Because of all the values presented, the articles that obtained the best results in the in vitro tests were those that worked with inorganic [22], organic [23], and polymeric [4,24] nanocarriers. They showed efficiency in reducing the toxicity and dose of the drug when coupled with nanocarriers. The articles that worked with liposomes and lipid nanocarriers did not obtain satisfactory results [1,2,10].

#### *3.2. In Vivo Data Discussion*

The discussion was carried out after analyzing the in vivo results of the articles selected in Table 5. Regarding the in vivo tests, the articles were evaluated regarding the results of the tests for reducing animals' parasite load, survival rate, and mortality. Some papers described tests only in the acute phase of the disease [1,8,27–29], and others only did so in the chronic phase [25,26], evaluating the appearance of cardiac and hepatic markers. Other articles obtained results for both phases of the disease [7,30,31]. The nanocarriers used in the in vivo tests were polymeric nanoparticles, lipid nanoparticles, polymersomes, and polymeric micelles.

#### 3.2.1. Polymeric Nanoparticles

Branquinho et al. [8] used polymeric nanoparticles for treating the acute phase of Chagas disease. Polymeric nanoparticles were synthesized with LYC encapsulated based on PCL and PLA-PEG and their in vivo effects were compared to the drug and to free BZN. Mice were separated into groups for four experiments and infected with different *T. cruzi* strains: CL (sensitive to BZN) and Y (partially resistant to BZN). All administrations were performed intravenously into the rear veins. Free BZN and free LYC were administered in a mixture of DMA-PEG 300 solution diluted in 5% glucose. In all experiments of this study, 2 mg/kg/day doses of free and encapsulated LYC in nanoparticles and 50 mg/kg/day doses of free BZN were administered [8]. In the analysis of the parasitemia peak of the groups infected by the CL strain (sensitive to BZN), in experiment I, initiated 24 h after the infection and lasting 10 days, the result was a reduction in parasitemia by 98.6% with free BZN, by 56.3% with free LYC, and by 96.3% for the LYC-loaded nanoparticles (LYC-PCL NC), when compared to the untreated control groups. As for the survival rate of animals infected by the CL strain (sensitive to BZN), those in the untreated control group survived for approximately 20 days. The groups treated with free BZN and with the LYC-loaded PCL nanoparticles had a 100% survival rate for 6 months after treatment and the animals that received only free LYC had a 75% survival rate in the same period. The remaining 25% survived for 24 days. Experiment II carried out with the CL strain, initiated 7 days after the infection and lasting 20 days, obtained a reduction during the parasitemia peak of 99.2% for free BZN, 57.7% for free LIC, 98.2% for the LYC-loaded PCL nanoparticles, and 99.30% for the LYC-loaded PLA nanoparticles. The results show that free LYC had a lower parasitemia reduction rate than the groups treated with free BZN and with LYC-loaded PLA-PEG and PCL nanoparticles. The survival rate was 21 days for the untreated groups. A total of 50% of the animals treated with free LYC survived for 34 days and 100% of the animals treated with free BZN and with both LYC-loaded nanoparticles survived during the entire acute phase for 6 months after the treatment, until they were necropsied [8].

The animals infected by the Y strain (partially resistant to BZN) were treated in experiment III, initiated 24 h after the infection and lasting 10 days. They obtained peak parasitemia reductions of 94.3% with free BZN, 37.8% with free LYC, and 96.9% by the LYCloaded PCL nanoparticles when compared to the untreated control group. This experiment did not use the PLA-PEG nanoparticles. Once again, the parasitemia of free LYC remained higher when compared to free BZN and LYC encapsulated in the nanoparticles, although it achieved a greater reduction when compared to the untreated control group [8].

The survival rate was 100% for the groups treated with free BZN and with the LYCloaded PCL nanoparticles. The animals in the untreated control group survived for only 16 days and treatment with free LYC did not improve survival. In experiment IV, the group began to be treated 4 days after the infection, for a total of 20 days, with PLA-PEG and PLC nanoparticles loaded with LYC and with free BZN, obtaining reductions in the parasitemia peak of 99.55% with the LYC-loaded PCL nanoparticles, of 95.76% for the LYC-loaded PLA-PEG nanoparticles, and of 98.20% for free BZN. The survival rate was the same as in the previous group [8].

Treatment efficacy was assessed by parasitological cure. In experiment I, of the animals infected by the CL strain, those treated with free BZN had a 100% cure, while those treated with the LYC-loaded PCL nanoparticles achieved a 50% cure. In experiment II, the animals treated with both nanoparticles and with free BZN had a 100% cure. In both experiments (I and II), none of the animals treated with free LYC or those in the untreated control group were cured. In experiment III, the animals treated with the LYC-loaded PCL nanoparticles achieved a 50% cure, while none of the mice treated with free LYC and free BZN were cured. In experiment IV, a 75% cure was obtained with free BZN, 100% with the LYC-loaded PLA-PEG nanoparticles, and 62.2% with the LYC-loaded PCL nanoparticles. No cure was achieved for the mice in the untreated control group. It is concluded that LYC was able to reduce parasitemia by 56% to 99% and by 37% to 99% in the CL and Y strains, respectively, with intravenous administration of 2 mg/kg/day, depending on the type of formulation and the schedule used [8]. The results could be even better with higher doses. The animals survived without signs of toxicity in the experimental tests with free LYC and with the LYC-loaded PLA-PEG and PCL nanoparticles. Free BZN in a DMA-PEG 300 solution was able to cure 75% of the animals with a 50 mg/kg/day dose, a better result than the one found with the standard dose of 100 mg/kg/day used for treating Chagas disease, which reached a 50% healing rate [8].

Despite the reduction in parasitemia and the survival rate of mice infected with the CL strain, experiment I achieved a cure rate of 50% using the LYC-loaded PCL nanoparticles, not outdoing free BZN, which reached 100%. The study explains that this result could be due to the high sensitivity of the CL strain to BZN and to the short treatment period, which was initiated 24 h after the infection and lasted only 10 days. The results of experiment II were better, achieving total survival and complete cure with the LYC-loaded PLA-PEG nanoparticles, with the LYC-loaded PCL nanoparticles, and with free BZN. The treatment in experiment II was initiated 7 days after the infection and lasted 20 days. It is believed that the results were better than experiment I because, after this period, the intracellular amastigote forms had already multiplied and ruptured the host cells, releasing the circulating trypomastigote form in the bloodstream, encountering the drugs and with the nanoparticles [8]. The same occurred in the experiments with the Y strain, partially resistant to BZN. The action of encapsulated LYC was better than that of free LYC due to the ability of the nanoparticles to carry out a prolonged release of the drug. As was the case in experiment I, experiment III carried out the treatment for 10 days, starting it 24 h after the infection, which resulted in a reduction in parasitemia and cure in 50% of the animals only with the use of the LYC-loaded PCL nanoparticles. Once again, the above shows the importance of longer treatment and waiting times to start after the infection. Experiment IV obtained a 100% parasitological cure with the LYC-loaded PLA-PEG nanoparticles, 75% with free BZN, and 62.5% with the LYC-loaded PCL nanoparticles. Free LYC ceased to be used in this experiment due to its lower results to the nanoparticles. In the entire treatment, 50% of the animals treated with free LYC did not survive and did not present a parasitological cure. The above shows the importance of encapsulation in nanocarriers [8].

De Mello et al. [30] also used PLA-PEG and PCL polymeric nanoparticles containing the LYC-encapsulated natural active ingredient for in vivo assays. The nanoparticles were prepared by the technique of interfacial deposition of the preformed polymer followed by solvent displacement. The paper carried out tests during the acute and chronic phases of the disease in mice infected with the *T. cruzi* Y strain (partially resistant to BZN) and compared the results. The animals were infected intraperitoneally with 10,000 circulating trypomastigotes for the acute phase and 500 circulating trypomastigotes for the chronic phase. In the acute phase, the animals were orally treated with both LYC-loaded nanoparticles and with free LYC at a dose of 5 mg/kg/day for 20 days, starting on the 4th day after the infection, as was performed in experiment IV of the article by Branquinho [8]. In the chronic phase, free LYC and both LYC-loaded nanoparticles were administered orally (5 mg/kg/day) and intravenously (2 mg/kg/day) for 20 days, starting 90 days after the infection. For comparison purposes, two groups of animals were treated with 100 mg/kg/day of free BZN orally and 50 mg/kg/day intravenously [30].

Fresh blood tests, PCR, blood culture, and enzyme-linked immunosorbent assay (ELISA) were performed to assess therapeutic efficacy. The cure rate achieved with the use of the LYC-loaded PLA-PEG nanoparticles was 62.5% in the acute phase by the oral route, 55.6% in the chronic phase by the oral route, and 50% in the intravenous route. With the LYC-loaded PCL nanoparticles, the cure rates were 57.0% in the acute phase by the oral route, 30.0% in the chronic phase by the oral route, and 33.3% by the intravenous route. As in the results obtained by Branquinho et al. [8], no animal was cured with the use of free LYC, despite managing to reduce parasitemia and increase the survival rate when compared to the control group without treatment. The animals treated with free BZN achieved a cure rate like those treated with nanoparticles during the acute phase. In the chronic phase, only the animals treated with the LYC-loaded nanoparticles were cured, both in oral and intravenous administration. Treatment with the LYC-loaded PLA-PEG nanoparticles and free BZN achieved total parasitemia suppression in the acute phase. The survival rate of the animals was 75% with free LYC, 87% with the LYC-loaded PCL nanoparticles, and 100% with the LYC-loaded PLA-PEG nanoparticles up to 6 months after treatment when they were necropsied. In the control groups, the animals survived for approximately 20 days [30].

The survival rate in the chronic phase was 70% (orally) and 40% (i.v.) with free LYC, 80% (orally) and 50% (i.v.) with free BZN, 100% (orally) and 90% (via i.v.) with the LYC-loaded PCL nanoparticles, and 90% (orally) and 100% (via i.v.) with the LYCloaded PLA-PEG nanoparticles. The survival rate of the untreated groups was 70% by the oral route. The results demonstrated greater efficacy of the LYC-loaded PLA-PEG nanoparticles in the treatment of the disease when compared to free LYC. The higher efficiency of nanoparticles containing LYC against the parasite is due to the controlled release of the active drug, as it has a smaller particle size, the ability to stabilize the drug in the gastrointestinal tract, and to influence tissue diffusion and LYC uptake into the blood [30].

Despite having good permeability, rapid absorption, and distribution in its free form, LYC has poor solubility. In addition to that, it requires the administration of repeated doses due to its rapid elimination. In oral administration, it shows chemical instability in contact with the pH of the gastrointestinal tract [30]. Encapsulation in polymeric nanoparticles controls the drug release in a slow and sustained way, which improves pharmacokinetics and biodistribution; in addition to reducing the need for multiple doses, which consequently reduces toxicity. Polymeric nanoparticles are more stable than other nanocarriers, such as lipid-based ones, liposomes, and micelles. The polymeric wall stabilizes the encapsulated drug and increases circulation time by reducing opsonization and release by the phagocytic system. Degradation of the polymeric wall and the release of LYC to the external environment are reduced by preventing the entry of proteins into the oil core. As they do not have elimination and accelerated metabolism like free LYC, LYC-loaded polymeric nanoparticles manage to accumulate in regions of inflamed tissues, improving their activity and being more effective [30]. Although the results achieved by the nanoparticles are close to those found with free BZN, it is important to emphasize that this shows the significant efficacy of these nanocarriers, as they do not compromise the action of the drugs but achieve excellent results by reducing the dose required for the treatment, which is essential to obtain the lowest possible level of adverse effects. In addition to that, the aqueous suspension of LYCencapsulated nanoparticles is a physiologically acceptable vehicle for oral administration. Having achieved a cure in the chronic phase by this study is encouraging, as it is a phase of the disease with virtually no effective conventional treatments.

Branquinho et al. [31] also evaluated in vivo assays of the action of the PLA-PEG polymeric nanoparticles loaded with LYC. Treatment was performed by administering higher doses orally to mice infected with a strain (VL-10) resistant to BZN and nifurtimox during the acute and chronic phases of the disease. During the acute phase, treatment was initiated 9 days after the infection and lasted 20 days. In the chronic phase, it was initiated 90 days after the infection, with the same duration. A total of 12 mg/kg/day of free LYC, 8

or 12 mg/kg/day of LYC-loaded PLA-PEG nanoparticles, or 100 mg/kg/day of free BZN were administered. With the tests in the acute phase of the disease, the LYC-loaded PLA-PEG nanoparticles managed to suppress parasitemia by 100% with a 12 mg/kg/day dose and by 50% with the lowest dose: 8 mg/kg/day. At a dose of 12 mg/kg/day of the LYCloaded PLA-PEG nanoparticles, the results obtained were 75% cure in the acute phase and 88% cure in the chronic phase of the disease. At a dose of 8 mg/kg/day, the cure was 43% both in the acute and chronic phases. The animals treated with free BZN and free LYC were not cured, as was also the case with untreated controls. In all groups, the survival rate was 87.5% in the acute phase and, with the LYC-loaded PLA-PEG nanoparticles, it was 100%. In the chronic phase, the survival rate with the use of 12 mg/kg/day of the nanoparticles or with 100 mg/kg/day of free BZN was 80%, while with the dose of 8 mg/kg/day of the nanoparticles and free LYC, it was 70% [31].

