Tumor targeting nanoparticle E7_49-57-HSP110-RGD elicits potent anti-tumor immune response in a CD8-dependent manner in cervical cancer-bearing mouse model

Figures & data

Table 1. Sequence of peptides used in the present study

Peptides	Sequence	Abbreviation
RGD	ACRGDMFFCA	RGD
E749-57	RAHYNIVTF	E7
GGG-K18	GGG-KKKKKKKKKKKKKKKKKK	GK
RGD-GGG-K18	ACRGDMFFCA-GGG-KKKKKKKKKKKKKKKKKK	AK

Table 2. Immunization protocols of nanoparticle vaccine in C57BL/6 mice

Group	Formulations	Dose/each injection
1	AK/E7-HSP110	99.8 μg peptide + 74 μg plasmid/100 μl PBS
2	GK/E7-HSP110	59.6 μg peptide + 73 μg plasmid/100 μl PBS
3	E7-HSP110 plasmid	74 μg plasmid/100 μl PBS
4	HSP110 plasmid	73 μg plasmid/100 μl PBS
5	E7 plasmid	50 μg plasmid/100 μl PBS
6	AK peptide	99.8 μg plasmid/100 μl PBS
7	PBS	100 μl

Figure 1. Identification of biological features of nanoparticles

(a) Assembly pattern of nanoparticles. (b) DNA retardation assay. (c) DNase I protection assay. M, DNA Marker; DNA, plasmid DNA without peptide. (d) The particle size at charge ratio 2.0 was determined by TEM. (e) Nanoparticles range analysis with a peak at 100 nm.

Figure 2. AK-nanoparticles targeted TC-1 cells and expressed exogenous protein

FITC-AK and FITC-GK nanoparticles were incubated with TC-1 cells or HAVSMC cells (a), or 2 μg/μl of AK-labeled Rhodamine and a series of the indicated concentration of free RGD-GGG cyclic peptides were co-incubated with TC-1 cells (b), and the fluorescence on cells was determined by fluorescence microscope. (c) TC-1 cells were incubated with nanoparticles AK/E7, AK/HSP110, AK/E7-HSP110 or PBS. The cells were then subjected to 10% SDS-PAGE and subsequent Western-blotting assays with an anti-HSP110 antibody respectively. GAPDH was included as an internal control. *P< .01 vs other controls.

Figure 3. Nanoparticle AK/E7-HSP110 elicited E7_49-57-specific lymphocyte proliferation and cytolytic capacity

(a) Proliferation of splenocytes from mice immunized with indicated immunogens was determined, respectively. (b) Lymphocyte cytolytic capacity was determined by LDH release assay at the indicated various E/T ratios. *P< .01vs all controls. ^#P < .05 vs GK/HSP110. These experiments were repeated three times and data was represented by the mean ± SD (n = 5 mice).

Figure 4. Specific CD8⁺T-cell immune response promoted by nanoparticle AK/E7-HSP110 in mice

FCM assay for MHC I tetramers tracing staining in E7_49-57-specific CD4⁺ T cells (a) and CD8⁺ T cells (b) from mice vaccinated with the indicated immunogens. (c) FCM assay for IFN-γ secretion of the E7_49-57-specific CD8⁺ T cells in each group. The bar graph reflected the percentage of E7-specific CD8⁺ IFN-γ⁺ T cells. ^# P < .05 vs GK/E7-HSP110 control, * P< .01vs other controls. The experiments were repeated three times independently and the data were represented by the mean ± SD (n = 5 mice).

Figure 5. Preventative and therapeutic effects of vaccines on mice inoculated with TC-1 tumor cells

The tumor preventative (a) and therapeutic (b) experiments were conducted in C57BL/6 mice that were inoculated with the indicated immunogens, respectively. *p < .01, vs all control groups at the indicated time points. (c) Tumor challenge experiment in CD4- and CD8-knockout mice. Results were recorded as mean tumor volume of 10 mice per group.