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Journal of Controlled Release2010ResearchNon-viral Gene Delivery

Targeted PLGA nano- but not microparticles specifically deliver antigen to human dendritic cells via DC-SIGN in vitro

Luis J. Cruz, Paul J. Tacken, Remco Fokkink, Ben Joosten, Martien Cohen Stuart, Fernando Albericio, Ruurd Torensma, Carl G. FigdorDOI 10.1016/j.jconrel.2010.02.013

Summary

Antibody-mediated targeting of vaccine components to dendritic cells (DCs) enhances vaccine efficacy, but linking multiple antigens and immune modulators to a single antibody is limited. Slow-release PLGA particles conjugated to DC-specific antibodies could overcome this by delivering large antigen payloads and enabling co-encapsulation of adjuvants. However, it was unknown whether nano- or micrometer-sized PLGA particles are better suited for. Particle characterization: MPs ~2 µm, NPs ~200 nm; PEG-lipid coating reduced zeta potential; antigen encapsulation 78–91%; antibody conjugation ~20–30 µg/mg PLGA. - Antigen degradation kinetics: Encapsulated antigen.

Keywords

PLGADendritic cellsT cellsAntigen presentationNanoparticlesPolymericVaccine delivery
Purpose: Antibody-mediated targeting of vaccine components to dendritic cells (DCs) enhances vaccine efficacy, but linking multiple antigens and immune modulators to a single antibody is limited. Slow-release PLGA particles conjugated to DC-specific antibodies could overcome this by delivering large antigen payloads and enabling co-encapsulation of adjuvants. However, it was unknown whether nano- or micrometer-sized PLGA particles are better suited for specific targeting to human DCs via DC-SIGN, and how particle size affects uptake specificity, antigen degradation, and presentation.
Hypothesis: If PLGA nano- and microparticles are coated with PEG-lipid and conjugated with the humanized anti-DC-SIGN antibody hD1, then targeted nanoparticles (NPs), but not microparticles (MPs), will specifically deliver antigen to human DCs via DC-SIGN, enhance antigen presentation, and induce antigen-dependent T cell responses at lower concentrations than non-targeted NPs. MPs will be taken up more nonspecifically due to their larger size and phagocytic mechanisms.
Aims: Primary aim: Engineer versatile nano- and micrometer-sized slow-release PLGA vaccine delivery vehicles that specifically target human DCs via DC-SIGN.
  • Secondary aim 1: Characterize particle size, zeta potential, PEG-lipid coating, antibody conjugation, and antigen encapsulation efficiency.
  • Secondary aim 2: Determine the kinetics and subcellular location of PLGA-encapsulated antigen degradation within human DCs.
  • Secondary aim 3: Compare specific uptake and processing of targeted NPs vs MPs by human DCs and nonspecific interactions with human blood cells in a mixed leukocyte population.
  • Secondary aim 4: Assess antigen presentation and T cell proliferation induced by targeted vs nontargeted NPs and MPs.
Delivery system:

Component: Polymer; Description: PLGA (Resomer RG 502 H, lactide:glycolide molar ratio 48:52 to 52:48)

Component: Particle Type; Description: Nanoparticles (NPs, ~200 nm) and microparticles (MPs, ~2 µm) prepared by emulsion solvent evaporation/extraction

Component: Targeting Ligand; Description: Humanized anti-DC-SIGN antibody hD1 (IgG2/IgG4 composite; Fc tail does not react with complement or Fc receptors)

Component: Isotype Control; Description: h5G1.1 (Eculizumab)

Component: Coating; Description: Lipid-PEG layer: DSPE-PEG(2000)maleimide and mPEG 2000 PE

Component: Conjugation Chemistry; Description: Sulfhydryl groups introduced into antibodies via SATP, conjugated to maleimide-functionalized PEG-lipids

Component: Payload; Description: FITC-labeled tetanus toxoid (TT) peptide 830–844 (FITC-KKQYIKANSKFIGITEL-NH₂) with cathepsin cleavage site; DQ-BSA (self-quenched model protein antigen) for degradation studies

Component: Particle Sizes; Description: MPs: ~2 µm (2099 ± 144 nm); NPs: ~200 nm (239 ± 14 nm)

Component: Zeta Potential; Description: PLGA-FITC-TT-PEG MP: −32.7 ± 1.6 mV; PLGA-FITC-TT-PEG NP: −28.6 ± 0.4 mV

Component: Antigen Encapsulation; Description: MPs: 89.8 ± 1.9%; NPs: 78.2 ± 1.8% (for FITC-TT)

Component: Antibody Content; Description: MPs: ~30.6 ± 2.1 µg/mg PLGA; NPs: ~20.0 ± 1.2 µg/mg PLGA