In all three articles discussed, the treatment with polymeric nanoparticles showed better results when compared to those achieved by the drugs in their free form. The result varies according to the *T. cruzi* strain, the dose used in the treatment, the length of the treatment time, and the start date. The article [8] tested a 2 mg/kg/day dose during treatment (10 and 20 days) and different strains in the acute phase. The results after 20 days were better, as well as in the studies by De Mello et al. [30] and Branquinho et al. [31]. The treatment was also more effective when initiated 4 to 9 days after the infection in the acute phase, as shown in experiments II and IV by Branquinho et al. [8] and in the tests by De Mello et al. [30] and Branquinho et al. [31]. Free LYC failed to cure any of the animals, while free BZN did not cure mice infected with the BZN-resistant VL-10 strain [31] but achieved high cure rates against sensitive (CL) [8] and partially resistant strains to BZN (Y) [30]. LYC-loaded polymeric nanoparticles achieved high cure rates against all strains at lower doses than those administered with free BZN.

The results of the tests carried out in the chronic phase of the disease were encouraging. The article [30] obtained a cure rate of approximately 55% with the LYC-loaded PLA-PEG nanoparticles and approximately 33% with the LYC-loaded PCL nanoparticles. There was no significant difference between the results with oral and intravenous administration. The study by Branquinho et al. [31] obtained a higher cure rate (88%) of mice in tests in the chronic phase with the administration of a higher dose: 12 mg/kg/day. In both studies, free BZN and free LYC failed to cure any animals in the chronic phase tests. In conclusion, the studies showed that LYC encapsulated in polymeric nanoparticles was more effective in reducing parasitemia and in achieving a parasitological cure, and increased animal survival, even when administered at low doses (2 mg/kg/day), when compared to free BZN (100 to 50 mg/kg/day). Encapsulated LYC presented no toxicity in any of all three studies, thus representing a promising alternative for the treatment of Chagas disease.

### 3.2.2. Lipid Nanoparticles

In addition to the aforementioned in vitro assays, the study by Carneiro et al. [10] carried out in vivo assays comparing the action against the circulating trypomastigote form of *T. cruzi* of the solid lipid nanoparticles loaded with compound H2bdtc (5-hydroxy-3 methyl-5- phenyl-pyrazoline-1-(S-benzyl dithiocarbonate) to its free form and to free BZN. The tests were performed on mice infected with the *T. cruzi* Y strain. The treatment was administered orally with 1.0 mg/kg/day of free BZN (100 times less than the standard concentration for the treatment of Chagas disease) and 1.4 mg/kg/day of free H2bdtc and H2bdtc-encapsulated in SLNs, for 10 consecutive days. The treatment was initiated on the fifth day after the infection, which reached its peak 9 days later. The results showed a 70% reduction in parasitemia with the administration of the 2bdtc-loaded solid lipid nanoparticle, 45% with free H2bdtc, and 15% with free BZN (positive control) when compared to the control group. The survival rate of the mice was 100% using the 2bdtcloaded solid lipid nanoparticles. This rate is comparable to the one obtained with the standard administration of free BZN at 400 µmol/kg/day, 100 times higher than the one used in this study with the nanoparticles. With free BZN and H2bdtc, the survival rate

compared to the control group was 57%. The study also evaluated whether there was heart and liver damage in the animals after the treatment [10]. The results showed that the animals treated with the nanoparticles did not show cardiac lesions, only reduced inflammation. The H2bdtc compound in its free form reduced cardiac damage by 50% when compared to free BZN in the standard treatment. The H2bdtc-loaded in SLNs also reduced liver toxicity and liver inflammation caused by the *T. cruzi* infection. Both in the in vitro and in vivo assays, the encapsulation of the H2bdtc compound obtained better results than those found with free BZN when administered at the same concentrations as in the study. The article shows that the nanoparticles were able to increase the oral bioavailability of the drug and improve solubility, which may facilitate access to the parasite. The study concludes that the compound is more efficient than the drugs currently used against the *T. cruzi* infection [10].

#### 3.2.3. Polymeric Micelles

The articles [26–28] studied BZN-loaded polymeric micelles (BZN-PMs). Rial et al. [27] and Scalise et al. [28] performed in vivo assays during the acute phase of the disease.

Rial et al. [27] administered polymeric micelles dispersed in olive oil by oral gavage at doses of 10, 25, and 50 mg/kg/day in different groups of mice, for 30 days, starting two days after infection, and compared the results obtained to free BZN dispersed in olive oil at a dose of 50 mg/kg/day by oral gavage. All mice treated with free BZN and with the BZN-loaded polymeric micelle survived until the end of the treatment. In the control group, only 15% of the animals survived. The saturation solubility of BZN encapsulated in polymeric micelles had a 10-fold increase (3.99 mg/mL) when compared to the solubility of free BZN (0.44 mg/mL) [27]. Improved solubility and absorption of BZN encapsulated in polymeric micelles (BZN-PMs) provided an increase in the animals' survival rate with a 10 mg/kg/day dose, much lower than the conventional 100 mg/kg/day free BZN dose. The decrease in the dose achieved by encapsulating BZN in polymeric micelles with sustained release improves the pharmacological action, can avoid possible adverse effects, and reduces the administration frequency [27].

Scalise et al. [28] carried out tests for 15 and 30 days with mice separated into groups, administering oral doses of 50, 25, and 10 mg/kg/day with BZN-loaded polymeric micelles (BZN-PMs). At doses of 50 and 25 mg/kg/day, all mice survived for at least 50 days. Mice infected and treated with a 10 mg/kg/day dose had a survival rate of 70%. Thus, it can be stated that the efficacy of the treatment is dose dependent. Furthermore, as in the study by Rial [27], BZN-loaded polymeric micelles had a significant antiparasitic effect and prevention of animal death even at lower doses.

Rial et al. [26] used BZN-loaded polymeric micelles (BZN-PMs) in 1-month-old female C57BL/6J model mice of similar weight infected with the Nicaragua strain of *T. cruzi* during the chronic phase of the disease. The treatment was carried out by separating the animals into different groups and comparing the efficacy of administering BZN-MP continuously with intermittently. For the continuous treatment, the animals received BZN-PM doses of 25 and 50 mg/kg/day for 30 days. For the intermittent treatment, they received 13 doses of 50 and 75 mg/kg every 7 days. The administration was via the oral route. The results showed that the intermittent doses were as effective as those administered continuously, which is beneficial because the total intermittent dose is smaller. All animals survived until the end of treatment. The evaluation of the efficacy of the treatment by qPCR showed that 80% of the animals continuously treated with a 50 mg/kg/day dose had negative results. All other groups treated with BZN-PMs had no parasite load detected. The authors compared the results with previous studies and revealed that 80% of the animals treated with continuous doses of 75 mg/kg did not present parasitemia, while for the group treated with intermittent doses of 100 mg/kg, 75% did not present parasitemia [26].

The study also evaluated levels of *T. cruzi*-specific antibodies. The results showed a reduction in the antibody levels in all treatment groups when compared to the untreated ones, as well as a better reduction with the intermittent BZN-PM administration. Cardiac electrical alterations in mice were evaluated by ECG, considering that the conduction alterations caused by the infection were reverted after BZN-PM administration continuously and intermittently. Despite this, free BZN showed the same efficacy as polymeric micelles. The study also provoked immunosuppression of the animals after the treatment to evaluate the reactivation of the parasite, not detecting blood parasitic load [26].

When compared to previous studies carried out by Rial et al. [26], a significant reduction was obtained concerning the standard dose of 100 mg/kg/day, being equally effective with a dose of 75 mg/kg/day. This equated to an approximately 70% reduction in the total dose. This improvement is due to the BZN encapsulation in micelles, which are smaller than standard BZN and increase solubility from 0.4 mg/mL to 3.99 mg/mL [26]. Good results were obtained in the administration of smaller BZN-PM doses, as shown in this study with intermittent doses, as they can be an alternative to long-term treatments with fewer adverse effects on the patient.

#### 3.2.4. Liposomes

In addition to the tests performed in vitro, the article [1] also evaluated the action of the vesicular nanocarrier (polymersome) with encapsulated BZN (poly (ethylene glycol) —block—poly (propylene sulfide) (BZN-PSs)) in in vitro assays in BALB/c mice infected with the *T. cruzi* Y strain, during the chronic phase of Chagas disease. The animals were separated into groups and the treatment was initiated 7 days after the infection with the administration of three different concentrations of the BZN-loaded vesicular nanocarrier (0.03, 0.15, and 1.5 mg/kg), 100 mg/kg for the group treated with free BZN, empty nanocarrier, and an untreated group. The results showed that the BZN-loaded vesicular nanocarrier obtained better reduction rates in cardiac parasitemia and myocarditis at a concentration of 1.5 mg/kg. The lowest dose of 0.03 mg/kg reduced parasitemia by 50%, while the mean dose of 0.15 mg/kg failed to reduce cardiac parasitemia but did reduce myocarditis (heart inflammation). At its standard treatment dose (100 mg/kg/day), free BZN was less effective in reducing parasitemia when compared to doses of the BZN-loaded vesicular nanocarrier. In addition to that, no hepatotoxicity was detected in mice using BZN-PS. The amount of BZN present in both nanocarriers administered during all 14 days of the treatment was 466 times lower than the one normally used with free BZN, achieving similar results in reducing parasitemia. The most important result achieved by the study was the reduction in myocarditis/cardiomyopathy (heart inflammation), the most serious sequel caused by infection with the parasite. Once again, using the encapsulated form of drugs in nanocarriers demonstrates significant efficacy in reducing the necessary doses for the treatment against the *T. cruzi* parasite, without causing undesirable adverse effects to the patients. Despite being promising, nanocarriers still need to be tested in clinical studies to evaluate their action in humans [1].

After analysis of the articles discussed, it is concluded that the best results were obtained with the use of drugs encapsulated in polymeric nanoparticles, as shown in the studies by Branquinho et al. [8], De Mello et al. [30], and Branquinho et al. [31]. The best length of the treatment in time was 20 days, starting 4 to 7 days after the infection. The studies analyzed the oral and intravenous routes, not showing significant differences in the results, with oral administration as the best choice for the patients' adaptation. In addition to BZN, the first-choice drug used against the *T. cruzi* parasite, LYC is a natural product encapsulated in polymeric nanoparticles that proved to be a great alternative for the treatment, obtaining high parasitemia reduction rates, as well as increased survival and cure rates for the animals during the acute and chronic phases of the disease, with doses much lower than those used in standard treatments with free BZN. Their studies can be improved and progress to future clinical trials.

Studies have shown that nanocarriers offer several advantages over traditional drugs for the treatment of Chagas disease. One of these advantages is that nanocarriers can improve the bioavailability of the drug, allowing it to reach specific sites more efficiently, improving the therapeutic index [13]. Nanocarriers administered orally need to be absorbed in the intestine by permeation for drug release into the blood or for cellular uptake. Furthermore, nanocarriers can slowly release the loaded actives which can reduce blood peaks of the drug and consequently adverse effects [1,23,24].

Nanocarriers can stabilize the active in the gastrointestinal tract and can promote the tissue diffusion and drug uptake into the blood [14,22,30]. Nanocarriers can also increase cellular uptake of the drug (Figure 2), which can kill the parasite in a shorter time compared to the free drug. In addition, nanosystems can accumulate in inflamed tissues, improving action and effectiveness. The use of nanocarriers for the treatment of Chagas disease also has some disadvantages, such as the difficulty in large-scale production, since the production of nanocarriers requires complex processes, which can increase the cost of treatment. In addition, toxicological and genotoxic studies must be performed to investigate the risk of human exposure to nanocarriers. The results showed that nanocarriers are also dose dependent and their effect varies according to the sensitivity of the *T. cruzi* strain in relation to the drug [28,30]. The advantages of nanocarriers as drug delivery systems are real, but there are many challenges to be overcome. It is necessary to evaluate the safety and efficacy of treatments using nanocarriers and to develop methods to produce them on an industrial scale to promote clinical studies.

#### 3.2.5. Nanocarriers of Drugs in the Chronic Phase of Chagas Disease

Chagas disease has two phases: acute and chronic. The acute phase exhibits high numbers of trypomastigotes in the blood and is controlled by the immune system with the help of drugs. After the acute phase, the parasite continues the life cycle being present outside and inside host cells. In the chronic phase, parasitemia in the blood is low and detection is difficult. In addition, the symptoms are not felt for years, but 4 out of 10 patients develop the chronic phase of Chagas disease. Intracellular amastigotes promote tissue damage to the heart, esophagus, and colon. Patients die due to heart failure caused by chronic chagasic cardiomyopathy [37,38].

There are two strategies for targeting nanocarriers to the target site: passive and active targeting. Both strategies work well for treating tumors and inflamed tissues. Cancer treatments involving nanocarriers depend on the enhanced permeability and retention effect (EPR effect). Tumor tissue grows quickly and releases a series of substances capable of stimulating neoangiogenesis. The vessels grow rapidly in a defective way and present defects such as holes allowing the passage of nanocarriers from the blood vessel into the tissue. Nanocarriers in the tissue interstitium can be endocytosed into the cytoplasm of cells. In active targeting, nanocarriers decorated with targeting molecules pass through the holes in the vessels and can bind to cells, facilitating the uptake. Inflamed tissues have permeable blood vessels, and this fact can be used in favor of the passive or active targeting of nanocarriers [37,39,40]. The use of nanocarriers to target tissue is a success in the treatment of tumors and inflammation; however, targeting these delivery systems to cardiac muscle in the chronic phase of Chagas disease is a challenge. Nanocarriers are effective in transporting drugs to cardiomyocytes when these systems escape from blood vessels into the interstitium of cardiac tissue. In the interstitium, nanocarriers can be recognized and taken up by cardiomyocytes by the mechanism of endocytosis, which is a route to reach the amastigotes. However, the number of amastigotes in cardiac tissue is small and the tissue lesions are slow. Thus, for every significant piece of damage to the cardiac tissue caused by amastigotes, local inflammation is established. Initially, inflamed tissue can facilitate the passage of the nanocarriers to the interstitium, but at the end of the inflammatory process there is a reduction in local circulation and hypoxia [37]. Thus, there is probability of success for the nanocarriers to reach cardiomyocytes at the beginning of the chronic phase or indeterminate phase where blood flow has not yet been compromised. In the late chronic phase, cardiac tissue has suffered extensive damage, compromising the blood flow and the effectiveness of nanocarriers.

