainability of the chemicals involved in their preparation.

The exploration of immunomodulatory polymers, initiated with the use of poly(methyl methacrylate) (PMMA) in 1976 for influenza vaccines has significantly evolved, addressing numerous challenges and expanding applications.[ 142 ] Early limitations, such as the non-degradability and potential toxicity of materials like PMMA and alum, underscored the need for biodegradable alternatives. These alternatives are crucial for ensuring biocompatibility and effective clearance from the body, thereby minimizing adverse effects such as frustrated phagocytosis and foreign body response. Furthermore, the use of complex biologics, cell therapies, and gene therapies in immunological indications has rapidly developed. Biodegradable polymers like those discussed herein provide platforms adept to novel therapeutics and optimize their safety and efficacy. The development of biodegradable polymers, including synthetic, semi-synthetic, and natural materials, has significantly advanced preclinical biomedical applications. Polymers like PLGA, PBAEs, Ace-DEX, chitosan, and alginate have demonstrated versatility and efficacy in forming nano- and microparticles for immunomodulation, offering controlled degradation rates, biocompatibility, and the ability to encapsulate and release therapeutic agents in a controlled manner. Among these, PLGA stands out due to its extensive use and FDA approval for various therapeutic applications, particularly in cancer vaccines. PBAEs are another class of polymers extensively used for immunomodulation, offering tunable degradation times. Their cationic nature allows them to complex with various nucleic acids, enhancing delivery efficiency and improving antigen presentation to MHC I MHCI for CD8 + T cell activation. Ace-DEX is notable for its pH responsiveness and biodegradability, making it ideal for targeted drug delivery and vaccine applications. Chitosan, with its mucoadhesive properties and ability to induce cytokine release, is valuable for mucosal immunomodulatory formulations and cancer therapies. Alginate displays a high amount of biocompatible in hydrogel form, which can facilitate delivery of immunomodulatory cell therapies. In conclusion, the advancements in immunomodulatory polymers have paved the way for innovative and effective preclinical and clinical delivery applications. Polymer choice can dictate many of the physicochemical properties, shape, and therapeutic efficacy. However, the shift towards biodegradable materials addresses the limitations of early non-degradable polymers, ensuring safer and more efficient therapeutic interventions. Additionally, sustainability considerations should be prioritized, with natural polymers generally being more sustainable due to their biodegradability and lower environmental footprint. Therefore, while synthetic polymers offer tailored properties for specific applications, the use of natural and semi-synthetic polymers presents a more sustainable approach, particularly in the context of environmental impact and the sustainability of the chemicals involved in their preparation.

In 1796, Edward Jenner hypothesized that exposure to cowpox would prevent future infection from smallpox [ 1 ]. By inoculating a young boy with pus from a cowpox lesion, the boy experienced no symptoms when inoculated with pus from a smallpox lesion later. This protective immunity from exposure to vaccinia virus antigen was hence called the first vaccine. In the 200+ years since, a vast array of infectious disease vaccines have been developed, and many diseases have been largely controlled or even eradicated, as in the case of smallpox[ 2 ]. As vaccine technologies have developed, protein subunit vaccines have provided a safer and more tailored therapeutic for vaccination than traditional live-attenuated or killed microbial vaccine methods, and the advantages of this approach were highlighted by the clinical success of the COVID-19 vaccines using mRNA lipid nanoparticle technology[ 3 , 4 ]. Despite the benefits of protein subunit and mRNA vaccines, they often require adjuvants to mount a robust immune response and face delivery and formulation challenges [ 5 ]. Similarly, a growing field of molecules and cell therapies has expanded the field of immunotherapy across new disease areas, but challenges often remain in their clinical translation. In cancer therapeutics, immunotherapy has changed the therapeutic paradigm, offering robust and powerful modalities such as chimeric antigen receptor (CAR) T cells, checkpoint inhibitor monoclonal antibodies (mAbs), and autologous or neoantigen