PLGA particulate delivery systems for subunit vaccines: Linking particle properties to immunogenicity

Figures & data

Table 1. Examples of analytical methods for characterization of antigen-containing PLGA particles.

Particle characteristic	Method
Particle size	Dynamic light scattering Nanoparticle tracking analysis Light obscuration Scanning electron microscopy Transmission electron microscopy Atomic force microscopy
Density	Density gradient centrifugation Helium compression pycnometry Resonant mass measurement
Crystallinity	X-ray diffraction Differential scanning calorimetry
Surface chemistry	X-ray photoelectron spectroscopy Nuclear magnetic resonance spectroscopy
Surface charge	Electrophoresis Laser Doppler velocimetry
Surface hydrophobicity	Hydrophobic interaction chromatography Contact angle measurement Two-phase partitioning
Antigen content, release and integrity	Bicinchoninic acid assay SDS-PAGE High performance size-exclusion chromatography Reverse-phase high performance liquid chromatography Enzyme-linked immunosorbent assay Fluorescence spectroscopy UV/VIS spectroscopy Fourier transform infrared spectroscopy Mass spectrometry

Table 2. Comparative studies about the effect of PLGA particle size on the observed immune response.

Formulation	Particle size	Antigen/TLRL	In vitro / in vivo	Adminstration route	Response	References
PLGA MPs	5 μm, 12 μm	HBsAg protein	In vitro and in vivo	Pulmonary	5 µm > 12 µm MPs uptaken by rat alveolar macrophages; Ab responses: 5 µm > 12 µm MPs	^49
PLGA NPs & MPs	200 nm, 500 nm, 1 μm	BSA protein	In vitro and in vivo	s.c.	Ab responses: 200 nm ˜500 nm < 1 μm particles.	^42
PLA NPs & MPs	200-600 nm, 2-8 µm	HBsAg protein	In vitro and in vivo	i.m.	NPs >> MPs uptaken by macrophages; MPs ↑ anti-HBsAg Ab responses and ↑ IL-4 secretion related to a Th2 response; NPs ↑ IFN-γ production and ↑ Ab isotype related to a Th1 response.	^50
PLA MPs	< 2 μm, 2-8 μm, 10-70 μm, 50-150 μm	TT	In vivo	i.m.	Ab responses ↑ by 2-8 µm MPs > > 10-70 μm ˜50-150 μm.	^48
PLGA NPs & MPs	500-600 nm, 3.5 μm	TT	In vivo	i.m.	NPs and MPs mixed together ↑ Ab responses > NPs ˜ MPs alone	^51
PLGA NPs & MPs	17 μm, 7 μm, 1 μm, 300 nm	OVA / Cpg ODN	In vitro and in vivo	i.p.	Particle uptake and upregulation of MHC class I and CD86 expression and ↑ OVA-specific CD8^+ T cells and ↑ IgG2a:IgG1 following the same size trend: : 17 μm << 7 μm < 1 μm < 300 nm	^34
PLGA NPs & MPs	300 nm,> 20 μm	OVA / poly(I:C)	In vitro and in vivo	s.c.	NPs >> MPs internalized by DCs and ↑ CD8^+ T cell activation in vitro; vaccination with NPs ↑ OVA-specific CD8^+ T cells & Ab production, MPs did not	^47
PLGA NPs & MPs	600 nm, 1 – 1.5 μm	OVA	In vitro	n/a	MPs > NPs induced in vitro MHC class I Ag cross-presentation	^52

Ab: antibody; Ag: antigen; <: less/lower than; >: more/higher than; <<: much less/lower than; >>: much more/higher than; ˜: similar; ↑: increased/high: ↓: decreased/low

Table 3. Examples of reports of PLGA formulations using Toll-like receptor ligands and their immunological effects.