There are articles published with promising results from the use of drugs encapsulated in nanocarriers for the treatment of Chagas disease in the chronic phase [7,25,26,30,31]. The articles use animal models and, probably, drugs encapsulated in nanocarriers are effective at the beginning of the chronic phase or indeterminate phase, where circulation in the cardiac tissue has not been compromised, with the possibility of escape of the nanocarriers from the blood vessels to the cardiac tissue.

#### **4. Materials and Methods**

To prepare this paper, a search of the literature was carried out in the PubMed and Web of Science scientific databases from January 2012 to May 2023, using the following combinations of keywords: "Chagas Disease and nanoparticles", "*Trypanosoma cruzi* and nanoparticles" and "Chagas Disease and *Trypanosoma cruzi* and nanoparticles". Subsequently, the articles found were assessed using the inclusion and exclusion criteria defined for this review. For the inclusion criteria, the articles considered were those that used nanocarriers in in vitro and in vivo studies in the search for new ways to improve the treatment of Chagas disease. According to the exclusion criteria established, review articles, patents, duplicates, book chapters, conference abstracts, articles published in journals with an impact factor less than 1.0, articles on vaccines, and articles that deviated from the topic proposed were excluded. The scoping review was conducted by three individuals to ensure the quality of study selection. For a better detailing of the search, specific spreadsheets were prepared using Microsoft Excel (2019) for the in vitro and in vivo assays, for the composition and physicochemical characterization of the nanostructured systems, as well as a final evaluation table for each article, regarding the presence of certain items, such as title appropriate to the content, abstract, contextualization and objective present in the introduction, ethical statement, study design, complete physicochemical characterization, size of the nanocarriers, animals, sample size, in vitro and in vivo studies, trypanocidal effect, statistics, interpretation of the results, limitations, and conclusion.

#### **5. Conclusions**

After a detailed analysis, this study showed that using nanocarriers capable of encapsulating drugs achieved efficient results in the treatment of the disease. In in vitro assays, the encapsulated drugs did not lose their trypanocidal activities against the different forms of *T. cruzi*. In the tests employed, they were able to reduce cellular toxicity and increase the selectivity of the drugs against the parasite. With the in vivo studies, carried out with mice, the tests in both phases of the disease (acute and chronic) showed a reduction in parasitemia and a high survival and cure rate in the animals with the administration of much smaller doses when compared to the standard treatment with free BZN. In addition to healing during the chronic phase, some results with drugs encapsulated in nanocarriers have shown a reduction in liver toxicity and heart inflammation, which is encouraging given the lack of treatments available for this clinical condition.

With the increased circulation of drugs provided by the prolonged release and the possible decrease in adverse effects by reducing the necessary doses, the nanocarriers showed physiological stability, efficacy, and safety in transporting and delivering drugs to the target.

The nanotechnological strategies for drug delivery could open a new horizon of clinical practices for the successful treatment of Chagas disease. One of the strategies, nanoparticles containing drugs, has shown promising results in inducing the host's immune response against the pathogen with few side effects; in addition, it showed promising results in reducing toxicity, elevating efficacy, and the bioavailability of the active compound against the pathogen, by prolonging release, thereby increasing the therapeutic index. However, there is a need for more clinical studies to predict whether the nanoparticles will offer improved effective antichagasic treatments since the benefits of the nanotechnology for this disease are excellent in relation to cellular assays which have stimulated the preliminary animal studies [6,13,37]. Moreover, the unique nanocarrier approved for clinical trials is liposomal amphotericin B (LAMB) with reasonable anti-*T*. *cruzi* activity. From this perspective, the pre-clinical studies of the nanocarriers containing anti-*T*. *cruzi* drugs

should be encountered, and this situation demands an approach from governmental and private agencies in conjunction with the pharmaceutical industry focusing on the efficient implementation of these nanosystems for drug delivery that will elevate the efficacy of anti-chagasic treatment procedures [6,13].

The limitations of nanocarriers are related to their ability to deliver drugs to tissues with low blood flow during the chronic phase of the disease. The targeting of nanocarriers to cardiac muscle in the chronic phase of Chagas disease is a challenge due to tissue fibrosis and low blood flow.

The results of preclinical studies analyzed by this review were important in the search for new alternatives for the treatment of Chagas disease. More studies can be carried out so that the treatment is perfected and can proceed to future clinical studies that may provide better adherence by the patients.

**Author Contributions:** D.d.F.P.: methodology, writing and editing; A.P.d.S.M.: methodology and review; D.d.A.G.: writing and review; T.d.N.: methodology, review and editing; M.S.d.S.d.B.M.: formal analysis and review; R.S.-O.: conceptualization and methodology; E.R.-J.: conceptualization, methodology and supervision. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by the: Carlos Chagas Filho Foundation for Research Support of Rio de Janeiro State (FAPERJ)—Cientista do Nosso Estado (CNE) [grant number: E-26/201.077/2021], Apoio aos Programas e Cursos de Pós-graduação Stricto Sensu do Estado do Rio de Janeiro [grant number: E-26/210.136/2021] and Rede NanoSaúde [grant number: E-26/010.000981/2019]; Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)—Bolsa Produtividade em Pesquisa PQ [grant number: 309522/2020-0]. This paper was also supported with a scholarship granted by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data sharing not applicable.

**Conflicts of Interest:** The authors declare that there are no conflicts of financial or non-financial interests that are directly or indirectly related to the paper submitted for publication.

#### **References**

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## *Review* **From Bench to Bedside: Implications of Lipid Nanoparticle Carrier Reactogenicity for Advancing Nucleic Acid Therapeutics**

**Tetiana Korzun 1,2,3,4 , Abraham S. Moses <sup>1</sup> , Parham Diba 3,4 , Ariana L. Sattler 4,5,6, Olena R. Taratula <sup>1</sup> , Gaurav Sahay <sup>1</sup> , Oleh Taratula 1,2,\* and Daniel L. Marks 4,5,6,\***

**Abstract:** In biomedical applications, nanomaterial-based delivery vehicles, such as lipid nanoparticles, have emerged as promising instruments for improving the solubility, stability, and encapsulation of various payloads. This article provides a formal review focusing on the reactogenicity of empty lipid nanoparticles used as delivery vehicles, specifically emphasizing their application in mRNAbased therapies. Reactogenicity refers to the adverse immune responses triggered by xenobiotics, including administered lipid nanoparticles, which can lead to undesirable therapeutic outcomes. The key components of lipid nanoparticles, which include ionizable lipids and PEG-lipids, have been identified as significant contributors to their reactogenicity. Therefore, understanding the relationship between lipid nanoparticles, their structural constituents, cytokine production, and resultant reactogenic outcomes is essential to ensure the safe and effective application of lipid nanoparticles in mRNA-based therapies. Although efforts have been made to minimize these adverse reactions, further research and standardization are imperative. By closely monitoring cytokine profiles and assessing reactogenic manifestations through preclinical and clinical studies, researchers can gain valuable insights into the reactogenic effects of lipid nanoparticles and develop strategies to mitigate undesirable reactions. This comprehensive review underscores the importance of investigating lipid nanoparticle reactogenicity and its implications for the development of mRNA–lipid nanoparticle therapeutics in various applications beyond vaccine development.

**Keywords:** empty lipid nanoparticles; reactogenicity; xenobiotics; ionizable lipids

### **1. Introduction**

Nanomaterial-based delivery vehicles are often praised for their ability to encapsulate both hydrophobic and hydrophilic payloads, which include small molecule drugs, imaging agents, and nucleic acids, resulting in their enhanced solubility, extended circulation time, and delayed degradation in the systemic circulation [1]. These properties make nanomedicines an attractive alternative to traditional therapeutic and diagnostic modalities, leading to their extensive use clinically and pre-clinically in various biomedical applications [2]. Extensive research was conducted on the efficacy of lipid nanoparticles (LNPs) for the delivery of messenger RNA (mRNA) as a vaccination tool for the prevention

**Citation:** Korzun, T.; Moses, A.S.; Diba, P.; Sattler, A.L.; Taratula, O.R.; Sahay, G.; Taratula, O.; Marks, D.L. From Bench to Bedside: Implications of Lipid Nanoparticle Carrier Reactogenicity for Advancing Nucleic Acid Therapeutics. *Pharmaceuticals* **2023**, *16*, 1088. https://doi.org/10.3390/ph16081088

Academic Editors: Ana Catarina Silva, João Nuno Moreira and José Manuel Sousa Lobo

Received: 7 July 2023 Revised: 24 July 2023 Accepted: 26 July 2023 Published: 31 July 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

of COVID-19 during the recent pandemic [3–7]. The potential applications of mRNA–LNPs for non-infectious diseases, including those necessitating protein supplementation or replacement therapies, were significantly enhanced by the progress made in this field [8]. These therapeutic interventions apply to a range of disorders, encompassing those linked to chronic inflammation, such as cancer, autoimmune diseases, cardiovascular disorders, diabetes, and other conditions. However, caution must be exercised when considering already reactogenic xenobiotic substances in the presence of pre-existing inflammation. Such a combination is likely to aggravate the underlying inflammation, hamper therapeutic mRNA translation, and interfere with the repeated administration of mRNA therapeutics.

Studies on the reactogenicity of mRNA–LNP formulations were conducted by leveraging the abundance of data derived from clinical trials of COVID-19 vaccines. Systemic and local reactogenic symptoms are known to occur after the administration of COVID-19 mRNA vaccines, including formulations developed by Pfizer-BioNTech and Moderna [4,9]. The observed side effects include fever, muscle aches, headaches, fatigue, chills, and pain at the injection site [3,6,7]. These symptoms are usually mild to moderate in severity and are more common after the second dose of the vaccine [10]. Individuals with underlying chronic inflammation may be at an increased risk of experiencing severe side effects from mRNA–LNP-based therapies and may also have a reduced response to the treatment itself [11]. Furthermore, as shown by COVID-19 vaccine reactogenicity studies, overactivation of immune responses triggered by mRNA–LNP formulations could lead to new-onset autoimmune disorders such as autoimmune liver diseases, Guillain–Barré syndrome, immune thrombotic thrombocytopenia, IgA nephropathy, rheumatoid arthritis, systemic lupus erythematosus, and others [11–13]. Despite the reported unfavorable outcomes, research demonstrates that the manifestation of vaccination side effects is linked to a more robust immune reaction and enhanced protection against COVID-19 [14,15]. However, to guarantee the safety and effectiveness of LNP carrier species for therapeutic applications beyond vaccines and infectious disease prevention, especially when immune activation is unnecessary, a thorough investigation of their toxicity and reactogenicity is indispensable during the formulation development and optimization stages.

The influence of multiple pro-reactogenic elements of mRNA–LNP formulations, such as ionizable lipids, the polyethylene glycol (PEG) coating, and mRNA cargo, should be studied with great caution. Extensive investigation concerning mRNA's reactogenicity, stability, and translatability creates a favorable environment for the current applications of modified mRNA in various therapeutic settings [16–18]. For LNP reactogenicity research, the current focus is now centered on investigating the reactogenic adjuvanticity of LNP formulations to enhance the immunogenicity of mRNA–LNP vaccine formulations [19]. Although a thorough investigation of the LNP carrier's reactogenicity is yet to be accomplished, the current data raise important questions revolving around LNP-associated side effects. For instance, the use of a greater mRNA–LNP dose in the mRNA-1273 vaccine and different ionizable lipids used in the formulation are potential explanations for the increased reactogenicity of mRNA-1273 compared with BNT162b formulations in the Moderna and Pfizer-BioNTech COVID-19 vaccines, respectively [20,21]. Therefore, further research and standardization in the area of reactogenic manifestations and their association with the LNP formulations is necessary to develop effective and safe LNP carrier formulations. By addressing the issue of reactogenicity, mRNA–LNP therapies hold promise for treating a wide range of non-infectious diseases, including those associated with chronic inflammation.

This review summarizes the current understanding of the reactogenicity of LNP carriers, including their potential to exacerbate underlying inflammation, impede the delivered mRNA translation, and potentially interfere with the repeated administration of mRNA therapeutics. Addressing a significant gap in the current literature regarding the reactogenicity of empty LNPs, our review seeks to bridge this critical void by providing a thorough and comprehensive analysis of the reactogenic manifestations associated with LNP carriers.

#### **2. Exploring LNPs as Xenobiotics in Reactogenic Responses**

Reactogenicity is a recognized phenomenon that manifests in individuals who are administered xenobiotics, which are defined as any foreign substances introduced into the body. This phenomenon encompasses a range of unfavorable events that may arise due to the immune system's response to such substances, including mRNA–LNP formulations, which are presently employed in COVID-19 vaccines. These reactogenic outcomes in the form of adverse reactions stem from the immune system's response to the LNPs themselves rather than the intended antigen of the vaccine. Although immunogenicity is an anticipated feature of LNP-based vaccines, the reactogenicity of mRNA–LNPs leads to undesirable therapeutic outcomes and unfavorable effects such as pain, fever, chills, and fatigue [4,9,22–24].

