Dipeptide, R∆F was synthesized using the solution-phase peptide synthesis method (Panda et al., 2013a; Varshney et al., 2018). The detailed procedure is provided in Supplementary Text S1. The synthesized R∆F was characterized using RP-HPLC (Shimadzu) on a C-18 column (Phenomenex) using a 5%–95% gradient (1 mL/min for 45 min) of acetonitrile-water containing 0.1% TFA (degassed and filtered) and electron spray ionization mass spectrometry (Applied Biosystems QStar (Q-TOF)).

The self-assembly of R∆F was studied at a concentration of 1–10 mg/mL. 1 mg R∆F was dissolved in 30–50 μL of hexafluoroisopropanol (HFIP) and the sample was sonicated for 15 min in a bath sonicator. To the above solution, 970–950 µL of Milli Q water (filtered, sterile) was added to make 1 mg/mL NPs solution and followed by incubation at room temperature for 60 min.

Hydrodynamic size measurements for R∆F nanoparticles were carried out using a zeta sizer (Malvern Zetasizer Nano Series Nano ZS90) at 37°C with laser (10 mW HeNe laser, 633 nm) and the experimental procedures reported earlier (Panda et al., 2013b; Thota et al., 2016; Varshney et al., 2018; Yadav et al., 2022).

The expression and purification of Fu24H have been described earlier (Mazumdar et al., 2010; Gupta et al., 2014). Briefly, Fu24H was cloned and expressed in an Escherichia coli system. The fusion protein was purified using Ni²⁺ affinity chromatography followed by anion-exchange chromatography. Protein purity was analyzed by RP-HPLC on a C-18 column (Phenomenex) using a 5%–95% gradient (1 mL/min) of acetonitrile-water containing 0.1% TFA and 15% SDS-PAGE electrophoresis, followed by a western blot. For the western blot analysis, Fu24H was separated on 15% SDS-PAGE and transferred to a nitrocellulose membrane. After blocking with 5% skimmed milk in PBS at 37°C for 2 h, the blot was first incubated with a PfMSPFu24-specific antibody and then with an HRP-conjugated anti-mouse IgG secondary antibody (Sigma, St. Louis, MO, United States) for 1 h. After washing, the bound protein in the immunoblot was detected with Pierce^™ ECL Western Blotting Substrate (Thermo Scientific, Rockford, IL, United States) and 1 μL/mL H₂O₂ in PBS.

The Fu24H was mixed with preformed R∆F NPs at a ratio of 1:10; 1:20; 1:30; 1:40; 1:50 (R∆F: Fu24H, w/w), followed by overnight incubation at 25°C with gentle mixing. The reaction mixture was ultracentrifuge at 1.2 lakh rpm for 3 h at 25°C. To measure entrapment efficiency, an aliquot of 25 μL supernatant was transferred to a 96-well plate having BCA reagent (Pierce^™ BCA Protein Assay Kit, Thermo Scientific, United States) and the reaction mixture was mixed well and incubated for 30 min at room temperature. Absorbance was measured at 562 nm using UV-visible spectroscopy (VersaMax, Molecular Devices, Sunnyvale, California, United States), and entrapment efficiency was calculated as follows: % Entrapment efficiency = Amount of Fu24H in R∆F NPs/Total amount of Fu24H added x 100.

The size and morphology of the R∆F NPs and R∆F-Fu24H complex were studied using TEM. An aliquot of 20 µL of R∆F NPs and R∆F-Fu24H complex was loaded onto carbon-coated copper grids, followed by incubation for 45 min at room temperature. The excess sample from the grids was removed using a Whatman filter paper, and the samples were negatively stained with 1% uranyl acetate (filtered with a 0.22 µm filter). The grids were visualized using the 120 kV mode of TEM (Tecnai 12 BioTWIN, FEI Netherlands) and micrographs were analyzed using Analysis II (Megaview, SIS, Germany) software.

HEK 293T and WRL 68 cell lines were maintained in DMEM with 3.7 g sodium bicarbonate, 3.7 g HEPES, 10% FBS, and 0.1% penicillin-streptomycin. All cell lines were maintained in a humidified incubator at 37°C with 5% CO₂ and passaged at a confluency of 70%–80%.