Receptor	Ligand	Formulation	Antigen	In vitro / in vivo	Adminstration route	Response	References
TLR 1/2	Pam3CSK4	PLGA NPs (˜350 nm)	OVA24 peptide	In vitro and in vivo	s.c.	TLR 2 stimulation ↑ MHC class I presentation of OVA24-NPs by DCs in vitro and ↑ prolonged Ag presentation and CD8^+ T cell activation in vivo after adoptive transfer of NP-loaded DCs	^96
	Pam3CSK4	PLGA NPs (˜500) and MPs (˜2 μm; μm)	CS252-260 coupled to Pam3CSK4 (Pam-CS252-260)	In vivo	i.p	Pam-CS252-260 particles ↑ cytolytic activity > CS252-260-MPs or sPam-CS252-260; 500 nm NPs > 2 μm ˜ μm MPs inducing CTL responses	^95
TLR 3	Poly(I:C)	(DEAE)–dextran- PLGA MPs (˜3 μm)	FITC-BSA	In vitro	n/a	poly(I:C) coated-MPs ↑ expression of CD80, CD86, and CD83 at the DC surface ˜ cytokine cocktail or ↑ concentrations of sPoly(I:C).	^84
TLR 4	MPLA	PLGA MPs (1 – 10 μm)	OVA323-39 peptide; MUC1 mucin peptide	In vivo	s.c.	Ag/MPLA-MPs ↑ T cell proliferative response and production of IFN-γ by T cells, eliciting a specific Th1 immune response > Ag-MPs or Ag mixed with alum	^87,88
	MPLA	PLGA NPs (350 – 450 nm)	OVA protein	In vitro and in vivo	i.p. or s.c.	OVA/MPLA-NPs ↑ CD8^+ T cell proliferative responses & IFN-γ in vitro and >13-folds increase in clonal expanded CD4^+ T cells in vivo > OVA-NPs	^89
	MPLA	PLGA NPs (˜300 nm)	HBcAg protein	In vivo	s.c.	HBcAg/MPLA-NPs ↑ Th1 cellular response with predominant IFN-γ profile > sHBcAg, sHBcAg/sMPLA, or HBcAg-NPs	^91
	MPLA	PLGA NPs (˜500 nm)	HBcAg129–140	In vivo	s.c.	HBcAg129–140/MPLA-NPs ↑ Th1-type response > control formulation of HBcAg129–140in CFA	^92
	MPLA	PLGA NPs (350 – 450 nm)	OVA; MUC1 lipopeptide (BLP25)	In vitro and in vivo	n/a	OVA/MPLA-NPs ↑ in vitro and in vivo antigen-specific primary Th1 immune responses > OVA-NPs or sOVA/sMPLA after adoptive transfer of antigen-pulsed DCs; MUC1/MPLA-NPs delivery to DCs ↑ MUC1 reactive T cells in vitro > MUC1-NPs, MPLA-NPs, sMUC1, or sMUC1 with MPLA-NPs	^10
	7-acyl lipid A	PLGA NPs (350 – 410 nm)	TRP2180-188peptide	In vivo	s.c.	TRP2180-188/7-acyl lipid A-NPs ↑ CD8^+ T cell-mediated anti-tumor immunity and therapeutic anti-tumor effect and levels of IFN-γ and pro-inflammatory Th1-related cytokines > TRP2180-188-NPs	^90
	MPLA	PLGA NPs (˜80 nm)	TRP2180-188peptide	In vitro and in vivo	i.d.	NP ↑ uptake in vitro and in vivo; TRP2180-188/MPLA-NPs ↓ growth of s.c. inoculated B16 melanoma cells in a prophylactic setting > TRP2180-188-NPs, sTRP2180-188/sMPLA	^93