vaccines to specifically target cancer cells and prevent off-target cell toxicity that is unachievable with chemotherapy or radiotherapy[ 6 ]. Many other immune system-related indications have benefited from such novel approaches including tolerogenic antigen-specific immunotherapies for autoimmunity and allergies as well as stem cell delivery for tissue repair [ 7 , 8 ]. Unfortunately, many of these innovative biologics and cell therapies have stalled in clinical translation due to issues such as stability, cell viability, manufacturing, and tissue specificity. Therefore, polymeric materials are being widely studied to tune the delivery and immunomodulatory properties of these immunotherapeutic drugs[ 9 ]. In 1976, one of the first reports on immunomodulatory polymers introduced poly(methyl methacrylate) (PMMA) as a vaccine platform. This polymer was utilized to absorb an inactivated virus and form an influenza vaccine ( Figure 1 ).[ 10 ] Despite the advanced nature of this concept at the time, the use of non-degradable biomaterials posed significant limitations for clinical applications including the toxicity associated with the in vivo persistence. This issue is not only relevant to PMMA but also is observed with alum, a non-degradable inorganic microparticulate used in subunit vaccines. In non-degradable systems like these, the overall size of the formulation is crucial for clearance by phagocytic cells and to prevent the occurrence of frustrated phagocytosis and a foreign body response ( Figure 2A ). Diffusion also dictates how most cargo is released from polymer systems, and when the carrier is non-degradable, it can limit drug release ( Figure 2B ). Even degradable polymers must have a molecular weight below 45,000 MW to ensure full body clearance via the kidneys ( Figure 2C ). Unlike non-degradable materials, biodegradable polymers can break down into monomer units over time in vivo , so there is greater concern with the size of non-degradable polymers for full renal clearance. These challenges highlight the need for improved and diverse biodegradable delivery systems. As gene therapies, cell therapies, biologics, and small molecule drugs, advance, developing and optimizing diverse biomaterial strategies becomes essential. A single approach cannot meet the needs of all immunomodulatory therapies, as highlighted by concerns over the widespread use of polyethylene glycol (PEG). PEG is ubiquitously found in drug delivery systems to increase circulation time and enhance shelf stability. PEG is notably used in the lipid nanoparticle mRNA COVID-19 vaccine formulations.[ 11 ] Unfortunately, upwards of 40% of people have immune memory for PEG. This highlights the need to diversify biomaterial carrier systems to prevent population-level immune responses. Additionally, PEG is a non-degradable synthetic polymer made from by-products of petroleum refining that can persist in the environment, leading to potential accumulation over time. There are reports highlighting bioaccumulation in aquatic organisms, potentially disrupting ecosystems.[ 12 ] Therefore, sourcing and disposal of polymers, monomers, reactants, and solvents must be considered to reduce ecological harm and promote sustainable drug delivery solutions. However, the development of biodegradable polymers often requires organic solvents, so it is important to evaluate the use of green solvents in both the polymer synthesis and fabrication of biomaterial-based nanoparticles commonly used in immunomodulatory applications ( Table 1 ). This should be viewed within the broader context of the environmental impact of pre-clinical research. However, even more environmentally friendly natural materials can have limited long-term sustainability due to production constraints and ecological changes, exemplified by the natural adjuvant QS-21. QS-21, while derived from the Quillaja saponaria tree, has an extraction process that can lead to deforestation and habitat destruction, undermining its eco-friendliness despite its biodegradability and biocompatibility [ 13 ]. Biodegradable options are essential for safely delivering therapies in various preclinical applications, including cancer vaccines, vaccines for infectious diseases, antigen-specific autoimmune therapies, immunotherapy, and cell-based therapies. Unlike nondegradable polymer alternatives, biodegradable polymers circumvent challenges such as toxicity and clearance. Investigating the generation, fabrication, and application of these polymer systems is vital to developing optimized therapeutic strategies for robust immunotherapies that support sustainable therapeutic pipelines.

