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European Journal of Pharmaceutics and Biopharmaceutics2013ResearchNon-viral Gene Delivery

Optimization of encapsulation of a synthetic long peptide in PLGA nanoparticles: low burst release is crucial for efficient CD8⁺ T cell activation

A.L. Silva, R.A. Rosalia, A. Sazak, M.G. Carstens, F. Ossendorp, J. Oostendorp, W. JiskootDOI 10.1016/j.ejpb.2012.11.006

Summary

Overlapping synthetic long peptides (SLPs) are promising for therapeutic cancer vaccination, but Montanide-based formulations have limitations including non-biodegradability, local side effects, poorly controlled release, and limited scalability. PLGA nanoparticles (NPs) offer a biodegradable alternative, but efficient encapsulation of moderately hydrophobic SLPs with low burst release had not been achieved. Standard acidic inner phase: Encapsulation efficiency was very low (1–30%) and burst release was extremely high (>70%) upon resuspension in PBS or IMDM. - Critical role of inner phase pH: Alkaline inner aqueous phase.

Keywords

T cellsPLGANanoparticlesAntigen presentationControlled releasePeptidesPolymeric
Purpose: Overlapping synthetic long peptides (SLPs) are promising for therapeutic cancer vaccination, but Montanide-based formulations have limitations including non-biodegradability, local side effects, poorly controlled release, and limited scalability. PLGA nanoparticles (NPs) offer a biodegradable alternative, but efficient encapsulation of moderately hydrophobic SLPs with low burst release had not been achieved.
Hypothesis: If formulation and process parameters — particularly the pH of the first emulsion — are optimized, then PLGA NPs can efficiently encapsulate the model SLP OVA24 with low burst release, and this low burst release is crucial for efficient MHC class I-restricted CD8⁺ T cell activation. Specifically, an alkaline inner aqueous phase (above the peptide’s pI) will yield ~330 nm NPs with ~40% encapsulation efficiency and <10% burst release, enabling enhanced antigen cross-presentation compared with high-burst NPs or soluble peptide.
Aims: Primary aim: Optimize PLGA NP formulation for efficient encapsulation of a synthetic long peptide (OVA24) and controlled release.
  • Secondary aim 1: Investigate the effect of formulation and process parameters — surfactant type, inner solvent, inner phase volume, and pH — on encapsulation efficiency, particle size, zeta potential, and burst release.
  • Secondary aim 2: Characterize the optimized NP formulation for size, polydispersity, zeta potential, antigen loading, and release kinetics.
  • Secondary aim 3: Evaluate the in vitro potency of OVA24-loaded PLGA NPs to induce MHC class I-restricted CD8⁺ T cell activation and compare low-burst vs high-burst formulations.
Delivery system:

Component: Polymer; Description: PLGA (Resomer RG 502H)

Component: NP Type; Description: Nanoparticles prepared by double emulsion/solvent evaporation (w₁/o/w₂)

Component: Payload; Description: OVA24, a 24-residue synthetic long peptide (DEVSGLEQLESIINFEKLAAAAAAK) covering the CTL epitope SIINFEKL of ovalbumin

Component: Targeting Ligand; Description: None

Component: Surfactant; Description: PVA (1% w/v) in outer aqueous phase; Tween 20 or sodium cholate tested as alternatives

Component: Inner Phase Optimization; Description: Alkaline inner aqueous phase critical: 50% ACN + 0.25 mM NaOH + 400 µL 50 mM Hepes pH 8.0; final apparent pH ~8.0

Component: Inner Phase Volume; Description: 500 µL used in optimized formulation

Component: Optimized NP Properties; Description: Size ~328 nm; PDI ~0.22; zeta potential −13.6 mV; encapsulation efficiency ~38%; burst release <10%

Component: Release; Description: ~30% cumulative release over 24 h in PBS at 37°C; no further release observed up to 24 h

Component: Key Design Feature; Description: Inner phase pH above peptide pI (4.3) prevents surface adsorption and enables true encapsulation with low burst release