Studies are underway to evaluate the reactogenicity of LNPs and their structural components (Table 1) independently of the intended cargo. The typical composition of LNP carriers consists of ionizable lipids, helper lipids, such as distearoylphosphatidylcholine (DSPC), as well as cholesterol and PEG-lipids (Figure 1) [25]. Ionizable lipids are key components of LNPs, as they provide the positive charge that facilitates the encapsulation of negatively charged nucleic acids [26]. Helper lipids, such as DSPC, are often used to optimize the biophysical properties of LNPs. Helper lipids can enhance the stability of the LNP formulation and promote cellular uptake and endosomal escape of nanoparticles [27]. Cholesterol is another essential component of LNPs, as it also influences the biophysical properties of the LNP formulation. The presence of cholesterol within the LNP membrane can enhance its fluidity, thereby promoting the efficient release of nucleic acids from the LNP and facilitating their uptake by cells via enhanced fusogenicity [28]. Lastly, the incorporation of PEG-lipids into LNP formulations serves to augment their stability, decrease the activation of phagocytic immune cells, and enhance their pharmacokinetic properties [26]. In the process of LNP self-assembly, PEG-lipids assume a crucial role by creating a hydrophilic steric barrier through the formation of PEG chains on the LNP surface [29]. Van der Waals forces between the hydrocarbon chains of DSPE, PEG-lipids, and cholesterol molecules are essential for holding the LNPs together and maintaining their structural integrity [30]. The collective result is the formation of a PEG layer around the intact LNP, with the PEG chains protruding from the particle's surface. This arrangement enhances the stability and integrity of the LNP structure, contributing to its overall functionality and effectiveness as a delivery vehicle. Hence, the incorporation of PEG-lipids to create a protective layer around the LNP serves to minimize its recognition by the immune system and enhances its in vivo circulation time. However, it is important to note that PEG-lipid incorporation can also lead to an increase in the immunogenicity of the formulation [31]. *Pharmaceuticals* **2023**, *16*, x FOR PEER REVIEW 4 of 28

**Figure 1.** Composition of LNP carriers used in drug delivery: Their composition comprises ionizable lipids, helper lipids such as distearoylphosphatidylcholine (DSPC), cholesterol, and polyethylene glycol (PEG)-conjugated lipids. Among these components, ionizable lipids and PEG-lipids are the **Figure 1.** Composition of LNP carriers used in drug delivery: Their composition comprises ionizable lipids, helper lipids such as distearoylphosphatidylcholine (DSPC), cholesterol, and polyethylene glycol

most reactogenic species, posing potential risks for unfavorable immune stimulation and adverse effects. PEG-lipids assume a significant role in LNP self-assembly, creating a hydrophilic steric barrier with protruding PEG chains surrounding the particle, while ionizable lipids bind to the nucleic

Among the components of LNP platforms, ionizable lipids and PEG-lipids are recognized as the most reactogenic elements [32]. Ionizable lipids such as Dlin-MC3-DMA (hereafter referred to as MC3), initially introduced in the therapeutic Onpattro (patirisan); SM-102, employed in the Moderna COVID-19 vaccine; and ALC-0315, utilized in the Pfizer-BioNTech COVID-19 vaccine are frequently evaluated and contrasted based on their reactogenicity and immunogenicity profiles, especially in the context of their implementation in vaccines (Table 1) [33–35]. Ionizable lipids are cationic at low pH and play a crucial role in encapsulating nucleic acids and promoting endosomal escape with the efficient release of cargo into the cytosol [34]. However, their positive charge can also interact with negatively charged endosomal membranes or proteins, resulting in cellular damage and undesired immune activation. Additionally, incorporating PEG-lipids into LNPs to enhance their stability, evade immune cell recognition, and impede opsonization by complement effector molecules can potentially lead to undesirable consequences. These may include the production of anti-PEG antibodies or the accumulation of PEG in various organs and tissues [31]. Importantly, the molecular weight of PEG was shown to play a crucial role in both the production of anti-PEG antibodies and their binding to the PEG moieties. A reduction in anti-PEG IgM formation was observed in mice when treated with LNPs conjugated to a fast-shedding PEG-lipid with short acyl chains, and hence lower molecular weight, in comparison with LNPs conjugated to a slow-shedding PEG-lipid with long acyl chains [36]. Moreover, the pretreatment of mice with slow-shedding PEGcontaining LNPs resulted in the suboptimal performance of LNP formulations containing nucleic acids [36]. Interestingly, the administration of high-molecular-weight PEG as a pre-treatment in mice, before introducing PEGylated liposomal formulation, effectively sequestered the existing pool of anti-PEG antibodies [37]. The decrease in available anti-PEG antibodies enabled the prolonged circulation of PEGylated liposomes, avoiding the induction of accelerated clearance and preserving their efficacy. Additionally, PEG with a molecular weight of 2000 Da and larger exhibited the highest affinity for binding anti-PEG

antibodies [38].

(PEG)-conjugated lipids. Among these components, ionizable lipids and PEG-lipids are the most reactogenic species, posing potential risks for unfavorable immune stimulation and adverse effects. PEG-lipids assume a significant role in LNP self-assembly, creating a hydrophilic steric barrier with protruding PEG chains surrounding the particle, while ionizable lipids bind to the nucleic acid payload through electrostatic interactions. Therefore, it can be inferred that LNPs themselves, independently of their cargo, possess structural components capable of inducing reactogenic and immunogenic responses. However, evaluating the reactogenicity of individual components of LNP carriers (hereafter referred to as empty LNPs, eLNPs) in isolation is often challenging due to the integral role played by the assembled eLNPs in delivering nucleic acids and the hydrophobicity of separate eLNP components. As such, the comprehensive assessment of the safety and Therefore, it can be inferred that LNPs themselves, independently of their cargo, possess structural components capable of inducing reactogenic and immunogenic responses. However, evaluating the reactogenicity of individual components of LNP carriers (hereafter referred to as empty LNPs, eLNPs) in isolation is often challenging due to the integral role played by the assembled eLNPs in delivering nucleic acids and the hydrophobicity Therefore, it can be inferred that LNPs themselves, independently of their cargo, possess structural components capable of inducing reactogenic and immunogenic responses. However, evaluating the reactogenicity of individual components of LNP carriers (hereafter referred to as empty LNPs, eLNPs) in isolation is often challenging due to the integral role played by the assembled eLNPs in delivering nucleic acids and the hydrophobicity can be their reactogenic However, evaluatingthe reactogenicity of individual components of LNP carriers (herereferred to as eLNPs) is due to role by assembled eLNPs delivering the such, comprehensive of role played by the assembled eLNPs in delivering nucleic acids and the hydrophobicity of separate eLNP components. As such, the comprehensive assessment of the safety and efficacy of the assembled product usually requires an evaluation of the reactogenicity of the intact eLNP platform independently of the intended cargo. **Table 1.** Key building blocks of representative FDA-approved mRNA–LNP formulations. played by the eLNPs delivering acids and the hydrophobicity of separate eLNP As such, the comprehensive assessment the and efficacy of the assembled product usually requires an evaluation of the reactogenicity of the intact eLNP platform independently of the intended cargo. **1.** Key building blocks of representative FDA-approved mRNA–LNP formulations.

Therefore, it can be inferred that LNPs themselves, independently of their cargo, possess structural components capable of inducing reactogenic and immunogenic responses. However, evaluating the reactogenicity of individual components of LNP carriers (hereafter referred to as empty LNPs, eLNPs) in isolation is often challenging due to the integral

Therefore, it can be inferred that LNPs themselves, independently of their cargo, possess structural components capable of inducing reactogenic and immunogenic responses.

evaluating the reactogenicity individual components LNP carriers referred as eLNPs) in isolation is often challenging to the integral

efficacy of the assembled product usually requires an evaluation of the reactogenicity of

efficacy of the assembled product usually requires an evaluation of the reactogenicity of

of separate eLNP components. As such, the comprehensive assessment of the safety and efficacy of the assembled product usually requires an evaluation of the reactogenicity of

of separate eLNP components. As such, the comprehensive assessment of the safety and efficacy of the assembled product usually requires an evaluation of the reactogenicity of

**Table 1.** Key building blocks of representative FDA-approved mRNA–LNP formulations. the intact eLNP platform independently of the intended cargo. the intact eLNP platform independently of the intended cargo. the intact eLNP platform independently of the intended cargo. intact eLNP independently of the **Category Common Name IUPAC Name Chemical Structure Category Name IUPAC Name Chemical Structure**

*Pharmaceuticals* **2023**, *16*, x FOR PEER REVIEW 5 of 28

*Pharmaceuticals* **2023**, *16*, x FOR PEER REVIEW 5 of 28

*Pharmaceuticals* **2023**, *16*, x FOR PEER REVIEW 5 of 28

*Pharmaceuticals* **2023** *16*FOR PEER 5

*Pharmaceuticals* **2023**, *16*, x FOR PEER REVIEW 5 of 28

When conducting studies that include reactogenic profiling of mRNA–LNP formulations, researchers use multiple controls beyond the eLNP carriers. These include phos-When conducting studies that include reactogenic profiling of mRNA–LNP formu-When conducting studies that include reactogenic profiling of mRNA–LNPformureactogenic Ionizable lipids used in <sup>a</sup>Onpattro therapy, <sup>b</sup>Moderna, and <sup>c</sup>Pfizer-BioNTech COVID-19 vaccines. used in <sup>a</sup>Onpattro therapy, <sup>b</sup>and <sup>c</sup>Pfizer-BioNTech vaccines.

phate-buffered saline, placebo mRNA–LNPs incorporating luciferase or GFP mRNA, dil-

lations, researchers use multiple controls beyond the eLNP carriers. These include phos-

lations, researchers use multiple controls beyond the eLNP carriers. These include phos-

uents, or naked nucleic acids [39–44]. Although LNPs are frequently employed as carriers for mRNA, utilizing eLNPs as a control for comparing their reactogenic effects to mRNAloaded LNPs and attempting to separate their reactogenic profiles may not always yield optimal results. This is due to the possibility that eLNPs differ from mRNA-loaded LNPs in their size, composition, surface interactions, and fusibility potential. The molar ratio of PEG-lipids, the lipid-to-mRNA mass ratio, and the N/P ratio (representing the positively charged nitrogen of ionizable lipids and the negatively charged phosphates of nucleic acids) are critical factors that influence the ultimate composition of LNPs containing ionizable lipids for mRNA complexation [45]. eLNPs with an altered concentration of helper lipids and interference from ionizable lipids on the surface often display variations in their physicochemical and biological properties compared with mRNA-loaded LNPs, including a positive surface charge, increased splitting dynamics, and a modified protein corona composition [45]. Consequently, eLNPs may display different organ tropism as a result of their differential interaction with homing proteins, such as ApoE, compared with mRNAloaded LNPs [45]. This, in turn, may augment the local and systemic eLNP reactogenicity. phate-buffered saline, placebo mRNA–LNPs incorporating luciferase or GFP mRNA, diluents, or naked nucleic acids [39–44]. Although LNPs are frequently employed as carriers for mRNA, utilizing eLNPs as a control for comparing their reactogenic effects to mRNAloaded LNPs and attempting to separate their reactogenic profiles may not always yield optimal results. This is due to the possibility that eLNPs differ from mRNA-loaded LNPs in their size, composition, surface interactions, and fusibility potential. The molar ratio of PEG-lipids, the lipid-to-mRNA mass ratio, and the N/P ratio (representing the positively charged nitrogen of ionizable lipids and the negatively charged phosphates of nucleic acids) are critical factors that influence the ultimate composition of LNPs containing ionizable lipids for mRNA complexation [45]. eLNPs with an altered concentration of helper lipids and interference from ionizable lipids on the surface often display variations in their physicochemical and biological properties compared with mRNA-loaded LNPs, including a positive surface charge, increased splitting dynamics, and a modified protein corona composition [45]. Consequently, eLNPs may display different organ tropism as a result of their differential interaction with homing proteins, such as ApoE, compared with mRNAloaded LNPs [45]. This, in turn, may augment the local and systemic eLNP reactogenicity. phate-buffered saline, placebo mRNA–LNPs incorporating luciferase or GFP mRNA, dilor LNPs are as carriers for mRNA, utilizing eLNPs as a control for comparing their reactogenic effects to mRNAloaded LNPs and attempting to separate their reactogenic profiles may not always yield optimal results. This is due to the possibility that eLNPs differ from mRNA-loaded LNPs in their size, composition, surface interactions, and fusibility potential. The molar ratio of PEG-lipids, the lipid-to-mRNA mass ratio, and the N/P ratio (representing the positively charged nitrogen of ionizable lipids and the negatively charged phosphates of nucleic acids) are critical factors that influence the ultimate composition of LNPs containing ionizable lipids for mRNA complexation [45]. eLNPs with an altered concentration of helper lipids and interference from ionizable lipids on the surface often display variations in their physicochemical and biological properties compared with mRNA-loaded LNPs, including a positive surface charge, increased splitting dynamics, and a modified protein corona composition [45]. Consequently, eLNPs may display different organ tropism as a result of their differential interaction with homing proteins, such as ApoE, compared with mRNAloaded LNPs [45]. This, in turn, may augment the local and systemic eLNP reactogenicity. researchers controls eLNP These phate-buffered saline, placebo mRNA–LNPs incorporating luciferase or GFP mRNA, diluents, or naked nucleic acids [39–44]. Although LNPs are frequently employed as carriers as comparing effects and reactogenicmay optimal results. This is due to the possibility that eLNPs differ from mRNA-loaded LNPs in their size, composition, surface interactions, and fusibility potential. The molar ratio of the ratio, the (representing ionizable lipids and ids) are critical factors that influence the ultimate composition of LNPs containing ionizable lipids for mRNA complexation [45]. eLNPs with an altered concentration of helper interference from on display physicochemical and biological compared LNPs, ing a positive surface charge, increased splitting dynamics, and a modified protein corona composition [45]. Consequently, eLNPs may display different organ tropism as a result of homing proteins,as ApoE, LNPs This, in the local eLNP When conducting studies that include reactogenic profiling of mRNA–LNP formulations, researchers use multiple controls beyond the eLNP carriers. These include phosphate-buffered saline, placebo mRNA–LNPs incorporating luciferase or GFP mRNA, diluents, or naked nucleic acids [39–44]. Although LNPs are frequently employed as carriers for mRNA, utilizing eLNPs as a control for comparing their reactogenic effects to mRNAloaded LNPs and attempting to separate their reactogenic profiles may not always yield optimal results. This is due to the possibility that eLNPs differ from mRNA-loaded LNPs in their size, composition, surface interactions, and fusibility potential. The molar ratio of PEG-lipids, the lipid-to-mRNA mass ratio, and the N/P ratio (representing the positively charged nitrogen of ionizable lipids and the negatively charged phosphates of nucleic acids) are critical factors that influence the ultimate composition of LNPs containing ionizable lipids for mRNA complexation [45]. eLNPs with an altered concentration of helper lipids and interference from ionizable lipids on the surface often display variations in their physicochemical and biological properties compared with mRNA-loaded LNPs, including a positive surface charge, increased splitting dynamics, and a modified protein corona composition [45]. Consequently, eLNPs may display different organ tropism as a result of their differential interaction with homing proteins, such as ApoE, compared with mRNAloaded LNPs [45]. This, in turn, may augment the local and systemic eLNP reactogenicity. When conducting that include profiling of formulations, researchers use multiple controls beyond the eLNP carriers. These include phosphate-buffered saline, placebo mRNA–LNPs incorporating luciferase or GFP mRNA, diluents, or nucleic acids Although LNPs are frequently employed as carriers for mRNA, utilizing eLNPs for effects to LNPs and to separate reactogenicmay always yield optimal results. This is due to the possibility that eLNPs differ from mRNA-loaded LNPs in their size, composition, surface interactions, and fusibility potential. The molar ratio of PEG-lipids, the lipid-to-mRNA mass ratio, and the N/P ratio (representing the positively charged nitrogen of ionizable lipids and the negatively charged phosphates of nucleic acids) are critical factors that influence the ultimate composition of LNPs containing lipids for complexation [45]. an altered concentration of helper lipids and interference from ionizable lipids on the surface often display variations in their physicochemical and biological properties compared with mRNA-loaded LNPs, includpositive surface increased splitting dynamics, and a modified protein composition [45]. may display different organ tropism a result of their differential interaction with proteins, as ApoE, compared with [45]. may the local and systemic reactogenicity.Ionizable lipids used in <sup>a</sup> Onpattro therapy, <sup>b</sup> Moderna, and <sup>c</sup> Pfizer-BioNTech COVID-19 vaccines. Among the components of LNP platforms, ionizable lipids and PEG-lipids are recognized as the most reactogenic elements [32]. Ionizable lipids such as Dlin-MC3-DMA (hereafter referred to as MC3), initially introduced in the therapeutic Onpattro (patirisan); SM-102, employed in the Moderna COVID-19 vaccine; and ALC-0315, utilized in the Pfizer-BioNTech COVID-19 vaccine are frequently evaluated and contrasted based on their reactogenicity and immunogenicity profiles, especially in the context of their implementation in vaccines (Table 1) [33–35]. Ionizable lipids are cationic at low pH and play a crucial role in encapsulating nucleic acids and promoting endosomal escape with the efficient release of cargo into the cytosol [34]. However, their positive charge can also interact with negatively charged endosomal membranes or proteins, resulting in cellular damage and undesired immune activation. Additionally, incorporating PEG-lipids into LNPs to enhance their stability, evade immune cell recognition, and impede opsonization by complement effector molecules can potentially lead to undesirable consequences. These may include the production of anti-PEG antibodies or the accumulation of PEG in various organs and tissues [31]. Importantly, the molecular weight of PEG was shown to play a crucial role in both the production of anti-PEG antibodies and their binding to the PEG moieties. A reduction in anti-PEG IgM formation was observed in mice when treated with LNPs conjugated to a fast-shedding PEG-lipid with short acyl chains, and hence lower molecular weight, in comparison with LNPs conjugated to a slow-shedding PEG-lipid with long acyl chains [36]. Moreover, the pretreatment of mice with slow-shedding PEG-containing LNPs resulted in the suboptimal performance of LNP formulations containing nucleic acids [36]. Interestingly, the administration of high-molecular-weight PEG as a pre-treatment in mice, before introducing PEGylated liposomal formulation, effectively sequestered the existing