Biodegradable polymers are critical in preclinical biomedical applications, however, non-degradable polymers are also used, so it is important to understand the challenges associated with non-degradable materials in the body. A non-degradable inorganic microparticle, alum, has been used clinically for over 100 years in subunit vaccines ( Figure 1 ).[ 27 ] Alum is a microparticulate aluminum salt that is 500nm to 10 microns in size.[ 35 ] Through primarily electrostatic interactions, alum is used to absorb subunit antigens to create a protective immune response in patients. The adjuvanticity of alum is thought to be linked to creating an antigen depot for sustained release, targeting of antigen to phagocytic antigen-presenting cells since they can only internalize particles of that size, and/or activation of the inflammasome.[ 36 ] Current FDA-approved vaccines with alum include those to prevent Anthrax (i.e. CYFENDUS, Biothrax), Hepatitis B (e.g. Engerix-B), Japanese Encephalitis Virus (i.e. Ixiaro), Human Papillomavirus (HPV; i.e. Gardasil, Gardasil 9), and Diphtheria, Tetanus & Acellular Pertussis (e.g. Infanrix, Pentacel).[ 37 ] Alum was also part of the GlaxoSmithKline (GSK) adjuvant system 4 (AS04) that also included monophosphoryl lipid A, but the only vaccine that contained this formulation (Cervarix for HPV) was pulled from the US market.[ 38 ] Among the six FDA-approved adjuvants, alum is by far the most widely used across vaccines. Since it was the only FDA-approved adjuvant from 1926 to 2015, there is substantial safety information regarding its use. Despite this breadth of safety characterization, there remains concern with the biocompatibility of non-degradable materials like alum. Vaccines are significantly safer than natural infection, extremely safe across a population, and the most effective way to prevent pandemics and the spread of disease.[ 39 – 44 ] However, like any medical intervention, there are side effects.[ 45 – 49 ] Some individuals may experience acute injection site irritation from alum-based vaccines. In rare cases, atypical responses, such as macrophagic myofasciitis (MMF), can occur. The highest number of MMF cases were in France, with over 250 reported across 10 years.[ 50 ] The condition is linked directly to the persistence of alum at the injection site for years, leading to muscle weakness, myalgia, and other symptoms.[ 51 , 52 ] Other conditions linked to alum, including granuloma formation, have occurred, although extremely rare.[ 53 ] While rare, these reports highlight the potential toxicity that can occur with persistent materials in the body and, thereby, the need to develop degradable biomaterials for immunomodulatory formulations. Just as alum is persistent in the body, it is also retained in the environment at large. While alum is derived from natural aluminum salts, its environmental impact stems from the energy-intensive mining and processing of aluminum, raising concerns about greenhouse gas emissions and potential long-term toxicity to ecosystems. As vaccine development shifts toward sustainability, there is a growing need for biodegradable alternatives that minimize environmental harm. One phenomenon that can occur with non-degradable particulates like alum or PMMA is frustrated phagocytosis and a foreign body response ( Figure 2A ). There are four primary phagocytic cells in the body: macrophages, dendritic cells, neutrophils, and monocytes. Phagocytes can internalize particles larger than 100 nm.[ 54 , 55 ] When a phagocytic cell cannot internalize particles, it undergoes ‘frustrated phagocytosis’, wherein instead of ingesting the particle, the cell spreads out over the particle surface. Over time, cytokines will be released, cueing other phagocytes to attempt to internalize the material. As attempts to internalize the particle continue, macrophages fuse into multinucleated giant cells, which attract T cells. Together, the multinucleated giant cells and T cells wall off the particles in a foreign body response granuloma.[ 56 ] Throughout this process, irritation like MMF and related conditions can occur. This issue is exacerbated with non-degradable particles, which have longer residence times and a higher proportion of particle aggregates compared to degradable particles. Therefore, non-degradable materials significantly increase the likelihood that phagocytic cells will be unable to internalize particles, leading to inflammation.