*2.3. Nanomaterials in Medicine*

At the nanoscale level, properties exist in all materials, both natural and synthetic, but only synthetic materials are generally considered to be part of "nanoscience and engineering" [7]. A wide variety of NPs are currently available, and many of the nanomaterials used can mimic the functions of globular biological macromolecules. These materials include lipid micelles, polymer nanostructures, protein constructions, ribonucleic acid NP, carbon dots, nanodiamonds, carbon nanotubes, graphene, and some inorganic materials such as mesoporous silica NP, superparamagnetic iron oxide NPs, and quantum dots. (Figure 3) [39–44]. These types of materials have unique optical, electronic and magnetic properties depending on size and shape [45].

**Figure 3.** Nanomaterials currently used in the design of NPs, and the available surface modifications.

In recent years, the understanding of MSD-mediated immunotherapy in cancer treatment has improved and a variety of nanomaterials have been developed to regulate MSD. The following is a description of multiple types of NPs composed of a variety of nanomaterials that are used to enhance some of the immunotherapies that are discussed in more detail in this review.

In the case of nanovaccines, for example, the size of these NPs is associated with the mechanism of cellular absorption and the subsequent endocytic pathway, which in turn determines the effect and outcome of the NPs on the cells. The smaller PNPs (25–40 nm) drain into the nodes through the tissue barrier faster than the larger NPs (100 nm), which have to be transported by dendritic cells (DCs). The shape of the NPs is also important in cellular uptake and bio-distribution [46]. Non-spherical NPs have been shown to prevent non-specific cellular phagocytosis by prolonging their systemic circulation, but spherical NPs are more easily transported by DCs [47]. Another important parameter is the charge of NPs, since it influences their internalization and further induction of immune response. Cationic NPs are absorbed more rapidly by macrophages or DCs and have a higher lysosomal escape potential, but they adsorb more serum proteins, reacting with negatively charged components, reducing the permeability of tumour tissues. The NPs that have the better circulation and best penetration into tumours are neutrally net charged NPs [48].

One of the most promising NPs are biodegradable NPs, which generally use poly (lactic-co-glycolic acid) (PLGA), which also has the advantage of a protective effect on antigens [49]. The size of these NPs is the same as that of pathogens, so they are better absorbed by antigen-presenting cells (APCs).

Inorganic and metallic NPs are also used as nano-vaccines. In this case, functional ligands are conjugated with mesoporous silica, calcium phosphate and gold NPs. Peptide micelles, dendrimers, oncolytic viruses and artificial exosomes are also being developed as DC-based nanovaccines [46].

Another type of NPs that allow for the improved recognition of TSAs by the immune system are polymeric NPs that contain large amounts of adjuvant and are membrane-coated by tumour cells with various types of TSAs [50]. Then, depending of properties of polymeric NPs and the type of immunotherapies, several applications have been developed which here are briefly described: i. In the case of aAPCs, dextran-conjugated superparamagnetic iron oxide NPs with major histocompatibility complex (MHC)-Ig dimer and anti-CD28 antibody are used. Magnetic field-induced aAPCs stimulate the activation and proliferation of antigen-specific T-lymphocytes [46]. ii. For cellular immunotherapy, polyNPs (β-amino ester) with a CAR-coding plasmid DNA load are used to enhance chimeric antigen receptor-modified T cells (CAR-T) cells [51]. iii. As for checkpoint inhibitors, zinc pyrophosphate (ZnP) NPs loaded with photosensitizing pyrolipid (ZnP @ pyro) for photodynamic therapy (PDT) have been shown to improve tumour sensitivity to PD-L1 (programmed death-ligand 1) blocking immunotherapy and induce immunogenic cell death [52]. iv. For cytokines, NPs with a self-assembly derived from PEGylated polylactic acid and cationic phospholipid have been designed for targeted administration of IL-12 plasmid DNA [53]. v. Another example is directed AuNPs loaded with endostatin, which blocks neovascularization and normalizes tumour vasculature [54].

Polymeric nano-carriers are used to deliver adjuvant, which accumulates at the site of the tumour through permeability and retention. An example is the use of polyethylene glycol (PEG)-PLGA NPs to encapsulate R837 and a near-infrared dye via an oil-in-water emulsion [55]. PLGA NPs are also used to improve the supply of monoclonal antibodies (mAb) and enhance the activation of T cells [56]. An example is the chemical conjugation of mAb against OX40 (tumour necrosis factor receptor) with PLGA NPs [57].