pool of anti-PEG antibodies [37]. The decrease in available anti-PEG antibodies enabled the prolonged circulation of PEGylated liposomes, avoiding the induction of accelerated clearance and preserving their efficacy. Additionally, PEG with a molecular weight of 2000 Da and larger exhibited the highest affinity for binding anti-PEG antibodies [38].

Therefore, it can be inferred that LNPs themselves, independently of their cargo, possess structural components capable of inducing reactogenic and immunogenic responses. However, evaluating the reactogenicity of individual components of LNP carriers (hereafter referred to as empty LNPs, eLNPs) in isolation is often challenging due to the integral role played by the assembled eLNPs in delivering nucleic acids and the hydrophobicity of separate eLNP components. As such, the comprehensive assessment of the safety and efficacy of the assembled product usually requires an evaluation of the reactogenicity of the intact eLNP platform independently of the intended cargo.

When conducting studies that include reactogenic profiling of mRNA–LNP formulations, researchers use multiple controls beyond the eLNP carriers. These include phosphatebuffered saline, placebo mRNA–LNPs incorporating luciferase or GFP mRNA, diluents, or naked nucleic acids [39–44]. Although LNPs are frequently employed as carriers for mRNA, utilizing eLNPs as a control for comparing their reactogenic effects to mRNA-loaded LNPs and attempting to separate their reactogenic profiles may not always yield optimal results. This is due to the possibility that eLNPs differ from mRNA-loaded LNPs in their size, composition, surface interactions, and fusibility potential. The molar ratio of PEG-lipids, the lipid-to-mRNA mass ratio, and the N/P ratio (representing the positively charged nitrogen of ionizable lipids and the negatively charged phosphates of nucleic acids) are critical factors that influence the ultimate composition of LNPs containing ionizable lipids for mRNA complexation [45]. eLNPs with an altered concentration of helper lipids and interference from ionizable lipids on the surface often display variations in their physicochemical and biological properties compared with mRNA-loaded LNPs, including a positive surface charge, increased splitting dynamics, and a modified protein corona composition [45]. Consequently, eLNPs may display different organ tropism as a result of their differential interaction with homing proteins, such as ApoE, compared with mRNA-loaded LNPs [45]. This, in turn, may augment the local and systemic eLNP reactogenicity.

Considering the major concerns surrounding the reactogenicity of eLNPs and their components, various efforts are underway to develop alternative lipid formulations that can minimize their adverse effects, while still maintaining the desirable properties of LNP platforms. It can be asserted with a high degree of confidence that, at present, the process of constructing LNPs entails adhering to comparable protocols with the primary variance arising from the application of different ionizable lipids (Table 1) but following similar LNP composition ratios (Table 2). For instance, the currently approved mRNA–LNP vaccines demonstrate a commonality in their structural compositions (Table 1). These compositions encompass a diverse range of ionizable lipids, each unique to the specific vaccine formulation, alongside the inclusion of diverse PEGylated lipids. Although the ionizable lipids utilized in each vaccine formulation differ, they all share certain structural features. These structural similarities, such as the amino-alcohol head group and branched hydrocarbon lipid tails characterized by ester linkages, contribute to the stability, mRNA encapsulation efficiency, and facilitation of cellular uptake of mRNA–LNP vaccines.

Despite the development of new formulations, the current Food and Drug Administration (FDA)-approved LNP platforms and their modified versions, which employ similar structural components, continue to serve as the established standard for delivering nucleic acids. Consequently, a comprehensive evaluation of their reactogenicity is imperative to expand the scope of their clinical applications. In light of this, it is essential to note that there are different types of reactogenicity assessments that can be used to evaluate the safety and efficacy of LNP-based therapies.

**Table 2.** eLNP composition and doses utilized in reactogenicity studies.

IL = ionizable lipid; Distearoylphosphatidylcholine = DSPC; ID = intradermally; IV = intravenously; IM = intramuscularly; SQ = subcutaneously; IN = intranasally.

#### **3. Assessment of Reactogenic Manifestations Following LNP Administration**

Evaluation of reactogenicity is a crucial component of preclinical safety studies for LNP formulations. Assessing reactogenicity can be carried out in several ways, depending on the administered formulation and the specific adverse events. While the assessment of reactogenicity in human subjects mainly involves evaluating the frequency and severity of local and systemic reactions, as well as monitoring for severe adverse events such as anaphylaxis, preclinical studies involving animal models typically employ a range of approaches. The employed methodologies in animal models encompass behavioral observations, hematological, immunological and biochemical analyses, histopathological examinations, and measurements of specific pro-inflammatory markers through immunohistochemistry and gene expression assays (Figure 2). The comprehensive utilization of these methods facilitates a more thorough understanding of the reactogenic manifestations associated with administered formulations.

*Behavioral observation* involves monitoring mice for signs of reactogenicity or toxicity, such as changes in food-motivated behavior, activity level, grooming, and respiratory distress. Food intake and body weight are essential parameters often closely examined in preclinical studies. Changes in these parameters provide valuable insights into the overall health of the animals and serve as early indicators of potential reactogenicity to xenobiotics. Therefore, in this review, food intake and body weight will be discussed further as important indicators of reactogenicity in mice.

**3. Assessment of Reactogenic Manifestations following LNP Administration**

tions associated with administered formulations.

Evaluation of reactogenicity is a crucial component of preclinical safety studies for LNP formulations. Assessing reactogenicity can be carried out in several ways, depending on the administered formulation and the specific adverse events. While the assessment of reactogenicity in human subjects mainly involves evaluating the frequency and severity of local and systemic reactions, as well as monitoring for severe adverse events such as anaphylaxis, preclinical studies involving animal models typically employ a range of approaches. The employed methodologies in animal models encompass behavioral observations, hematological, immunological and biochemical analyses, histopathological examinations, and measurements of specific pro-inflammatory markers through immunohistochemistry and gene expression assays (Figure 2). The comprehensive utilization of these methods facilitates a more thorough understanding of the reactogenic manifesta-

**Figure 2.** Methodological approaches for investigating reactogenic manifestations in animal models: By integrating different methodologies, such as behavioral observations, histopathological studies, and hematological, biochemical and inflammation analyses, a more comprehensive understanding of the cause-and-effect relationship between treatments and reactogenic manifestations can be achieved, aiding in the development of improved LNP platforms. **Figure 2.** Methodological approaches for investigating reactogenic manifestations in animal models: By integrating different methodologies, such as behavioral observations, histopathological studies, and hematological, biochemical and inflammation analyses, a more comprehensive understanding of the cause-and-effect relationship between treatments and reactogenic manifestations can be achieved, aiding in the development of improved LNP platforms.

*Behavioral observation* involves monitoring mice for signs of reactogenicity or toxicity, such as changes in food-motivated behavior, activity level, grooming, and respiratory distress. Food intake and body weight are essential parameters often closely examined in preclinical studies. Changes in these parameters provide valuable insights into the overall health of the animals and serve as early indicators of potential reactogenicity to xenobiotics. Therefore, in this review, food intake and body weight will be discussed further as important indicators of reactogenicity in mice. The *hematological analysis* focuses on assessing blood samples for parameters such as platelet, white blood cell (WBC), and red blood cell (RBC) counts (i.e., complete blood count (CBC) with differential), hemoglobin concentration, and RBC characteristics. For instance, in rats and monkeys treated with MC3 eLNPs, no change was observed in RBC counts, hemoglobin, hematocrit, and RBC distribution width compared with PBS administration [49]. However, an increase in all these parameters was observed in response to mRNA-loaded LNPs. Additionally, rats exhibited a systemic increase in total WBC count resulting from mRNA-LNP treatment but not from MC3 eLNPs [49]. The systemic The *hematological analysis* focuses on assessing blood samples for parameters such as platelet, white blood cell (WBC), and red blood cell (RBC) counts (i.e., complete blood count (CBC) with differential), hemoglobin concentration, and RBC characteristics. For instance, in rats and monkeys treated with MC3 eLNPs, no change was observed in RBC counts, hemoglobin, hematocrit, and RBC distribution width compared with PBS administration [49]. However, an increase in all these parameters was observed in response to mRNA-loaded LNPs. Additionally, rats exhibited a systemic increase in total WBC count resulting from mRNA-LNP treatment but not from MC3 eLNPs [49]. The systemic increase in WBCs found in that study mirrored the increasing mRNA concentration per administered dose. Interestingly, despite greater WBC counts in the mRNA-LNP treatment group, neutrophils and monocytes were increased in both the mRNA–LNP and MC3 eLNP treatment groups [49]. A transient increase in monocytes and neutrophils in the MC3 eLNP treatment group was observed for a shorter period (specifically, at day 9 postadministration), in comparison with mRNA–LNP treatment groups, where this increase was sustained (up to day 16 post-administration) [49].

In addition to CBC with differential, *immunological investigations*, including immune profiling and complement activation studies, play a crucial role in identifying specific components or characteristics of eLNPs that contribute to their reactogenic properties. Despite the absence of specific studies examining the activation of complement by eLNPs, ample evidence exists regarding complement activation through classical, mannose-binding lectin, and properdin-mediated pathways involving liposomes [57–61]. The activation of the complement cascade can occur through the recognition of structural components of liposomes or antibodies that target these components on the liposomal surface, including cholesterol, PEG, or structural lipids [62–64]. Additionally, the complement cascade can be initiated by the interactions of specific proteins that form the protein corona surrounding nanoparticles [61,65].