[ 57 , 58 ] Non-degradable materials can also impose significant diffusional barriers for vaccine adjuvants and stop the ability of antigen-presenting cells (APCs) from reaching the antigens – overall negating the effects of the vaccine or immunomodulatory carrier ( Figure 2B ). The body’s handling of non-degradable materials and their environmental impacts highlight the need for immune-modulatory biomaterials to be degradable within the body and produced in an eco-friendly manner. Several of these have already been used for a range of preclinical and clinical immunomodulatory applications. Polymeric particles are typically made from biodegradable polymers and can range in size from nano- to microparticles, ( Table 1 ). The particles can have a variety of forms, including hydrogels, matrix polymeric particles, membrane-coated particles, polyplexes, and polyethylene glycol-coated (PEGylated) nanoparticles ( Figure 1 ). Over the last 40+ years, these particles have been developed, brought to clinics, and been FDA-approved. The polymers used in these particles often include synthetic polyesters such as polylactic acid (PLA), polyglycolic acid (PGA), poly(lactic-co-glycolic acid) (PLGA), polycaprolactone (PCL), polyanhydrides, and poly(β-amino esters) (PBAE). Semi-natural or natural polymers like acetalated dextran (Ace-DEX), polyamino acids, chitosan, alginate, gelatin, cellulose, or hyaluronic acid (HA) can also generate drug delivery carriers. Of these materials, polyesters (e.g PLGA), Ace-DEX, and chitosan are hydrophobic polymers that are often formed into particles composed of a continuous matrix wherein the antigen and/or adjuvant are dispersed throughout, including the particle surface [ 59 ] ( Figure 3 ). The encapsulate is then released from the matrix through diffusion into an aqueous environment and particle degradation. Fabrication methods such as emulsion, spray drying, and industrial coacervation are the most used for hydrophobic polymers. Natural polymers alginate, cellulose, gelatin, and HA are hydrophilic and often have hydrogel properties when prepared as particles or scaffolds ( Figure 3 ) [ 60 ]. Typically formed through ionic crosslinking, alginate particles will swell in water and release the encapsulate through diffusion. PBAEs are also hydrophobic polymers, but their commonly cationic nature allows the formation of polyplexes with nucleic acids such as antigen-encoding mRNA and DNA. Biomaterial choice can influence the formulation size and route of administration of polymeric particles, which significantly alter their biodistribution once injected in vivo ( Figure 4 ). Phagocytic cells can internalize particles larger than 100 nm, while other cells generally cannot engulf particles of this size.[ 54 , 55 ] Studies have shown that polymeric microparticles ranging from 1 to 2 μm are four times more likely to be phagocytosed by CD11c + cells than CD11c − cells. Within this size range, approximately fifty times more CD11c + CD11b − or CD11c + CD11b + cells (DCs) from local draining lymph nodes phagocytosed the microparticles compared to CD11c − CD11b + cells (macrophages).[ 61 ] These results indicate that larger particles (1–2 μm) are predominantly transported by DCs from the injection site to the lymph node, whereas smaller particles (<1 μm) are cleared by resident macrophages. Moreover, nanoparticles below 80nm in diameter will typically traffic directly to lymph nodes.[ 62 ] Hence, it is vital to optimize the size profile of a carrier system to target the desired immune cell trafficking mechanism for the disease and drug. One of the major factors influencing particle size is the fabrication method, and there are technical and environmental tradeoffs with each. For instance, microfluidic devices make highly monodisperse nanoparticles but often require harsh organic solvents [ 63 ]. Organic solvents can have a larger environmental impact, and they may not be compatible with naturally derived polymers. Consequently, polymer selection must consider compatibility with the desired fabrication method. Narrow ranges of polymer molecular weight (MW) are optimal for size homogeneity, so tuning the MW range is another consideration in polymer selection, especially when comparing differences in controlled natural versus synthetic reactions. Another increasingly studied factor contributing to nano/microparticle size in vivo is the protein corona[ 64 ]. In a study by Alberg et al., three hydrophilic, PEGylated nanoparticles with formulations similar to a drug in phase II clinical trials had negligible protein corona formation when incubated with human serum compared to other nanoparticles [ 65 ]. They claim that protein corona is not an unavoidable phenomenon but one that is specific to the particle. With the obvious biological importance of protein corona formation in vivo , this study highlights that there are differences between polymers and particle formulations in their protein corona formation. Moreover, it is important to consider how a polymer’s surface charge, size, hydrophobicity, and other properties will influence its protein corona and size in vivo . Immunostimulatory polymeric particles offer several advantages, including controlled release of encapsulated agents that result in extended immune cell activation. They are also more stable than micelles and lipid particles when stored, which can contribute to sustainability by reducing waste from expired or unstable products. This minimizes the need for frequent production and lowers resource use and disposal. Additionally, polymeric particles can be fabricated to cross-present antigens to both MHCI and MHCII, whereas soluble antigen delivery typically presents only on MHCII, as cytosolic delivery is required for MHCI presentation.