Approach: In vitro model only; no in vivo studies. - Cell lines: D1 dendritic cells (immature splenic DC line from C57BL/6 mice) and B3Z CD8⁺ T-cell hybridoma specific for H-2Kᵇ-restricted SIINFEKL, expressing β-galactosidase under the IL-2 promoter/NF-AT element. - Antigen presentation assay: DCs incubated for 2.5 h with formulations, washed, then co-cultured overnight with B3Z T cells. T-cell activation measured by CPRG colorimetric assay (OD 590 nm). - Formulations compared: Low-burst releasing (LBR) PLGA-OVA24 NP; high-burst releasing (HBR) PLGA-OVA24 NP; soluble OVA24; empty NP; soluble OVA24 + empty NP. - Release media: PBS, IMDM cell culture medium, 5% glucose; long-term release in PBS at 37°C under shaking. - n = 3 independent batches for most experiments; statistical analysis by t-test and ANOVA.
Key methods:

Technique: Dynamic light scattering (DLS); Purpose: Z-average size and polydispersity index

Technique: Laser Doppler electrophoresis; Purpose: Zeta potential

Technique: Reversed-phase HPLC; Purpose: Quantify OVA24 content, encapsulation efficiency, and release

Technique: Burst release assay at t₀; Purpose: Measure immediate peptide release upon resuspension in physiological media

Technique: Release kinetics over 24 h; Purpose: Assess sustained release profile

Technique: B3Z T-cell activation assay; Purpose: Measure MHC class I antigen cross-presentation and CD8⁺ T cell activation

Technique: Statistical analysis; Purpose: Two-tailed unpaired/paired Student’s t-test; two-way ANOVA

Key results: Standard acidic inner phase: Encapsulation efficiency was very low (1–30%) and burst release was extremely high (>70%) upon resuspension in PBS or IMDM. - Critical role of inner phase pH: Alkaline inner aqueous phase (apparent pH above peptide pI of 4.3) was essential. Optimized formulation (50% ACN + 0.25 mM NaOH + 400 µL 50 mM Hepes pH 8.0) yielded NPs of ~328 nm, PDI ~0.22, zeta potential −13.6 mV, encapsulation efficiency ~38%, and burst release <10%. - Release kinetics: Optimized low-burst NPs showed ~30% cumulative OVA24 release over 24 h in PBS at 37°C, with no further release thereafter. Free OVA24 aggregated in PBS at 37°C, limiting longer release studies. - CD8⁺ T cell activation: OVA24 encapsulated in PLGA NPs significantly enhanced B3Z CD8⁺ T cell activation compared with soluble OVA24. Low-burst releasing NPs induced significantly higher T cell activation than high-burst releasing NPs. Soluble OVA24 mixed with empty NPs did not improve activation, demonstrating that encapsulation is required.
Interpretation: The authors conclude that efficient entrapment of SLP in PLGA NPs with low burst release is crucial for potent MHC class I-restricted CD8⁺ T cell activation. The pH of the first emulsion — specifically an alkaline inner phase above the peptide’s pI — is the key parameter controlling encapsulation and release. This method may be a promising basis for SLP-based cancer immunotherapy and is potentially applicable to other amphiphilic/hydrophilic peptides.
Limitations: No in vivo studies; only in vitro antigen presentation and release. - One model peptide (OVA24) tested; broader applicability only preliminary/unpublished. - Release study limited by OVA24 aggregation in PBS at 37°C; no long-term release profile beyond 24 h. - No co-encapsulation of adjuvants (e.g., TLR ligands) or targeting ligands tested. - Particle size ~330 nm; optimal size for DC uptake and in vivo trafficking not evaluated. - Mechanism of encapsulation at higher pH not fully resolved; hypothesized based on peptide charge/hydrophobicity. - No toxicity, stability, or scalability data reported.

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Optimization of encapsulation of a synthetic long peptide in PLGA nanoparticles: low burst release is crucial for efficient CD8⁺ T cell activation | Brilliant Blue Biosciences