For toxicity studies, 5 × 10³ cells/well (HEK 293T and WRL 68 cell lines) were seeded in a flat-bottomed 96-well plate and incubated at 37°C with 5% CO₂. After 12 h, cells were treated with RΔF NPs, R∆F-Fu24H complex at different concentrations (10 μg/mL to 50 μg/mL) for 24 h, followed by the addition of 20 μL of MTT reagent (5 mg/mL in PBS). After 3 h of incubation, media was removed and formazan crystals were dissolved in 100 µL of dimethyl sulfoxide (DMSO). The cell viability was measured on a microplate reader VersaMax ELISA reader (Molecular Devices, Sunnyvale, California, United States) at 570 nm and cell viability (%) was calculated using the equation: (Absorbance of treated cells/Absorbance of control) x100.

Human O+ Red blood cells (RBC) stored in 10% citrate-phosphate-dextrose were obtained from the Rotary Blood Bank (New Delhi, India). RBCs were washed three to five times using phosphate-buffered saline (PBS) and centrifuged at room temperature at 1,500 g for 10 min. The packed cell volume obtained was used to make a 10% (v/v) suspension in PBS. Aliquots of 100 μL of the packed RBC suspension were transferred to a 96-well microtiter plate (Corning, New York, United States) and 100 μL of different concentrations of R∆F NPs alone (10–50 μg/mL) and R∆F-Fu24H complex (25–50 μg/mL) were added to the respective wells. The plate was incubated at 37°C for 3 h and then centrifuged at room temperature at 1,500 g for 10 min. An aliquot of 100 μL supernatant was transferred to a new microtiter plate and absorbance was measured at 540 nm using a microplate reader VersaMax ELISA reader (Molecular Devices, Sunnyvale, California, United States) to measure RBC lysis. Cells incubated with PBS alone acted as the negative control, and RBCs treated with 0.2% Triton X-100 were used as a positive control.

Mice were intravenously administered with R∆F NPs at 60 mg/kg concentration and continuously monitored for any behavioural and body weight changes. For the in vivo toxicity study, a dose of 60 mg/kg for R∆F NPs was used which was 1.2 times excess to the dose utilised for in vivo immunisation studies (50 mg/kg). Post NPs administration, animals were kept under observation for 30 min. After 6 h of dosing, mice were euthanized using CO₂ inhalation and blood with other major organs including liver, kidney, spleen, and heart were collected for further analysis by complete blood count (CBC) and histopathology studies. All observations were systematically recorded and animals with any moribund condition or severe pain or enduring signs of severe distress were humanely killed without delay.

DCs and T cells (>90% pure) were purified from splenocytes of BALB/c mice (6–8 weeks) using 30% BSA in PBS, anti-CD11c-biotin and anti-CD3-biotin (from Biolegend) antibodies, and Streptavidin MicroBeads as described earlier as per the manufacturer’s instructions (Miltenyi Biotec) (Tamura et al., 2005; Verma et al., 2021). DCs and T cells (>90% pure) fractions purified from separate spleens were incubated in the ratio of 1:5 (1 × 10⁴ DCs and 5 × 10⁴ T cells) in the U bottom 96 well plate. After 12 h of incubation, the cells were stimulated with a serial dilution of R∆F NPs from 5 μg/mL to 20 μg/mL and MAGE 3 at 10 μg/mL, which was used as a positive control. After 72 h, the cells were centrifuged at 2000 rpm for 10 min at 4°C. Supernatants were collected, and T cells responses were measured by quantifying IFN-γ using an enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s instructions (R&D Systems, United States).