[ 66 ] This cross-presentation enhances the education of cytotoxic T cells and can improve vaccine efficacy. Not only can polymeric particles effectively deliver immunostimulatory agents, but some have inherent immunomodulatory properties, such as chitosan, which can activate the innate immune system via NLRP3 inflammasome activation.[ 67 ] In addition to immunostimulation, biomaterial systems can be designed to suppress the immune system via the delivery of immunosuppressive payloads or through inherent polymer or formulation properties. Broad immune suppression is often a design goal for biomaterials in implants to prevent foreign body response, but similar biopolymers are used in delivery systems for tissue repair and regeneration [ 68 ]. One of the primary mechanisms for suppressing the immune response in the context of tissue repair involves the polarization of macrophages. M1 macrophages exhibit a phagocytic phenotype, but they can be polarized via chemical and physical cues, such as extracellular matrix (ECM) stiffness towards an M2-like phenotype that promotes tissue repair and growth, commonly after injury[ 68 , 69 ]. Similarly, polymeric scaffolds such as those based on hyaluronic acid (HA), can exhibit stiffness softening through thermoresponsive-elasticity that promotes M2-like macrophage polarization [ 69 ]. Both delivery and immunomodulatory attributes of a biomaterial can be advantageous to elicit desired immune responses, but the choice of polymer and optimization of synthesis and fabrication of polymeric carriers require specificity to the application for therapeutic safety and efficacy.

Acetalated dextran (Ace-DEX) is a semi-synthetic polymer that has a facile synthesis via an acetalation reaction, where the hydroxyl groups of dextran are modified with cyclic or acyclic acetal groups. This process renders the hydrophilic polysaccharide dextran insoluble in water but soluble in organic solvents such as short-chain alcohols and other common solvents. Alcohols are biodegradable, and their environmental impact is much lower than other solvents, especially if managed through proper recycling. Ace-DEX can be processed similarly to many polyesters yet is often more crystalline in structure than polyesters. During synthesis both cyclic and acyclic acetals are formed, which degrade through hydrolysis at different rates, providing a tunable degradation profile.[ 18 ] The acetalation reaction can be adjusted to produce either methanol or ethanol as a byproduct, depending on the specific conditions used.[ 103 , 104 ] The degradation tunability of Ace-DEX has been shown to influence the humoral and cellular responses of subunit vaccines formulated with the polymer.[ 105 , 106 ] The properties of acetalated dextran are particularly notable for their pH responsiveness and biodegradability. Ace-DEX degrades more rapidly in acidic environments, such as those found in the phagolysosomes of macrophages or DCs, making it ideal for targeted drug delivery and vaccine applications. [ 105 , 106 ] The degradation products of Ace-DEX are non-toxic, biodegradable, and sustainable, including dextran, acetone, and either methanol or ethanol.[ 103 , 104 ] Additionally, Ace-DEX’s ability to form particles further expands its utility in biomedical applications, including drug delivery, tissue engineering, and vaccine delivery.[ 18 , 19 ] Like other polymeric platforms, Ace-DEX particles have been used for many applications, primarily infectious disease vaccines and autoimmune therapies[ 107 , 108 ]. For autoimmune therapeutic vaccines, the desire is to switch an aberrant autoreactive immune response or lessen it in an antigen-specific fashion. Current treatments for autoimmune diseases suppress the overall immune system, opening the patient up to opportunistic infections. For example, anti-IFN-γ antibodies suppress the cytokines signaling non-specifically,[ 109 , 110 ] Copaxone ® induces T cell tolerance via induction of Th2 cytokine production, and Tecfidera ® modifies inflammatory responses via induction of anti-oxidant effects.