	MPLA or RC529	PLGA MPs (3 – 5 μm)	gp120 protein; MenB	In vivo	i.p.	Ag adsorbed on TLRL-MPs ↑ IgG serum titers > Ag adsorbed-MPs with sTLRL.	^94
TLR 9	CpG ODN	PLGA NPs (˜300 nm)	Tetanus toxoid (TT)	In vitro and in vivo	s.c.	TT/CpG-NPs ↑ antigen-specific T cell proliferation ex vivo & IFN-γ secretion and 16-fold IgG titers > sTT/sCPG; co-encapsulation ↑ Th1 and Th2 immune responses toward Th1 type bias.	^80
	CpG ODN	PLGA MPs (μm)	OVA protein; CpG-OVA conjugate	In vitro and in vivo	s.c.	OVA/CpG-MPs were uptaken by DCs in vitro; OVA/CpG-MPs ↑ Ag-specific CD4^+ and CD8^+ T cells ˜ CPG-OVA conjugates in vivo. In a tumor challenge, MPs caused complete tumor regression in 4 out of 5 mice.	^82
	CpG ODN	PLGA MPs (μm)	PLA2 protein	In vivo	s.c.	PLA2/CPG-MPs ↑ PLA2-specific Ab responses and ↑ Th1-associated isotype IgG2a. The effect of CpG ↑ when protamine was co-encapsulated for complexation of CpG.	^76
	CpG ODN	bare, chitosan-coated, and protamine-coated PLGA MPs (μm)	SIINFEKL peptide	In vivo	s.c.	Only uncoated SIINFEKL-MPs with adsorbed CpG ↑ IFN-γ secreting and SIINFEKL-specific CD8^+ T cells.	^77
	CpG ODN	PLGA MPs (˜1 – 1.5 μm) coated with CTAB or DSS	p55 gag or gp120 env proteins	In vitro and in vivo	i.m.	CpG adsorbed to PLGA-CTAB MPs co-administered with gp120 env or p55 gag proteins adsorbed to PLGA-DSS MPs ↑ Ag-specific serum IgG titers, as well as CTL responses against p55 gag > sCp/sAg,	^102
	CpG ODN-chitosan complexes	PLGA 502 and 752 MPs (˜1 – 2 μm)	OVA protein	In vivo	i.d.	OVA/CpG-MPs ↑ Ab response and isotype shifting to Th1 > OVA- MPs.	^81
TLR 9 & TLR 3	CpG ODN or Poly(I:C)	PLGA MPs (μm)	OVA protein	In vivo	s.c.	CpG/OVA- or poly(I:C)/OVA-MPs ↑ (i) SIINFEKL/H-2Kb tetramer positive CTLs, (ii) IFN-γ production, (iii) in vivo cytotoxicity and (iv) protection from vaccinia virus > to OVA-MPs with sTLRL or OVA-MPs with TLRL-MPs.	^79
	CpG ODN & Poly(I:C)	PLGA MPs (˜0.5 - 5 μm)	OVA protein	In vivo	s.c.	OVA/CpG-MPs with MP-poly(I:C) ≥ IFA in eradication of preexisting tumors and suppression of lung metastases	^85
	CpG ODN or/and Poly(I:C)	PLGA NPs (˜1 μm)	OVA protein	In vitro		poly(I:C)/OVA- or CpG/OVA-NPs ↑ prolonged MHC class I- & II-restricted presentation and ↑ OVA-specific CD8^+ and CD4^+ T cells; combination of both TLRLs synergistically ↑ MHC class I-restricted, but not class II, Ag presentation.	^86

