**7. Delivery of DAMPs and ICD-Inducers to Tumor Tissues**

As commented previously, ICD is triggered by the combined release of a specific set-up of DAMPs that includes calreticulin, HMGB-1, and ATP, among others. These molecules are exposed to the external cell membrane, and/or released to the external media in dying cells. All of these events are part of a cyclic process which is finely regulated during this specific sub-type of apoptosis. In terms of cell biology, as previously mentioned, several therapeutic procedures can trigger this event and have been investigated in both pre-clinical and clinical applications [88].

However, due to the variety of these different types of stimuli, the exposure or release of DAMPs can vary among the therapeutic strategies. Moreover, it is possible that, for clinical applications, the control of DAMPs exposure and release can be different among patients and tumor stages due to internal sub-tumoral tissue organizations and the different phenotypes of cancer cells. This situation makes the clinical translation difficult, delaying this therapy for patients.

To reduce this potential drawback, some authors have discussed alternative strategies to burst the DAMPs present inside tumor tissues (Figure 3), thus ensuring the presence of these molecular triggers for ICD induction. In terms of efficacy, the simple delivery strategy of DAMPs to target tumor tissues may not be efficient because ICD induction depends both on the release of chemotactic agents and on the presence of recognition molecules, such as calreticulin, on the cell membrane surface. Moreover, as discussed earlier, calreticulin exposure is a key factor for specific cell recognition. *Pharmaceutics* **2022**, *14*, x FOR PEER REVIEW 12 of 18

**Figure 3.** The induction of immunogenic cell death (ICD), the exposure of damage-associated molecular patterns (DAMPs), and the activation of antigen-presenting cells (APC) can be enhanced by delivery strategies based on nanotechnology. **Figure 3.** The induction of immunogenic cell death (ICD), the exposure of damage-associated molecular patterns (DAMPs), and the activation of antigen-presenting cells (APC) can be enhanced by delivery strategies based on nanotechnology.

Alternatively, some authors proposed the use of gene therapy to deliver DNA sequences that could increase DAMPs expression in target tumor cells. The rationale for this strategy is supported by the fact that some tumor types have a reduced expression of DAMPs, such as calreticulin. For instance, Garg et al. (2015) described a pre-clinical tumor

immunization against cancer cells. In terms of microevolution, it makes sense, as calreticulin is a molecule that activates tumor cell phagocytosis by APC. In this situation, selected tumor clones could reduce the expression of DAMPs, thus impairing ICD

Due to the challenge of tumor targeting, nanoparticles or nanocarriers could be used to concentrate ICD inducers and DAMP molecules close to tumor tissues [26,27,108]. This is the classical argument for using nanotechnology for tumor therapy. Within this approach, nanocarriers are passively or actively delivered to the tumor regions and release the carried ICD activators to induce cell death and initiate the immune recognition and then the immune surveillance against malignant cells. This approach is somewhat different from the direct delivery of DAMPS to tumor tissues, but has been used

In the ICD approach widely proposed in the literature, conventional drugs, such as doxorubicin or mitoxantrone, for example, will lead tumor cells to succumb to ICD and release DAMPS. The preclinical results are promising; however, there are some concerns about the translation possibilities for this strategy, especially for the passive delivery of nanocarriers for tumor tissues. The main problems are the structural differences between preclinical induced tumor tissues and natural tumors that are developed in clinical conditions [109]. The argument is that passive targeting is not reproduced in clinical conditions. Despite all this discussion, there is some evidence that, at least in part, nanocarriers can increase the delivery of drugs and immunoadjuvant molecules to tumor

In the work by Zhou et al. (2022), murine melanoma (B16F10) cells were subjected to different treatments, such as hypoxia, cisplatin, radiotherapy, photodynamic therapy, and hypochlorous acid (HOCl). The cell-delivered secretions (CDS) of melanoma cells treated with HOCl activated dendritic cells and macrophages and produced the best antitumor

activation.

successfully [108].

tissues.