[ 111 ] Also, blanket immune suppression occurs with glucocorticoids.[ 112 ] Several companies (e.g. Cartesian Therapeutics, formally Select and Barinthus Biotherapeutics, formally Vaccintech) are working on antigen-specific tolerance, but most are focused on food allergies and celiac disease, overlooking very common autoimmune diseases such as multiple sclerosis and type 1 diabetes. Further, of these antigen-specific therapies only ex vivo cell-based therapies have enrolled patients in clinical trials ( NCT02903537 , NCT04530318 , NCT02283671 , NCT02618902 ). Unfortunately, not only are cell-based therapies often costly and difficult to translate, but they are also generally less sustainable than polymeric particles due to the need for complex infrastructure, extensive resources for cell maintenance, and higher logistical and storage demands (e.g., cryopreservation and transportation). In contrast, polymeric particles are easier to scale, more stable, and involve simpler production processes, resulting in a lower environmental and resource footprint. Ace-DEX microparticles encapsulating an autoimmune peptide and a tolerizing agent have been used to create a therapeutic multiple sclerosis (MS) vaccine.[ 113 , 114 ] This relies on the generation of regulatory T cells that are specific to an MS-associated immune response. During MS autoimmunity, effector T cells (CD8 + ) and B cells attack the myelin on nerve cells. Tolerance is a natural immune response to help mediate aberrant immune responses, and it is a break in tolerance that results in autoimmunity. By generating MS-specific Tregs, that break in immunity can be treated. MS-specific Tregs will secrete the potent anti-inflammatory cytokine IL-10 in response to myelin protein, which reduces or stops the effects of autoreactive T cells and B cells. Tregs can be formed when a DC presents antigen on MHCII to a CD4 + T cell in absence of co-stimulation ( Figure 6 ). Using Ace-DEX particles, Chen et al.[ 113 ] encapsulated antigenic peptides and the tolerogenic agent rapamycin. The particles ranged from 378 to 649nm, too large to be taken up by non-phagocytic cells, and thereby passively targeted phagocytic cells when injected SC. The particles were loaded at approximately 5% (μg drug/mg particle) with both rapamycin and peptide. When particles were co-cultured with DCs and T cells, there was a significant increase in FOXP3 + CD4 + T cells, reduction of IFNγ and IL-2, and decrease in IFNγ:IL-10 production with the Ace-DEX MP group containing rapamycin and antigen. When the rapamycin and antigen Ace-DEX particles were given as a footpad delayed-type hypersensitivity treatment, reduced foot swelling was noted in the co-therapy group. This response was dose-sparing compared to the soluble controls. In a relapse and remitting model of experimental autoimmune encephalomyelitis (EAE), mice were treated three times at disease onset. Compared to all controls, the PLP (EAE-relevant antigen) and rapamycin Ace-DEX particle-treated mice were the only group that did not have severe onset of paralysis, as indicated by an increased clinical score. In splenocytes isolated from the treated mice, only the rapamycin and PLP-treated mice had reduced IL-17 and IFNγ cytokine levels after antigen recall. Later work highlighted that this antigen-specific approach did not alter protective influenza vaccine responses.[ 115 ] Ace-DEX particles have also been shown to treat T1D aberrant immune responses with an antigen-specific therapy by illustrated reduced expansion of autoreactive T cells, generating FoxP3 + CD4 + T cells, reducing pro-inflammatory signaling and reducing T1D onset in a mouse model of disease.[ 116 ] Taken together, this data indicated that Ace-DEX particles with antigen and rapamycin generated an antigen-specific tolerogenic response. Polyamino acids are a class of biodegradable polymers that can be generated naturally by microbes or synthetically. Poly-L-lysine is a widely used polyamino acid because its positive charge enhances complexation with nucleic acids, endosomal escape via the ‘proton-sponge effect’ ( Figure 5 ), and cell membrane disruption [ 117 ]. Moreover, PLL can form or be incorporated into biodegradable dendrimers. In a study by Joubert et al. at AstraZeneca, they found that PLL addition to poly(amidoamine) dendrimers improved endosomal escape and cytosolic mRNA expression[ 118 ]. mRNA and other nucleic acid payloads provide powerful immunostimulatory payloads for both cancer and infectious disease vaccination, indicating that cationic poly amino acids like PLL present a highly biocompatible and advantageous material for nucleic acid delivery, especially with regard to diversifying away from PEGylated LNPs.