In addition to hematological and immunological assessment, blood samples for reactogenic studies can be evaluated for *biochemical parameters*, including liver enzymes, kidney function, and electrolyte balance. The most common biochemical approach utilized to

assess systemic reactogenicity in animal models involves the evaluation of hepatic and renal function, as these organs are integral to the metabolism and elimination of foreign substances, including nanoparticles [66]. Exposure to reactogenic substances can result in liver or kidney damage, which is generally reflected by changes in the level of liver enzymes such as alanine and aspartate aminotransferases (AST, ALT) or by changes in markers of renal function, such as increased blood urea nitrogen (BUN) or plasma creatinine levels [67,68]. For instance, AST (but not ALT or alkaline phosphatase) was the only parameter significantly elevated in mice after MC3 eLNPs administration compared with placebo treatment [46]. In rats, the same type of eLNP containing MC3 ionizable lipids increased AST and ALT levels. Moreover, these parameters were increased to the same extent in the rats treated with the highest dose of the mRNA–LNP under investigation [49].

In addition to systemic manifestation, administration of eLNPs also yields local reactogenic reactions. Rodents typically manifest edema, redness, and infiltration of innate immune cells at the injection site. Additionally, innate immune cell infiltration is observed in organs penetrated by LNP-based formulations, such as the lung in studies with intranasal drug administration, mirroring similar responses observed in humans [23,49]. To evaluate the impact of xenobiotics on affected tissues, reactogenicity studies employ an assessment method involving *histopathological examination*, which entails the collection and microscopic analysis of tissue samples from various organs for signs of tissue damage, inflammation, or immune cell infiltration. For instance, histopathological assessment of liver samples showed that MC3 eLNPs induced individual cell necrosis in the livers of rats [49]. Similarly, histopathological approaches are employed to assess the infiltration of immune cells into organs and tissues. In mice, injection-site immune cell infiltrates consist of neutrophils, macrophages, and dendritic cells, and demonstrate the localized release of chemokines in response to eLNPs [23]. The intranasal delivery of labeled LNPs resulted in leukocytic infiltration of lung tissues, with a predominant presence of neutrophils and eosinophils [23]. Additionally, in rhesus macaques, injections with eLNPs and, to a greater extent, mRNA-loaded LNPs led to CD45-positive cell infiltration that denotes non-specific infiltration with hematopoietic cells [48]. Liang et al. also reported that neutrophil and monocyte infiltration in rhesus macaques was similar for mRNA-loaded LNP and eLNP treatments, regardless of administration route, when intramuscular and intradermal routes of injection were compared from immunogenic and reactogenic perspectives [48].

Finally, the assessment of pro-inflammatory effector molecules released in response to eLNP administration encompasses a range of *studies targeting inflammation* that include a wide array of methodologies, including ELISA, flow cytometry, cytokine profiling, quantitative PCR, and microarray analysis. Specifically, chemokine and cytokine expression studies can provide valuable insights into the reactogenicity pathways of eLNPs as a drug delivery system. For instance, the expression of chemokines at the injection site is known to facilitate the trafficking and recruitment of more circulating innate immune cells to the site of active inflammation [69]. Specifically, eLNPs induce the local release of multiple chemokines, including CCL2, CCL3, CCL4, CCL7, CCL12, CXCL1, and CXCL2, that further attract macrophages and monocytes [23]. The potent chemokine CCL2 is also overexpressed by monocytes in response to Acuitas eLNP administration in human peripheral blood mononuclear cells (PBMCs) ex vivo [50]. Collectively, eLNP carriers devoid of payload elicit the recruitment of monocytes and neutrophils, which subsequently amplify this cascade through the local expression of multiple effector molecules. However, eLNP administration elicits a response of shorter duration when contrasted with mRNA-loaded LNP treatments [49]. Our observation further suggests that this disparity may stem from the unique molecular-level responses to mRNA payloads and eLNP carriers, which can be recognized as danger- (DAMPs) or pathogen-associated molecular patterns (PAMPs).

#### **4. Cellular and Molecular Responses to LNP Carriers**

Within the intricate landscape of molecular interactions involving LNPs, innate immune cells emerge as the primary first responders to these therapeutics. Among these

cellular "arenas", macrophages and neutrophils take center stage as pivotal participants in the dynamic interplay with LNP-based therapies. Employing complex molecular processes, innate immune cells orchestrate a series of reactions that significantly impact the efficacy and safety of LNP formulations. Neutrophils play a critical role in the initiation of acute inflammation and are among the earliest immune cells to be recruited into tissues during such inflammatory processes [70]. Functioning as polymorphonuclear cells, they demonstrate remarkable versatility and contribute significantly to the innate immune response against invading pathogens and tissue damage [71]. Their rapid mobilization to inflamed sites is regulated by a diverse array of signaling molecules, including chemokines and cytokines, which guide their directed migration from the bloodstream to the sites of tissue inflammation [72–74]. Neutrophils interact with different nanoparticles, including LNPs and cationic liposomes [75–78]. Studies reveal that the surface characteristics of lipid-containing nanoparticles, such as PEG density and surface charge, play a pivotal role in modulating the neutrophils' capacity to internalize LNPs [76,78]. Monocytes, the second most abundant cell type responding to LNPs, assume an important role in the context of tissue inflammation. This significance is attributed to their selective recruitment and guided migration into inflamed tissues, which is regulated by an array of inflammatory mediators encompassing cytokines, chemokines, and complement effector molecules [69]. Such an extravasation mechanism enables monocytes to actively participate in immune surveillance and host defense, as they engage with inflamed tissues to facilitate pathogen clearance, tissue repair, and the maintenance of tissue homeostasis [79,80]. Monocytes demonstrate proficient phagocytic capabilities and are equipped with pattern recognition receptors (PRRs), enabling them to actively interact with microbial pathogens, cellular debris, and diverse xenobiotics [81]. Macrophages are also widely recognized to be one of the initial and principal cell types responsible for nanoparticle processing [82]. As nanoparticles interact with the bloodstream, they become coated with opsonins, such as antibodies to PEG or complement effector molecules, which mark them for recognition by the mononuclear phagocytic system [82]. The mononuclear phagocytic system, including monocytes and macrophages, identifies opsonized nanoparticles, facilitating their efficient uptake and clearance from circulation [83–85]. This finely tuned process is essential for the immune surveillance and removal of foreign particles, contributing significantly to the body's defense against potential threats posed by xenobiotics in the bloodstream.

Hematologic studies revealed that the administration of LNP formulations, including eLNPs, leads to a systemic increase in WBC levels. However, a more comprehensive understanding of the biological responses to these formulations can be gained by focusing on the innate immune cells' reactions at the immediate site of administration. For instance, regardless of the eLNP administration mode, be it intravenous (IV), intramuscular (IM), intradermal (ID), or intranasal (IN), the cellular administration sites undergo infiltration by diverse innate immune cells. Among these cellular constituents, neutrophils, monocytes, macrophages, and, to a lesser extent, eosinophils and dendritic cells are identified as the predominant species in areas of infiltration [23,48,50]. This infiltration pattern highlights the robust and multifaceted response of the innate immune system to the presence of administered LNPs and signifies the importance of understanding the specific roles played by these immune cells. Furthermore, a localized approach that defines innate immune cell populations offers a comprehensive view of the early molecular events and interactions that shape the subsequent immune responses, thereby facilitating the development and optimization of LNP-based therapies.

The broad and diverse molecular pathways that underlie eLNP-related reactogenic manifestations as part of DAMP- and PAMP-mediated signaling involve the activation of toll-like receptors (TLRs), primarily TLR4 and TLR2 [50,86]. The interactions between eLNPs and TLRs are studied using downstream effectors and adaptor proteins that facilitate further signal transduction. Signal transduction from TLR4 and TLR2 necessitates the presence of the myeloid differentiation primary response 88 (MyD88) adaptor protein, which is essential for downstream signaling [87]. Additionally, some TLRs, such as TLR4,

are known to modify their homophilic interactions with adjacent adaptor proteins based on the acidity of the local environment [87,88]. When TLR4 is internalized along with the forming endosome, the decrease in endosomal pH influences the exchange of the MyD88 adaptor protein for the TIR-domain-containing adaptor-inducing interferon-β (TRIF) adaptor protein [89]. It remains unclear which component of the LNP construct or its protein corona is responsible for TLR ligation, and it is also unknown whether the ligation occurs on the plasma membrane, where TLR4 associates with MyD88, or in the endosome, where TLR4 associates with TRIF for further signal transduction. Furthermore, TLR ligation can be facilitated in the endosome by both LNP surface structures and released LNP components (Figure 3). The acidification of the endosomal environment as endosomes undergo maturation induces the degradation of LNPs, releasing ionizable lipids from the LNP core [90,91]. In the case of LNP-mediated cargo delivery, this conversion occurs during the LNP processing within the endosomal compartment, followed by their escape from the endosomes to facilitate the release of their cargo into the cytosol. It is facilitated by the interactions between the surface of LNPs and the negatively charged membrane of the endosomes in addition to the decrease in ambient pH or by the action of helper lipids or cholesterol or its derivatives [92,93]. Apart from the destabilization of the endosomal membrane caused by LNPs, additional factors, such as the elevation of osmotic pressure or the swelling of particles within the endosome, can contribute to the facilitation of LNP disintegration and subsequent escape from the endosomes [91]. This process allows for the liberation of LNP contents into the cytosol, inevitably compromising the integrity of the intact particles. *Pharmaceuticals* **2023**, *16*, x FOR PEER REVIEW 11 of 28

**Figure 3.** TLR4 signal propagation mediated by MyD88 and TRIF adaptor proteins: Converging pathways involving MyD88 and TRIF adaptors activate the cytokines TNF-α, IL-6, and IL-1β through nuclear translocation of the NF-κB, IRF3, and IRF7 transcription factors. Positive feedback loops and type I interferons enhance cytokine expression, activating additional pro-inflammatory effectors, and inducing cessation of mRNA expression. Complement cascade activation exacerbates cytokine-dependent reactogenicity, with C5a and C3a promoting inflammation via their receptors. TLR4 and C3aR/C5aR activation converge on MAPKs, increasing cytokine production by APCs. **Figure 3.** TLR4 signal propagation mediated by MyD88 and TRIF adaptor proteins: Converging pathways involving MyD88 and TRIF adaptors activate the cytokines TNF-α, IL-6, and IL-1β through nuclear translocation of the NF-κB, IRF3, and IRF7 transcription factors. Positive feedback loops and type I interferons enhance cytokine expression, activating additional pro-inflammatory effectors, and inducing cessation of mRNA expression. Complement cascade activation exacerbates cytokinedependent reactogenicity, with C5a and C3a promoting inflammation via their receptors. TLR4 and C3aR/C5aR activation converge on MAPKs, increasing cytokine production by APCs.

Considering that TLR signaling, specifically TLR4-dependent signal propagation, can be categorized into two main pathways, namely MyD88- and TRIF- facilitated path-

togenic manifestations. Therefore, considering the involvement of these proteins in propagating pro-inflammatory signals, researchers utilized genetic ablation approaches such as MyD88 and TRIF knock-out (KO) mouse models to investigate the impact of eLNPs on TLR receptors. For instance, MyD88 KO mice showed no response to empty LNPs in terms of follicular T-helper and germinal center B-cell upregulation, indicating that the adjuvant properties and, possibly, the reactogenicity of eLNPs rely on signal transduction via MyD88 adaptors [52]. Moreover, the convergence of signaling pathways dependent on MyD88 and TRIF adaptors results in the activation of a common set of cytokines, namely tumor necrosis factor-alpha (TNF-α), interleukin 6 (IL-6), and interleukin 1 β (IL-1β). This activation occurs through the induction of nuclear translocation of transcription factors such as nuclear factor-kappa B (NF-κB) and interferon regulatory factors 3 (IRF3) and 7 (IRF7). The expression of these cytokines is further enhanced by positive feedback loops involving type I interferons and interferon-stimulated gene factors (ISGFs) (Figure 3). Consequently, this amplification of cytokine expression leads to the activation of additional effectors, ultimately resulting in the global cessation of mRNA expression and the maturation of cytokines such as IL-1β that is associated with physiological reactogenic responses such as fever and painful sensations. Importantly, within the context of investigations concerning MyD88 and TRIF adaptors in LNP reactogenicity studies, mice with a dual knockout of MyD88 and TRIF exhibited reduced expression of downstream

Considering that TLR signaling, specifically TLR4-dependent signal propagation, can be categorized into two main pathways, namely MyD88- and TRIF- facilitated pathways (refer to Figure 3), it is crucial to employ both adaptor proteins in reactogenicity studies and to investigate their involvement in the eLNP-mediated induction of reactogenic manifestations. Therefore, considering the involvement of these proteins in propagating pro-inflammatory signals, researchers utilized genetic ablation approaches such as MyD88 and TRIF knock-out (KO) mouse models to investigate the impact of eLNPs on TLR receptors. For instance, MyD88 KO mice showed no response to empty LNPs in terms of follicular T-helper and germinal center B-cell upregulation, indicating that the adjuvant properties and, possibly, the reactogenicity of eLNPs rely on signal transduction via MyD88 adaptors [52]. Moreover, the convergence of signaling pathways dependent on MyD88 and TRIF adaptors results in the activation of a common set of cytokines, namely tumor necrosis factor-alpha (TNF-α), interleukin 6 (IL-6), and interleukin 1 β (IL-1β). This activation occurs through the induction of nuclear translocation of transcription factors such as nuclear factor-kappa B (NF-κB) and interferon regulatory factors 3 (IRF3) and 7 (IRF7). The expression of these cytokines is further enhanced by positive feedback loops involving type I interferons and interferon-stimulated gene factors (ISGFs) (Figure 3). Consequently, this amplification of cytokine expression leads to the activation of additional effectors, ultimately resulting in the global cessation of mRNA expression and the maturation of cytokines such as IL-1β that is associated with physiological reactogenic responses such as fever and painful sensations. Importantly, within the context of investigations concerning MyD88 and TRIF adaptors in LNP reactogenicity studies, mice with a dual knockout of MyD88 and TRIF exhibited reduced expression of downstream effectors, specifically TNF-α and IL-6, in comparison with wild-type (WT) mice when exposed to mRNA-loaded LNPs [94]. The study has a limitation from the perspective of reactogenicity investigations in that it did not distinguish between the effects of mRNA-loaded LNPs and empty LNPs on the MyD88/TRIF dual KO model. This is further complicated by the fact that both TRIF and MyD88 proteins also function as adaptors to TLRs that detect nucleic acids, such as TLR3, which recognizes double-stranded RNA (dsRNA) molecules as PAMPs and DAMPs [95]. Moreover, the MyD88 adaptor is involved in signal transduction from TLR7 that is activated by single-stranded RNA (ssRNA) [89]. As such, it is unclear whether the observed response is due to the nucleic acid payload or the LNP carrier or both. A further puzzle that arises in the decoding of reactogenicity of mRNA-loaded LNPs and eLNPs is that both the induction of TLRs by nucleic acids and eLNPs result in the expression of a similar subset of cytokines and chemokines involved in reactogenic symptoms. Additionally, multiple positive feedback loops and feeder pathways can exacerbate the expression of reactogenicity-related effectors.