BALB/c mice were obtained from the Jackson Laboratory and housed in the animal house facility of the International Centre for Genetic Engineering and Biotechnology (ICGEB) under pathogen-free conditions. 6–8 weeks old female mice, were randomly allocated into two groups with 6 mice/group. Mice were immunized with an aliquot of 25 µg of Fu24H formulated with Alhydrogel^® and R∆F NPs. As reported in our earlier published work, the dose of 25 µg of the protein antigen in mice has been optimised and has been used in different formulations, including Alhydrogel and nanostructures like FΔF and LΔF hydrogels (Mazumdar et al., 2010; Gupta et al., 2014; Anand et al., 2021). Animals were immunized intramuscularly at 0 days, 28 days, and 56 days. Sera were collected before immunization at day −2 and at day 70 after the last immunization.

Sera collected from individual immunized mice (n = 6/group) were measured for anti-Fu24H antibody response by ELISA. Briefly, flat-bottom 96 well, microtiter ELISA plates (MaxiSorp, Nunc) were coated with Fu24H (2 μg/mL) in a carbonate-bicarbonate buffer and incubated overnight at 4°C. Plates were washed three times with phosphate-buffered saline (PBS) containing 0.05% Tween 20 (PBST) and blocked with 2% skimmed milk powder in PBS (pH 7.2) for 2 h at 37°C. The immune sera were three-fold serially diluted in PBST containing 0.25% skimmed milk powder and added to antigen-coated plates and incubated for 1 h at 37°C. Following three washes with PBST, goat anti-mouse IgG conjugated with horseradish peroxidase (Sigma) was added to the wells and incubated for 1 h at 37°C. After final washings with PBST followed by PBS, 1 mg/mL o-phenylene diamine dihydrochloride (Sigma) as chromogen and hydrogen peroxide as substrate were added to the plate. The color was allowed to develop for 20 min in dark and the reaction was stopped with 2.0 N sulfuric acid. The absorbance was measured at 490 nm using a VersaMax ELISA reader (Molecular Devices, Sunnyvale, California, United States). End-point IgG titers were established as the mean plus three standard deviations of absorbance of pre-immune sera. The end-point titer was calculated using a 4-parameter curve fitting model (GraphPad Prism Software, version 6.01, San Diego, CA, United States). The endpoint titer is defined as the reciprocal of the highest dilution of a serum that gives an absorbance above the cut-off OD value. And the cut-off value is defined as the mean plus three standard deviations of absorbance of pre-immune sera.

Cytokines were quantified in sera collected from individual mice (n = 6/group) before immunization on day −2 and 2 weeks after the last immunization at 70 days. The levels of pro-inflammatory cytokines: IFN-γ, TNF-α, IL-12, IL-6; and anti-inflammatory cytokines: IL-10 and IL-4 in the serum were measured using a capture ELISA according to the manufacturer’s protocol (R&D System, United States). Before the assay, sera samples were diluted 1:4 in sample diluent. Concentrations were determined by generating standard curves for each cytokine using the cytokine standard provided with the kit.

Sera collected from immunized mice (n = 6/group) were tested for their ability to inhibit the P. falciparum parasite (3D7 lab strain) replication by an in vitro growth inhibition assay (GIA), as described previously (Mazumdar et al., 2010; Gupta et al., 2014; Anand et al., 2021). For the parasite culture, human O+ RBCs were procured from Rotary blood bank, New Delhi, India. P. falciparum parasites were cultured in human O+ RBCs and synchronized through treatments with Percoll and sorbitol. For the invasion inhibition assay, synchronized parasites were adjusted to 2% hematocrit and 0.3% parasitemia in a late-trophozoites/early-schizonts stage and incubated in the presence or absence of sera. The sera were added to the parasite culture at a final dilution of 1:10 and incubated for one cycle (44 h post-invasion) under a mixed gas environment (90% N_2, 5% CO₂, 5% O₂). Parasite growth was assessed by staining parasite-infected RBCs with ethidium bromide (EtBr) at a concentration of 10 μg/mL in PBS and measured by flow cytometry. Invasion inhibition was calculated with respect to pre-immune sera as % inhibition = 1—[(percent invasion from immune sera)/(percent invasion from preimmune sera)] × 100.

Data were analysed using GraphPad Prism Software, version 6.01(San Diego, CA, United States). Data are shown as mean ± standard deviation (SD). For antibody titers, the log10-transformation of the titers were statistically compared. Differences between the groups were compared using unpaired Student’s t-test after confirming the assumptions of normality. Differences were considered significant at a p-value < 0.05 and n = sample size (biological repeat).