Ab: antibody; Ag: antigen; <: less/lower than; >: more/higher than; <<: much less/lower than; >>: much more/higher than; ≥: equal or higher than; ˜: similar; ↑: increased/high: ↓: decreased/low; CFA: complete Freund's adjuvant; sX: soluble X

Table 4. Examples of studies of PLGA particles targeted to DCs.

Receptor	Formulation	Antigen / adjuvant	In vitro /in vivo	Administration route	Response compared to untargeted particles	References
Integrin, lectin and mannose receptors	PLGA MPs (˜2.5 μm) c.c. to RGD peptide; WGA; mannose-PEG3-NH2	–	In vitro	n/a	↑ uptake of targeted MPs	^108
Integrin receptor	PLGA NPs (˜200 nm) c.c. to RGD peptide	OVA	In vitro and and in vivo	Oral	↑uptake by M cells and ↑ IgG responses in vivo	^107
	PLGA MPs (˜1 μm) containing alginate or c.c. RGD-alginate	SPf66; S3	In vivo	i.d.	↑ Ab and cellular responses and more balanced Th1/Th2 responses; ↑ IFN-γ secretion and splenocyte proliferation	^109
Mannose receptor	Mannan c.c. to PLGA NPs (˜400 nm)	OVA	In vitro and and in vivo	s.c.	↑ antigen-specific CD4^+ and CD8^+ T cell responses in vitro and and vivo	^113
	Mannan-coated on or c.c. to PLGA NPs (˜400-500 nm)	–	In vitro	n/a	↑ DC uptake and cell surface markers (CD40, CD86) and secretion of inflammatory cytokines (IL-12, IL-6 and TNF-α)	^111,114
DC-SIGN	PLGA MPs (2 μm) and NPs (200 nm) c.c. to humanized hD1 anti-DC-SIGN antibody	BSA; TT	In vitro	n/a	MPs were taken up nonspecifically; NPs effectively targeted DCs: ↑ uptake & Ag-specific T cell responses at 10–100 fold lower concentrations	^12
DEC-205	PLGA NPs (˜200 nm) c.c. to bfFp containing anti-DEC-205 antibody fragment	OVA	In vitro and and in vivo	s.c.	2-fold ↑ receptor-mediated uptake of bfFp functionalized NPs in vitro; ↑ OVA-specific IgG responses in vivo	^117
DEC-205	PLGA NPs (˜200-250) c.c. to anti-DEC-205 mAb	OVA / KRN	In vitro and and in vivo	Footpads	↑ antigen-specific humoral & CTL responses and promoted potent antitumor responses	^119
DEC-205; CD40; CD11	PLGA NPs (200 nm) c.c. either with anti-DEC-205, -αCD40 or -CD11 mAbs	OVA / poly(I:C) & R848	In vitro and and in vivo	s.c.	↑ uptake of targeted NPs & IL-12 production and expression of IFN-γ in vitro; ↑ OVA-specific CD8^+ T cell responses in vivo	^11
CD40	PLGA NPs (200 nm) c.c. with anti-αCD40 mAb	OVA; HPV-E7 / poly(I:C) & Pam3CSK4	In vitro and and in vivo		↑ selective delivery to DCs and ↑ CD8^+ T cell priming in vitro; ↑ tumor control and prolonged survival of tumor-bearing mice in vivo	^14

Ab: antibody; Ag: antigen; <: less/lower than; >: more/higher than; <<: much less/lower than; >>: much more/higher than; ≥: equal or higher than; ˜: similar; ↑: increased/high: ↓: decreased/low; CFA: complete Freund's adjuvant; sX: soluble X; c.c.: chemically conjugated; bfFp: bifunctional fusion protein of strepatividin

Table 5. Summary of the main advantages and disadvantages of PLGA-based particulate vaccine delivery systems.

Advantages

Disadvantages

• PLGA polymers are biodegradable, widely available and approved by regulatory agencies such as FDA • PLGA particles for delivery of several different agents are on the market • PLGA particles can be administered via various routes • PLGA particles may decrease toxicity of vaccine components • Particle size, surface and/or release characteristics can be tailored • PLGA particles allow controlled Ag release • PLGA particles protect Ag from degradation and elimination • PLGA particles enhance Ag uptake by APCs by mimicking size and shape of pathogens • PLGA particles enhance and prolong Ag cross-presentation efficiency • PLGA particles allow concomitant delivery of multiple vaccine components • Large surface area and surface functional groups allow conjugating of targeting moieties • PLGA particles may lead to Ag dose sparing

• Negative charge of PLGA particles is disadvantageous for particle uptake • PLGA particle preparation process must be tailored to the properties of the Ag • PLGA particles cannot be sterile filtered • Ag degradation may occur during preparation, storage and release • Ag release is often incomplete • Particle aggregation may occur • Particle size may limit crossing of biological barriers

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