Cytokine-dependent reactogenic manifestation can be exacerbated following complement cascade activation, where pro-inflammatory effects of complement effector molecules acting on their respective receptors result in the upregulation of IL-6, TNF-α, and IL-1β expression (Figure 3). The anaphylatoxins, C5a and C3a, are produced through proteolytic processes during the complement cascade activation, which involves the previously mentioned liposome-dependent activation of the classical, mannose-binding lectin, and properdin-mediated complement pathways [57–61,96]. The administration of LNPs possibly results in similar activation patterns, resulting in the generation of C3a and C5a effectors through the action of proteolytic convertases [97,98]. While functioning as anaphylatoxins, attracting innate and adaptive immune cells, and promoting the maturation of adaptive immune cells, C3a and C5a also interact with the C3a receptor (C3aR) and the C5a receptor (C5aR), propagating and amplifying the TLR-dependent inflammatory cascade [99–105]. For instance, the activation of TLR4 and C3aR/C5aR converges to the same mitogen-activated protein kinases (MAPKs), leading to the nuclear translocation of NF-κB, which results in a significant upregulation of IL-6, TNF-α, and IL-1β by antigen-presenting cells (APCs) [106,107].

#### **5. Enhanced Cytokine Gene Expression in Response to eLNP Administration** the challenge of how the initial TLR-dependent expression of *IL1β* can be distinguished from its subsequent expression that is self-potentiated by IL-1β binding to IL-1R. Further-

activation of NF-κB, further emphasizing the convergence of TRIF- and MyD88-dependent pathways [114]. The converging nature of these pathways is elucidated in Figure 4. Overall, the pathway involving TLR activation leading to NF-κB nuclear translocation, regardless of whether it is MyD88- or TRIF-dependent, leads to the expression of cytokines such as TNF-α, IL-6, interleukin 8 (IL-8), IL-1β, and others, although the timing of their expression differs. In this context, the mechanism of transcriptional activation of IL-1β is somewhat complicated, because the interleukin-1 receptor (IL-1R) requires MyD88 as an adaptor molecule to transmit signals following IL-1R ligation by IL-1β (Figure 4) [115]. Therefore, the expression of *IL1β* in cells and tissues that encounter eLNPs presents

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Reactogenic symptoms such as fever, chills, and localized inflammation at the injection site of the LNP-based vaccines are known to be driven by cytokine expression in the resident and migratory immune cells attracted to the injection site [108]. Recent research shed light on the mechanisms underlying these reactogenic manifestations, revealing that they are driven by certain cytokines, including IL-6, IL-1β, and TNF-α [109]. These cytokines are part of the body's natural immune response and are responsible for inducing inflammation and fever, which help to mobilize the immune system to fight off the perceived target pathogen. The regulation of pro-inflammatory cytokine expression is tightly controlled by the previously mentioned transcription factors NF-κB, IRF3, and IRF7, which play crucial roles in the molecular mechanism of acute and chronic inflammation (Figure 4) [110,111]. more, at the protein level, post-translational modifications are necessary to activate the precursor of IL-1β, pro-IL-1β, and release the mature, biologically active form of IL-1β. Proteolytic activation of IL-1β is induced by the enzyme caspase-1 that is activated by the multi-protein complex inflammasome, NLRP3, which is assembled in response to TLR activation (Figure 4) [116]. Once activated, caspase-1 cleaves pro-IL-1β into its mature, active IL-1β form, which can then be secreted by the cell to exert its pro-inflammatory effects. In summary, the ligation of TLRs with the subsequent activation of different signaling pathways leads to the expression and post-translational activation of cytokines, including IFN-β, IL-6, TNF-α, and IL-1β, which are commonly used as outcome measures to study eLNP-induced reactogenicity.

**Figure 4.** Integration of TLR4 signaling, inflammasome activation, and complement anaphylatoxin receptors in cytokine regulation: Upon TLR4 stimulation on the plasma membrane, a MyD88-dependent pathway is initiated, leading to the phosphorylation of interleukin-1 receptor-associated kinases (IRAKs), particularly IRAK1 and IRAK4. The recruited TNF-Receptor-Associated Factor 6 (TRAF6) undergoes ubiquitination, facilitating the formation of polyubiquitin chains that recruit transforming growth factor-β-activated kinase 1 (TAK1), TGF-beta-activated kinase 1 (MAP3K7), binding protein 1/2/3 (TAB 1/2/3), and inhibitor of nuclear factor-κB kinases (IKKs). These molecules are crucial for activating NF-κB and mitogen-activated protein kinase (MAPK) signaling. NF-κB translocates to the nucleus, acting as a transcription factor for pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6, signifying completion of the "early phase" of NF-κB activation. Activation of TLR4 in the endosome triggers a TRIF-dependent pathway, where translocating chain-associating membrane protein (TRAM) serves as an adaptor molecule connecting TLR4 and TRIF. TRAF6, recruited via TRIF, undergoes ubiquitination and interacts with receptor-interacting serine/threonineprotein kinase 1 (RIP1), facilitating the recruitment and activation of IKK for the "late phase" of NFκB activation. Regardless of MyD88 or TRIF dependence, TLR activation induces NF-κB nuclear translocation, resulting in the expression of cytokines such as TNF-α, IL-6, IL-1β, and others, with temporal variations. Additionally, TNF-Receptor-Associated Factor 3 (TRAF3) interacts with TRIF and recruits TANK-binding kinase 1 protein (TBK1) and IκB kinase epsilon (IKKε) kinase, which phosphorylate and activate IRF3/7. IRF3/7 translocates to the nucleus, promoting the expression of **Figure 4.** Integration of TLR4 signaling, inflammasome activation, and complement anaphylatoxin receptors in cytokine regulation: Upon TLR4 stimulation on the plasma membrane, a MyD88-dependent pathway is initiated, leading to the phosphorylation of interleukin-1 receptor-associated kinases (IRAKs), particularly IRAK1 and IRAK4. The recruited TNF-Receptor-Associated Factor 6 (TRAF6) undergoes ubiquitination, facilitating the formation of polyubiquitin chains that recruit transforming growth factorβ-activated kinase 1 (TAK1), TGF-beta-activated kinase 1 (MAP3K7), binding protein 1/2/3 (TAB 1/2/3), and inhibitor of nuclear factor-κB kinases (IKKs). These molecules are crucial for activating NF-κB and mitogen-activated protein kinase (MAPK) signaling. NF-κB translocates to the nucleus, acting as a transcription factor for pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6, signifying completion of the "early phase" of NF-κB activation. Activation of TLR4 in the endosome triggers a TRIF-dependent pathway, where translocating chain-associating membrane protein (TRAM) serves as an adaptor molecule connecting TLR4 and TRIF. TRAF6, recruited via TRIF, undergoes ubiquitination and interacts with receptor-interacting serine/threonine-protein kinase 1 (RIP1), facilitating the recruitment and activation of IKK for the "late phase" of NF-κB activation. Regardless of MyD88 or TRIF dependence, TLR activation induces NF-κB nuclear translocation, resulting in the expression of cytokines such as TNF-α, IL-6, IL-1β, and others, with temporal variations. Additionally, TNF-Receptor-Associated Factor 3 (TRAF3) interacts with TRIF and recruits TANK-binding kinase 1 protein (TBK1) and IκB kinase epsilon (IKKε) kinase, which phosphorylate and activate IRF3/7. IRF3/7 translocates to the nucleus, promoting the expression of type I interferons. Post-translational modifications are necessary to activate pro-IL-1β, with caspase-1 and apoptosis-associated speck-like protein containing CARD (ASC) adaptor activation by the NOD-, LRR-, and pyrin-domain-containing protein 3 (NLRP3) inflammasome complex playing a key role in this process. TRAF3, through IKKε and TBK1, can interact with NLRP3 inflammasome components in certain cellular contexts. Activated IL-1β, via interaction with IL-1R, can upregulate NF-κB nuclear translocation via a MyD88-dependent mechanism. Furthermore, C3aR and C5aR receptors ligated by C3a and C5a complement anaphylatoxins, lead to activation of the AP-1 and NF-κB transcription factors, either by interacting with MAPKs or IKKs, further propagating pro-inflammatory cytokine expression.

The activation of transcription factors IRF3/7 is governed by the TRIF-dependent signaling via TLR4 upon ligation by eLNPs and via TLR3 upon ligation by dsRNA structures as integral components of secondary mRNA confirmations. [50,112,113]. The activation through the phosphorylation and subsequent nuclear translocation of IRF3/7 results in the expression of interferon β (IFN-β) and interferon-stimulated genes factors (ISGFs). Moreover, TLR stimulation with the subsequent MyD88-dependent signal transduction results in the expression and phosphorylation of another transcription factor, NF-κB, indicating the initiation of the "early" NF-κB pathway [114]. The phosphorylated NF-κB protein translocates to the nucleus, where it acts as a transcription factor to regulate the expression of pro-inflammatory effector molecules, including TNF-α, IL-1β, and IL-6 [110]. Together with IRF activation, the TRIF-dependent signaling pathway also guides the "late" activation of NF-κB, further emphasizing the convergence of TRIF- and MyD88-dependent pathways [114]. The converging nature of these pathways is elucidated in Figure 4. Overall, the pathway involving TLR activation leading to NF-κB nuclear translocation, regardless of whether it is MyD88- or TRIF-dependent, leads to the expression of cytokines such as TNF-α, IL-6, interleukin 8 (IL-8), IL-1β, and others, although the timing of their expression differs. In this context, the mechanism of transcriptional activation of IL-1β is somewhat complicated, because the interleukin-1 receptor (IL-1R) requires MyD88 as an adaptor molecule to transmit signals following IL-1R ligation by IL-1β (Figure 4) [115]. Therefore, the expression of *IL1β* in cells and tissues that encounter eLNPs presents the challenge of how the initial TLR-dependent expression of *IL1β* can be distinguished from its subsequent expression that is self-potentiated by IL-1β binding to IL-1R. Furthermore, at the protein level, post-translational modifications are necessary to activate the precursor of IL-1β, pro-IL-1β, and release the mature, biologically active form of IL-1β. Proteolytic activation of IL-1β is induced by the enzyme caspase-1 that is activated by the multi-protein complex inflammasome, NLRP3, which is assembled in response to TLR activation (Figure 4) [116]. Once activated, caspase-1 cleaves pro-IL-1β into its mature, active IL-1β form, which can then be secreted by the cell to exert its pro-inflammatory effects. In summary, the ligation of TLRs with the subsequent activation of different signaling pathways leads to the expression and post-translational activation of cytokines, including IFN-β, IL-6, TNF-α, and IL-1β, which are commonly used as outcome measures to study eLNP-induced reactogenicity.

The upregulation of pro-inflammatory cytokines in response to eLNP treatment has been studied in both ex vitro experiments and in vivo murine studies, with results showing increased expression over time and across different proprietary lipid formulations. eLNPs containing Acuitas' proprietary lipids were assessed in ex vitro experiments with human PBMCs and in in vivo murine studies [50,52]. Expression of IL-1β and IL-6 was upregulated in monocytes and increased in a time-dependent manner after treatment with eLNPs [50]. Furthermore, Tahtinen et al. and Alameh et al. showed that the eLNPs containing SM-102 utilized in the Moderna COVID-19 vaccine and eLNPs containing the Alnylam ionizable lipid led to an increase in IL-1β levels in purified monocytes and draining lymph node lysate, respectively [52,53]. Levels of IL-1β and IL-6 were also increased in the sera of mice treated with eLNPs containing the MC3 and YK009 ionizable lipids compared with a control group treated with phosphate-buffered saline (PBS) [46]. Furthermore, serum TNFα in this study was higher in mice treated with MC3 eLNPs than with mRNA-containing MC3 LNPs in the first 6 h post-treatment, although no information was provided about dosing considerations of eLNPs in comparison with mRNA–LNPs [46]. Interestingly, in contrast to TNF-α, levels of both IL-1β and IL-6 were increased in the eLNP treatment group, similarly to the mRNA-loaded LNP treatment group [46]. Parhiz et al. also found that MC3 eLNPs upregulated serum IL-6 in mice but were less reactogenic in terms of IL-6 levels compared with C12-200 ionizable-lipid-containing eLNPs [56]. In contrast, Tahtinen et al. reported that MC3 eLNPs did not change IL-1β levels; however, the doses of eLNPs administered to the mice were not reported in the study [53]. Sedic et al. found no change in IL-6 and TNF-α expression in rats subjected to MC3 eLNPs in comparison with the control group [49].