Another polymer under study is acetylated dextran, which enhances the properties of traditional polymers by allowing for the loading of hydrophilic drugs in a very efficient way, and it is biodegradable and pH-responsive, dissolving under acidic conditions but remaining stable under physiological conditions [56].

Liposomes are also nano-carriers, which allow for a more specific delivery of cytokines and mAb to the site of the tumour. The payloads can be conjugated on the liposomal membrane or charged in the center of the particle. An example is IL-2 and anti-CD137 sticky liposomes [58].

Water-in-oil emulsions are also used, which are large in size and provide a reservoir for the local release of therapeutic agents [56]. An example is the use of these water-in-oil emulsions to deliver anti-CTLA-4 antagonistic antibodies and anti-CD40 agonist antibodies [59].

Another type of material used is hydrogels, which are particularly suitable for delivering biomolecules [56]. They can be generated by the self-assembly of amphiphilic polysaccharides, and cholesterol-bearing pullulan (CHP)-based platforms are also being studied in immunotherapy [60]. Hydroxypropyl cellulose (HPC) nanogels have been shown to drain nearby lymph nodes after skin administration and release their antigen payload into the APCs, enhancing antitumour immunity [61]. Another example is the bioreducible cationic alginate-polyethylenimine nanogel, used to encapsulate ovalbumin as a vaccine that is absorbed by dendritic cells, facilitating antigenic presentation and activating immune responses [62]. Nanogels can also be used in the administration of cytokines, such as murine IL-12 that is incorporated into a CHP nanogel, allowing its sustained release into the bloodstream [63].

AuNPs show great promise due to their safety and adjustable nature, and increase the potency and decrease the toxicity of immunotherapeutics through improved patency and retention [56]. AuNPs conjugated to a tumour peptide that binds to CD13 in the tumour endothelium have been shown to transport and release TNF-α more effectively in vivo [64]. AuNPs can also be used as contrast agents in CT. As an example, the administration of anti-PD-L1-conjugated AuNPs in mice generated a CT signal that correlated with tumour growth, so these NPs can be used to predict responses to immunotherapy treatments [65].

Because of their porous structures, mesoporous silica NPs (MSNs) have a high intrinsic payload encapsulation capacity [56]. An example is the use of liposome-coated MSNs loaded with doxorubicin and oxoplatin (apoptosis inducers) and indoximod (an adjuvant that interferes with immunosuppressive pathways in MSDs), increasing their half-life in circulation and tumour targeting [66]. MSNs have also been designed with large pores that induce a potent immune response when it is combined with photothermal agents and model antigens [56,67].

Other nanoplatforms that are starting to be used are biomimetic nano-carriers, which further improve delivery efficiency and subsequent immune responses. Natural debris can be used to design these NPs, modifying their surface and improving their absorption by the target cells [56]. An example is mannose modification, which has an affinity for receptors present in APC [68]. Galactose modification is another example of biomimetic targeting [69]. These natural carriers also include virus-like particles (VLPs), e.g., cowpea mosaic virus (CPMV)-based VLPs, which combined with an antigenic peptide of human epidermal growth factor receptor 2 (HER2) protein can be used as a vaccine in the treatment of cancer of HER2+ tumours [70].

Heat shock proteins (HSP) also interact with APC receptors and improve antigenic presentation. An example is the use of HSP96-bound antigenic peptides, which are used as a vaccine in colorectal liver metastases [71].

Lipoprotein-based nanoporters are also used, such as the synthetic high-density lipoprotein-mimicking nanodisc that has been used in the targeted vaccination of neo-antigens [72].

Briefly, delivery platforms and their biomimetic modifications provide different advantages in cancer immunotherapy. In addition, many of these nanoplatforms are located at the interface of the natural and the synthetic nanomaterials. Despite the advantages, there are several challenges for these nano-carriers, which include the cost-effective supply of biological nanomaterials, their large-scale production at the pharmaceutical level and the optimisation of long-term storage conditions [56].

The great development of these nanomaterials and the importance they have acquired in the field of immuno-oncology makes it necessary to study both disciplines simultaneously. Furthermore, these disciplines currently have enormous potential for development, and therefore the feedback of knowledge between them must be constant in order to achieve common objectives. The following is a more detailed description of fundamental aspects of immuno-oncology, which helps us to understand its relationship with nanomedicine and also might aid in finding novel applications and new actors in the field.