R∆F (Figure 1A) was synthesized using the standard solution-phase synthesis procedure (Supplementary Text S1). The dipeptide was characterized by RP-HPLC and mass spectrometry. RP-HPLC profile of R∆F showed a single peak at 19 min and a purity of more than 95% (Figure 1B). Mass spectrometry analysis indicated that the observed molecular mass of R∆F was 320.2 Da (319.3 Da + 1H⁺) (Figure 1C). Self-assembly of the dipeptide was initiated by solubilising R∆F in HFIP and then adding water to the mixture. R∆F at 1 mg/mL self-assembled to form homogeneous nanospheres having a hydrodynamic diameter of 368.62 ± 108.4 nm with a PDI of 0.22 ± 0.06 as assessed by DLS (Figure 1D). Zeta sizer analysis showed that R∆F NPs acquired a surface charge of ∼5.16 ± 1.01 mV (Figure 1E). TEM images revealed that R∆F NPs formed well-defined spherical structures with a diameter in the range of ∼50–100 nm (Figure 1F).

The purity of Fu24H was analyzed by RP-HPLC. Fu24H eluted as a single peak at a retention time (RT) of 29 min with more than 98% purity (Figure 2A). Purified Fu24H was observed as a single band on 15% SDS-PAGE and by western blot (Figures 2B, C and Supplementary Figure S1).

We determined whether R∆F NPs form a stable complex with Fu24H NPs. Fu24H (25 µg) were mixed with different ratios of R∆F NPs (Fu24H: R∆F NPs; 1:10, 1:20, 1:30, 1:40, 1:50; w/w) and incubated overnight at 25°C with gentle mixing. At a ratio of 1:50, (Fu24H: R∆F NPs) optimum entrapment (61.44% ± 1.40%) was observed and this ratio was used in all further experiments (Figure 3A). R∆F-Fu24H complex showed a surface charge of ∼19.87 ± 1.11 mV in the zeta sizer (Figure 3B). Whether the morphology of RΔF nanoparticles changed upon complexation with Fu24H was investigated by TEM. TEM images of RΔF showed no significant morphological changes in the shape after loading with Fu24H antigen (size < 150 nm), but a nanoparticle cluster formation was observed (Figure 3C).

After confirming the physicochemical characteristics of R∆F NPs and R∆F-Fu24H complex, we next investigated their potential biological properties, including cellular toxicity, hemocompatibility, and anti-inflammatory effects. The cellular toxicity of R∆F NPs and R∆F-Fu24H complex were tested on different cell lines, HEK 293T, and WRL 68 cells, using the standard MTT assay. No cytotoxicity was observed in any of the cell lines when treated with R∆F NPs alone or with R∆F-Fu24H complex (Figures 4A–C). Since injected nanoparticles would inevitably come into contact with blood, it is critical to assess the potential toxicity of R∆F NPs and R∆F-Fu24H complex on RBCs (Chen et al., 2015). In this context, we investigated whether R∆F NPs and R∆F-Fu24H complex caused hemolysis in RBCs. When used at the highest concentration of 50 μg/mL, both R∆F NPs and R∆F-Fu24H complex resulted in less than 5% hemolysis and caused no damage to RBCs (Figure 4D). This result confirms the hemocompatibility of the nanoparticles. Nanoparticles have been shown to influence inflammatory cell responses. We, therefore, investigated whether R∆F NPs activate an inflammatory response when murine dendritic cells and T cells (DC-T) were cocultured in the presence of RΔF NPs. Our result showed that RΔF NPs did not stimulate production of the inflammatory cytokine IFN-γ in the DC-T coculture (Supplementary Figure S1). These findings indicated the high biocompatibility of R∆F NPs in vitro.

