Amino-functionalized poly(l-lactide) lamellar single crystals as a valuable substrate for delivery of HPV16-E7 tumor antigen in vaccine development

Figures & data

Figure 1 Scanning electron micrographs of PLLA_sc (A) and APLLA_sc (B).

Notes: A drop of PLLA_sc and APLLA_sc suspensions in isopropanol was deposited on the sample holder. The magnitude scale bars are indicated.

Abbreviations: PLLA_sc, poly(l-lactide) single crystals; APLLA_sc, amino-functionalized poly(l-lactide) single crystals.

Figure 2 Attenuated total reflection Fourier transform infrared spectra of PLLA_sc and APLLA_sc (A). Differential scanning calorimetry thermograms of PLLA_sc and APLLA_sc recorded at 10°C per minute (B).

Note: The arrow indicates the amide C=O stretching and N-Hbending large peak (A).

Abbreviations: PLLA_sc, poly(l-lactide) single crystals; APLLA_sc, amino-functionalized poly(l-lactide) single crystals.

Table 1 Advancing and receding contact angles of pristine and APLLA films in water measured using a stage speed of 60 μm per second

Sample	θa(°)	θr(°)	Δθ = θa–θr(°)
Pristine PLLA film	96.2±0.2	29±5	67±5
APLLA film	85±2	5±3	80±5

Note: The data are reported as the mean ± standard deviation (n=5).

Abbreviations: PLLA, poly(l-lactide); APLLA, amino-functionalized poly(l-lactide).

Figure 3 Protein release experiment.

Notes: E7-PLLA_sc and E7-APLLA_sc suspensions were centrifuged and rinsed in phosphate-buffered saline five times. The released E7 was quantify in the supernatant after each rinse step. The y axis reports the cumulative quantity of E7 released with respect to that initially adsorbed on the PLLA substrates. The x axis indicates each single wash step. The values are the mean of three determinations.

Abbreviations: PLLA, poly(l-lactide); E7-PLLA_sc, E7-containing poly(l-lactide) single crystals; E7-APLLA_sc, E7-containing amino-functionalized poly(l-lactide) single crystals.

Figure 4 Scanning electron micrographs of E7-PLLA_sc (A) and E7-APLLA_sc (B) aggregates.

Notes: A drop of either E7-PLLA_sc or E7-APLLA_sc suspension, with the same concentration employed for subcutaneous mouse immunization, was deposited on the sample holder. The magnitude scale bars are indicated.

Abbreviations: E7-PLLA_sc, E7-containing poly(l-lactide) single crystals; E7-APLLA_sc, E7-containing amino-functionalized poly(l-lactide) single crystals.

Table 2 Sera pool titration by endpoint dilution ELISA of antibodies against E7 after the second and third doses of vaccine

Antigen	Dose 2	Dose 3
E7-APLLAsc	1:16,000	1:32,000
E7-PLLAsc	1:2,000	1:4,000
E7	1:4,000	1:8,000
APLLAsc	1:1	1:1
PLLAsc	1:1	1:1

Note: The endpoint titer was an optical density of 0.1.

Abbreviations: PLLA_sc, poly(l-lactide) single crystals; APLLA_sc, amino-functionalized poly(l-lactide) single crystals.

Figure 5 Determination of immunoglobulin isotypes by enzyme-linked immunosorbent assay.

Notes: The E7-specific IgG1, IgG2b, IgG2c and IgG3 immune reactivity is shown in OD₄₅₀ values on the y axis. The bars represent pools from sera of mice groups immunized with free E7 (E7, white bars), E7-PLLA_sc (gray bars), and E7-APLLA_sc (black bars). The four isotypes are indicated on the x axis along with the result of a total IgG enzyme-linked immunosorbent assay performed in parallel as control using the same serum pools. *Statistically significant result (P<0.05).

Abbreviations: E7-PLLA_sc, E7-containing poly(l-lactide) single crystals; E7-APLLA_sc, E7-containing amino-functionalized poly(l-lactide) single crystals; Ig, immun oglobulin.

Figure 6 INF-γ-secreting cells from mice immunized with E7-APLLA_sc, E7-PLLA_sc and free E7 (E7).

Notes: The cells were stimulated with either an unrelated mixture of peptides (unp, black bars) or two CTLs E7 peptides (pE7, grey bars). *P=0.009; **P=0.01.

Abbreviations: unp, unrelated mixture of peptides; pE7, E7 peptide; IFN-γ, interferon gamma; E7-PLLA_sc, E7-containing poly(l-lactide) single crystals; E7-APLLA_sc, E7-containing amino-functionalized poly(l-lactide) single crystals.

Figure 7 Tumor protection experiment.

Notes: Mice naïve or vaccinated with three doses of free E7 (E7), E7-APLLA_sc, E7-PLLA_sc, APLLA_sc and PLLA_sc were challenged with 1×10⁵ TC-1 tumor cells and tumor growth was monitored weekly. The x axis indicates weeks of monitoring after tumor challenge and the y axis indicates the percentages of animals without tumor.

Abbreviations: E7-PLLA_sc, E7-containing poly(l-lactide) single crystals; E7-APLLA_sc, E7-containing amino-functionalized poly(l-lactide) single crystals.