Component: Key Design Feature; Description: PEG-lipid layer provides stealth properties and enables oriented antibody conjugation; DC-SIGN targeting for specific delivery to human DCs

Approach: In Vitro Only — Human Cells: - DCs: Monocyte-derived DCs from healthy donors, cultured with IL-4 and GM-CSF for 6 days. - PBLs: Peripheral blood lymphocytes from TT-responsive donors, restimulated once with TT peptide. - Leukocytes: Mixed blood cell population from buffy coats, co-cultured with DCs at 1:20 ratio. - Groups compared: hD1-targeted NPs and MPs; isotype control (h5G1.1) NPs and MPs; no antibody NPs and MPs; soluble antigen controls. - Doses: Binding/uptake: 100 µg/mL particles; degradation: 0.5 µg DQ-BSA; antigen presentation: various particle concentrations (0.01–1000 ng/mL peptide equivalent). - Time points: Binding at 4°C for 1 h; uptake at 37°C for 0, 1, 18 h; degradation up to 6 days; antigen presentation 2 days + 4 days co-culture.

No in vivo studies.

Key methods:

Technique: Scanning electron microscopy (SEM); Purpose: Particle morphology and size

Technique: Dynamic light scattering (DLS); Purpose: Particle diameter and size distribution

Technique: Zeta potential measurement; Purpose: Surface charge and PEG-lipid coating confirmation

Technique: Flow cytometry; Purpose: Antibody conjugation, particle binding/uptake, DC maturation markers

Technique: Confocal laser scanning microscopy; Purpose: Particle uptake, subcellular localization, antibody surface presence

Technique: DQ-BSA degradation assay; Purpose: Fluorescence dequenching to quantify antigen degradation within DCs

Technique: Live cell imaging with LysoTracker Red; Purpose: Lysosomal localization of antigen degradation

Technique: T cell proliferation assay (³H-thymidine); Purpose: Antigen presentation to autologous TT-responsive T cells

Technique: Coomassie protein assay; Purpose: Antigen encapsulation efficiency

Key results: Particle characterization: MPs ~2 µm, NPs ~200 nm; PEG-lipid coating reduced zeta potential; antigen encapsulation 78–91%; antibody conjugation ~20–30 µg/mg PLGA. - Antigen degradation kinetics: Encapsulated antigen degraded ~13% after 1 day and ~38% after 6 days in DCs; no significant difference between NPs and MPs. Soluble antigen degraded much faster: 27% at 2 days and 94% at 6 days. Degradation occurred within lysosomal compartments within 1 h. - DC uptake: Targeted hD1-NPs showed specific binding and rapid uptake by DCs; isotype and no-antibody NPs showed minimal binding. Targeted hD1-MPs showed high nonspecific uptake, with only marginal enhancement by targeting. In mixed leukocyte populations, targeted NPs were more specifically delivered to DCs (only 5% relative uptake by leukocytes vs DCs), while MPs were scavenged more by leukocytes (11% relative uptake). - Antigen presentation: Targeted NPs induced T cell proliferation at 10–100 fold lower concentrations than nontargeted NPs. Targeted MPs did not significantly enhance antigen presentation over nontargeted MPs. - DC maturation: Particle uptake did not enhance expression of CD80, CD83, or CD86 (data not shown).
Interpretation: The authors conclude that DC-SIGN targeting of PLGA nanoparticles, but not microparticles, effectively and specifically delivers antigen to human DCs and enhances antigen presentation. Nanoparticles are more suitable for in vivo DC targeting due to their specific uptake, reduced nonspecific interactions with blood cells, and ability to induce T cell responses at lower antigen doses. This is the first study assessing antibody-mediated delivery of PLGA NPs and MPs to human DCs and supports targeted NP-based vaccine therapeutics.
Limitations: In vitro only: No in vivo validation, biodistribution, or efficacy in animal models. - Monocyte-derived DCs: May not fully reflect in vivo DC subsets (e.g., CD1c⁺ and CD141⁺ myeloid DCs). - Limited donors: Antigen presentation experiments used donors responding to TT antigen; donor variability not fully addressed. - No DC maturation/activation assessment: Particles did not induce maturation markers; co-encapsulation of adjuvants was not tested. - No toxicity or safety data reported for targeted particles. - Only one targeting receptor (DC-SIGN) and one antigen (TT peptide) tested; generalizability to other targets/antigens not established. - Particle stability and storage not evaluated. - Scale-up and GMP manufacturing not addressed.

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Targeted PLGA nano- but not microparticles specifically deliver antigen to human dendritic cells via DC-SIGN in vitro | Brilliant Blue Biosciences