To determine the toxicity of R∆F NPs in vivo, mice were injected intravenously with the NPs, and the toxicity was evaluated by analyzing histological and haematological data, as well as animal behavioural patterns. Compared to the PBS-treated control group, the mice treated with R∆F NPs showed no abnormal behaviour. At the end of the experiment, major organs (heart, liver, kidney, and spleen) were harvested, and a microscopic examination of the tissue was performed after staining with hematoxylin and eosin (H&E). Histopathological images revealed there was no tissue damage or abnormalities in mice treated with the NPs compared to control group (Supplementary Figure S2). We further evaluated potential hematotoxicity of the nanoparticles in mice and blood was collected after euthanasia for complete blood count (CBC). CBC is a panel of blood tests used to know important details about the blood’s cellular composition. CBC parameters, such as White blood cells, Red blood cells, Haemoglobin, Haematocrit, Total Leucocyte Count, Platelet Count, Neutrophils, and Lymphocytes, were all within the normal range for NPs and PBS groups (Table 1). Results of in vivo toxicity study indicated that R∆F NPs did not produce any behavioural changes and there was no clinical toxicity in mice treated with NPs compared to the non-treated group. These results were in line with the earlier studies (Yadav et al., 2022).

Six BALB/c mice per group were given three intramuscular injections of 25 µg of Fu24H formulated with either R∆F NPs or Alhydrogel^® as an adjuvant as per the schedule shown in Figure 5A . Serum samples were collected prior to immunization and after the third immunization on day 70, and Fu24H-specific IgG titers were determined. Pre-immune sera from both groups showed no antibody titre to Fu24H. Fu24H-R∆F complex induced high Fu24H-specific IgG titers (4.3 × 10⁵), which is comparable to the antibody titre induced by FDA-approved adjuvant Alhydrogel®-Fu24H (4.9 × 10⁵) (p > 0.05 by Student’s t-test) (Figure 5B).

Next, we tested the invasion inhibitory activity of the antibodies present in the day 70 immune sera by performing a growth inhibition activity (GIA) assay on P. falciparum laboratory line 3D7. The GIA assay was used to compare the effectiveness of immune sera from R∆F-Fu24H complex and Alhydrogel®-Fu24H in blocking P. falciparum parasite invasion of erythrocytes. Figure 5C depicts the growth-inhibitory properties of the resulting antiserum. Immune sera from the R∆F-Fu24H complex and Alhydrogel®-Fu24H groups were able to inhibit erythrocyte invasion of P. falciparum by 40.1% and 37.8%, respectively, when tested at a 1:10 serum dilution. There was no statistically significant difference in the efficiency of inhibition by sera from R∆F-Fu24H complex and Alhydrogel®-Fu24H groups. These immunogenicity results suggest that R∆F NPs can effectively deliver antigens and elicit functional antibodies which partially inhibit parasite growth in vitro.

To determine the type of cytokine response induced by R∆F-Fu24H complex or Alhydrogel^®-Fu24H, sera from day 70 post-immunization were tested for Th1 (IFN-γ, IL-12p70), Th2 (IL-4, IL-10), and inflammatory (IL-6, TNF-α) cytokines. Compared to Alhydrogel®-Fu24H, the R∆F-Fu24H complex induced significantly higher levels of IFN-γ (131.76 ± 32.75 pg/mL vs. 205.61 ± 44.26 pg/mL; p = 0.0082), TNF-α (131 ± 14.27 vs. 159.95 ± 15.18 pg/mL; p = 0.0079), IL-6 (17.01 ± 4.95 pg/mL vs. 41.39 ± 19.49 pg/mL; p = 0.004), IL-4 (49.98 ± 18.12 pg/mL vs. 71.84 ± 13.59 pg/mL; p = 0.039) and IL-10 (40.70 ± 14.08 pg/mL vs. 73.75 ± 9.28 pg/mL; p = 0.0038). There was no significant difference in the level of IL-12p70 (60.34 ± 26.31 pg/mL vs. 46.81 ± 17.27 pg/mL; p = 0.31) in mice immunized with Fu24H-R∆F complex or Alhydrogel®-Fu24H (Figure 6). These findings suggest that the cytokine response induced by the R∆F-Fu24H complex is comparable to that induced by Alhydrogel^®.