Alternatively, some authors proposed the use of gene therapy to deliver DNA sequences that could increase DAMPs expression in target tumor cells. The rationale for this strategy is supported by the fact that some tumor types have a reduced expression of DAMPs, such as calreticulin. For instance, Garg et al. (2015) described a pre-clinical tumor model that reduces the constitutive calreticulin expression, thus reducing the effectiveness of immunogenic protocols [104]. The authors defined this tumor model as resistant to immunization against cancer cells. In terms of microevolution, it makes sense, as calreticulin is a molecule that activates tumor cell phagocytosis by APC. In this situation, selected tumor clones could reduce the expression of DAMPs, thus impairing ICD activation.

Due to the challenge of tumor targeting, nanoparticles or nanocarriers could be used to concentrate ICD inducers and DAMP molecules close to tumor tissues [26,27,108]. This is the classical argument for using nanotechnology for tumor therapy. Within this approach, nanocarriers are passively or actively delivered to the tumor regions and release the carried ICD activators to induce cell death and initiate the immune recognition and then the immune surveillance against malignant cells. This approach is somewhat different from the direct delivery of DAMPS to tumor tissues, but has been used successfully [108].

In the ICD approach widely proposed in the literature, conventional drugs, such as doxorubicin or mitoxantrone, for example, will lead tumor cells to succumb to ICD and release DAMPS. The preclinical results are promising; however, there are some concerns about the translation possibilities for this strategy, especially for the passive delivery of nanocarriers for tumor tissues. The main problems are the structural differences between preclinical induced tumor tissues and natural tumors that are developed in clinical conditions [109]. The argument is that passive targeting is not reproduced in clinical conditions. Despite all this discussion, there is some evidence that, at least in part, nanocarriers can increase the delivery of drugs and immunoadjuvant molecules to tumor tissues.

In the work by Zhou et al. (2022), murine melanoma (B16F10) cells were subjected to different treatments, such as hypoxia, cisplatin, radiotherapy, photodynamic therapy, and hypochlorous acid (HOCl). The cell-delivered secretions (CDS) of melanoma cells treated with HOCl activated dendritic cells and macrophages and produced the best antitumor immune response when compared to the other treatments. Aiming to increase the effectiveness of the treatment, the HOCl-CDS produced in vitro was then associated to nanofibers of a scaffold hydrogel containing melittin and RADA24 peptides. This nanosystem was then injected into the subcutaneous melanoma in vivo (C57BL/6 mice). The results indicated that the obtained hydrogel induced cell death, cytotoxicity in T lymphocytes, and increased the antitumor effect of the immune checkpoint inhibitors.

Another example of a successful use of nanocarriers for increasing the immunogenicity of tumors was published by Sethuraman et al. (2020) [105]. In this work, the authors describe a liposome nanocarrier with a DNA plasmid encoding calreticulin. They observed that this strategy increased the expression of calreticulin in target tumor cells, reducing tumor growth due to immune activation. Interestingly, when they combined this liposomal formulation with the application of focused ultrasound treatment, the results were better. This improvement is probably related to the temperature increase provided by the ultrasound. In higher temperatures, some amount of cell death could be triggered, which in combination with calreticulin superexpression could increase the immune activation.

The authors did not evaluate the modality of cell death induced by the treatment. However, it shows the potential of DAMPs delivery for immune system activation. As a potent phagocytosis inducer, calreticulin is a key factor for alternative types of immune activation. As noted previously, this protein was included as a molecular signature for the ICD, but its presence in external spaces may also promote tumor recognition by APC, thus contributing to immunological surveillance [106].

In this section, we presented potential uses of drug delivery strategies to optimize ICD induction. First, nanoparticles could deliver DAMPs directly to target tissues; second, nanocarriers could deliver ICD inducers; and third, nanoparticles could be used during gene therapy to increase the expression of DAMPS in the target tumor tissues. There is

certainly no ideal strategy, but an effective therapy could be based on the combination of different approaches, and eventually on other strategies to optimize ICD induction in immunotherapies.