Despite the limited availability of data concerning eLNP reactogenicity in inflamed tissues, there is evidence indicating that pre-treatment with lipopolysaccharide (LPS), which is known to induce a TLR4-dependent innate immune response and acute inflammation, exacerbates existing inflammation following subsequent eLNP administration [56]. The exacerbation of inflammation induced by LPS may arise from various factors, such as heightened eLNP internalization or the self-potentiation of chemokine and cytokine expression via parallel or converging reactogenic pathways induced by LPS and eLNPs. Furthermore, LPS induces not only peripheral inflammation but also hypothalamic inflammation, thereby introducing a brain axis that leads to the induction of sickness behavior components, such as fatigue, fever, weight loss, decreased food intake, and anhedonia [117–119]. These findings emphasize the need to mitigate the reactogenic effects of LNPs to ensure their safe and effective application.

Abundant evidence from the studies demonstrates the utilization of not only diverse ionizable lipids but also different doses, emphasizing the significance of a comprehensive assessment of these factors to gain a more informed understanding of their impact on therapeutic applications. Table 2 showcases a selection of representative studies that vividly demonstrate the significant differences in eLNP doses employed. It emphasizes the wide range of doses utilized in the administered formulations. Therefore, the introduction of standardization in the doses and dosage regimens of eLNPs is crucial for facilitating a comparative evaluation of eLNP reactogenicity. Furthermore, standardization is particularly important for evaluating the safety profile of eLNPs to identify potential adverse reactions and to determine the optimal dose range for clinical use. Since pre-existing inflammation can increase the risk of adverse reactions to eLNPs, utilizing standardized dosing in research settings could help to identify the optimal dose range for minimizing the risk of exacerbating inflammation or of inducing further damage to cells and tissues.

### **6. LNP-Inducible Expression of Cytokines Modulating Sickness Behavior**

The physiologic responses to eLNPs include a typical sickness response, consisting of attenuation of food intake and loss of body weight (Figure 5). This is driven, at least partially, by the action of pro-inflammatory cytokines on the hypothalamic centers regulating appetite and metabolism. Cytokines such as IL-6, IL-1β, and TNF-α play a role in the negative regulation of body weight in rodents as part of their sickness behavior in response to inflammatory conditions [120–125]. IL-6 is mainly expressed by innate immune cells, such as monocytes, but it is also produced by skeletal muscle and adipose tissues and has both pro- and anti-inflammatory effects [126–129]. In terms of body weight regulation, IL-6 exhibits a biphasic effect, where acute elevation of IL-6 levels decreases food intake and increases energy expenditure, while chronic elevation of IL-6 levels is associated with increased body weight and insulin resistance [123,130]. IL-1β is produced by a variety of cells, including innate immune cells such as macrophages, monocytes, and dendritic cells, and is known to be a potent pro-inflammatory cytokine [131,132]. Both IL-6 and IL-1β act on the hypothalamus, a key regulatory center in the brain that controls feeding behavior and energy expenditure [133]. IL-6 acts on the hypothalamus to increase the expression of anorexigenic (appetite-reducing) neuropeptides [134,135]. Although IL-1β increases the expression of orexigenic (appetite-stimulating) neuropeptides, such as neuropeptide Y (NPY) and agouti-related protein (AgRP), its potent induction of hypothalamic inflammation leads to a reduction in food intake and a consequent decrease in body weight [136,137].

The findings suggest that the elevated levels of IL-6 and IL-1β induced by eLNPs lead to decreased body weight through the modulation of hypothalamic function. IL-6 and IL-1β were shown to be increased at both the mRNA and protein levels in mice treated with eLNPs containing non-disclosed proprietary ionizable lipids [23]. Importantly, IL-6 and IL-1β overexpression was also associated with a decrease in murine body weight [23]. Of note, the authors also observed high mortality at the 10 µg eLNP dose delivered intranasally in this study, with just 20% survival on day 1 post-treatment [23]. Therefore, the observed weight reduction could be due to TLR-dependent septic shock involving a cytokine storm with the possible overexpression of IL-6, IL-1β, and TNF-α, as discussed

in [138]. Importantly, the results of studies utilizing MyD88/TRIF KO mice that were administered mRNA–LNP injections suggest that MyD88/TRIF dual KO mice did not exhibit weight loss [94]. These findings suggest that effector molecules produced due to the LNP activation of TRIF- or MyD88-dependent reactogenic pathways play a pivotal role in inducing sickness behavior. Therefore, incorporating food intake and body weight as key indicators of systemic reactogenicity in the routine evaluation of eLNP is crucial, as they not only form an integral part of sickness behavior but are also paramount in assessing the efficacy and safety of LNP therapies for conditions that afflict patients' frailty, such as cancer and other chronic diseases. immunity against intracellular pathogens [150]. Liang et al. reported that, in contrast to mRNA-loaded LNPs, eLNPs do not increase Type I IFN-inducible myxovirus resistance gene A (MxA) expression, which is used as a marker for the induction of endogenous type I interferons [48]. However, in the study by Connors et al., protein levels of IFNα and IFNγ were significantly increased after 24 h of Acuitas eLNP stimulation [50]. Connors et al. also report that eLNPs induced increased levels of phosphorylated IRF7, leading to the expression of ISGs [50]. Although there is a slight increase in IRF7 activation when TLR4 is ligated, significant activation only occurs when TLR7 or TLR9 are engaged, such as in the case of an LNP-encapsulated payload of nucleic acids acting on these receptors [149].

eLNPs acting on TLR2 or TLR4 [50,148,149]. Interferon-gamma (IFN-γ), or type II interferon, has lower pro-inflammatory potency and is important for the induction of adaptive

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**Figure 5.** Mechanistic insights into reactogenic manifestations and therapeutic challenges in LNP administration: Complex mechanisms underlie reactogenic manifestations of LNP administration. Expression of cytokines, including IL-6, IL-1B, TNF-a, and interferons, initiate core sickness behaviors such as decreased food-motivated behaviors, fatigue, and lethargy. Concurrent LNP-dependent **Figure 5.** Mechanistic insights into reactogenic manifestations and therapeutic challenges in LNP administration: Complex mechanisms underlie reactogenic manifestations of LNP administration. Expression of cytokines, including IL-6, IL-1B, TNF-a, and interferons, initiate core sickness behaviors such as decreased food-motivated behaviors, fatigue, and lethargy. Concurrent LNP-dependent complement

activation can result in CARPA symptoms, including anaphylaxis, chills, headache, and cardiopulmonary distress. Moreover, complement activation-dependent MAC formation potentially eliminates cells that are actively internalizing LNPs, leading to the release of additional DAMP signals and additional cytokine expression. CTLs also contribute to removing cells presenting LNP-related antigens from the treated cell pool. Complement opsonization of LNPs, along with anti-PEG antibodies, may increase systemic and local reactogenicity by sequestering particles and reduce their therapeutic efficacy. Additionally, activated PKR and RNase L enzymes can hinder mRNA translation or degrade the mRNA therapeutic payload, further decreasing the therapeutic efficacy.

Together with a decrease in food-motivated behavior, fatigue is an important indicator of the severity of the reactogenic insult (Figure 5). Type I and Type II interferons play a critical role in developing disease-associated and iatrogenic fatigue syndromes [139–144]. Hence, the assessment of interferon expression holds significant importance due to their role in causing symptomatic reactogenic manifestations, including fatigue, in response to mRNA–LNP formulations. This is particularly relevant in the context of COVID-19 vaccine administration, where fatigue is a frequent side effect [145,146]. The role of interferons, along with other pro-inflammatory cytokines, is considered significant in both localized and systemic inflammation and in the development of fever [147]. Type I interferons, including IFN-α subtypes and IFN-β, are expressed as part of the pro-inflammatory cascade induced by unmodified mRNA acting on TLR3, TLR7, and TLR9 and by eLNPs acting on TLR2 or TLR4 [50,148,149]. Interferon-gamma (IFN-γ), or type II interferon, has lower proinflammatory potency and is important for the induction of adaptive immunity against intracellular pathogens [150]. Liang et al. reported that, in contrast to mRNA-loaded LNPs, eLNPs do not increase Type I IFN-inducible myxovirus resistance gene A (MxA) expression, which is used as a marker for the induction of endogenous type I interferons [48]. However, in the study by Connors et al., protein levels of IFNα and IFNγ were significantly increased after 24 h of Acuitas eLNP stimulation [50]. Connors et al. also report that eLNPs induced increased levels of phosphorylated IRF7, leading to the expression of ISGs [50]. Although there is a slight increase in IRF7 activation when TLR4 is ligated, significant activation only occurs when TLR7 or TLR9 are engaged, such as in the case of an LNP-encapsulated payload of nucleic acids acting on these receptors [149].

### **7. Reactogenicity Interference with Translation of mRNA Delivered by LNP Carriers**

The immune response triggered by the injection of mRNA–LNP formulations can result in the downregulation of protein translation from the delivered mRNA, which limits the efficacy of mRNA-based therapies (Figure 5) [151,152]. The production of proinflammatory effector molecules downregulates expression of the delivered mRNA through various mechanisms, including the inhibition of translation initiation and the degradation of mRNA [153,154]. The immune system may recognize the eLNP carriers or eLNP structural components as a threat and mount an immune response, even in the absence of an mRNA payload. Significantly, compelling evidence suggests that mRNA–LNP formulations exhibit a substantial abundance of eLNPs that lack the incorporation of any therapeutic payload [45]. Hence, it is imperative to explore the reactogenic effects of eLNPs that may trigger immune responses and ultimately diminish mRNA expression in order to ensure optimal protein expression.

The delivery of mRNA using LNPs was observed to result in reduced mRNA translation when mice were simultaneously exposed to mild doses of LPS [152]. This finding holds significance as both eLNPs and LPS are capable of binding to TLR4. Lokugamage et al. further showed that administering a TLR4 inhibitor increased mRNA translation; however, it did not fully restore it to the levels observed in the absence of LPS treatment [152]. Furthermore, TLR4 activation by mRNA–LNPs themselves led to a decrease in mRNA translation. Importantly, the reduction in mRNA expression was not attributed to a decline in the particle endocytosis or endosomal escape of LNPs, but rather to the action of RNA-dependent protein kinase R (PKR) subsequent to TLR4 ligation. PKR, which reduces

mRNA translation in the cell, is an intermediary component downstream of TLR signaling that, upon activation, phosphorylates eukaryotic initiation factor 2 (eIF2) [16,155,156]. This phosphorylation event prevents the recruitment of the initiator transfer RNA (tRNA) to the ribosome and the activation of eIF2 by binding to guanosine triphosphate (GTP), thereby inhibiting the initiation of translation [156–159]. Furthermore, one of the innate immune mechanisms by which effector molecules such as TNF-α, IL-1 family cytokines, type I IFNs, and other pro-inflammatory cytokines can inhibit translation initiation is by controlling the phosphorylation of the alpha subunit of eIF2, eIF2α [159,160]. Consequently, the initial event of TLR4 ligation, along with the subsequent expression of downstream molecules such as TNF-α, members of the IL-1 family, and type I interferons, contributes to the decreased expression of both therapeutic mRNA and the entire pool of mRNA within the cell as part of a reactogenic outcome.

In addition to inhibiting translation initiation, cytokines can also induce the degradation of mRNA molecules (Figure 5). One mechanism by which this occurs is through the activation of ribonuclease L (RNase L), an enzyme that degrades mRNA in response to exogenous nucleic acid insults, such as those arising from viral infection [161]. The activation of RNase L by pro-inflammatory cytokines can lead to both viral and host mRNA degradation, thereby reducing the overall level of mRNAs available for translation [162,163]. Moreover, pro-inflammatory cytokines can activate signaling pathways that lead to the induction of cell death, which also contributes to the downregulation of mRNA expression by the cells actively internalizing mRNA–LNP formulations [164]. Despite the low-reactogenicity modified mRNA formulations used in clinical practice, [17,18] the use of eLNP carriers may still cause reactogenic manifestations and subsequently lead to cytokine-dependent downregulation of mRNA expression.

The reduction in exogenous mRNA expression can be further modulated by activating complement and adaptive immunity by actively removing the cells internalizing mRNA–LNPs. Activation of the complement pathway by LNPs can initiate the formation of membrane attack complexes (MACs), which often inflict cellular damage. The formation of MACs as a result of activation through classical, mannose-binding lectin, and properdin-mediated complement pathways [165] not only poses a direct threat to the targeted cells but can also impact the uptake and internalization of LNP formulations, leading to a decrease in their therapeutic effectiveness. Additionally, anti-PEG antibodies and complement effector molecules play a critical role in the opsonization of LNPs, resulting in their accelerated clearance from the system and subsequently reducing their therapeutic efficacy. Furthermore, PEG incorporated into the LNP coating induces an anaphylactoid, complement-activation-related pseudoallergy (CARPA) rection [166,167]. CARPA is a harmful immune reaction triggered by the activation of the complement system, leading to adverse symptoms and potentially life-threatening complications in response to certain medications or therapeutic agents [166,168]. In their study, Ndeupen et al. put forth the hypothesis that PEGylated lipids present in LNPs may elicit CARPA in individuals who possess pre-existing PEG-specific antibodies. These antibodies could arise from the previous administration of mRNA–LNP formulations, including COVID-19 vaccines [23]. Of significant importance, extensive evidence exists regarding the activation of the adaptive immune system by LNP delivery, resulting in the generation of antibodies against immunogenic LNP components, including the formation of anti-PEG antibodies [20,169].