Antigen-Loaded Upconversion Nanoparticles for Dendritic Cell Stimulation, Tracking, and Vaccination in Dendritic Cell-Based Immunotherapy

Component: Nanoparticle Core; Description: NaY/GdF₄:Yb:Er upconversion nanoparticles (UCNPs) — composition: Y:Gd:Yb:Er = 58%:20%:20%:2%; synthesized by thermal decomposition; average diameter ~170 nm; hexagonal crystal structure (JCPDS No. 28-1192)

Component: Polymer Coating; Description: • OA-PAA (octylamine-grafted polyacrylic acid) — phase transfer to aqueous<br>• PEG (six-armed amine-terminated, 10 kDa) — biocompatibility, stability<br>• PEI (branched, 10 kDa) — positive charge, antigen loading via electrostatic interaction

Component: Final Nanoparticle; Description: UPP (UCNP-PEG-PEI) — dual-polymer coated; highly positively charged (+40.1 mV); excellent stability in saline, medium, and serum

Component: Antigen; Description: Ovalbumin (OVA) — model antigen; isoelectric point 4.7 (negatively charged at physiological pH); loaded onto UPP via electrostatic interaction

Component: Antigen Loading Capacity; Description: ~10% (w/w) OVA (saturated loading)

Component: Particle Properties; Description: • Size: UCNP 170 nm → UPP ~180 nm → UPP@OVA ~190 nm<br>• Zeta potential: UCNP (+12.5 mV) → UPP (+40.1 mV) → UPP@OVA (+29.4 mV)<br>• UCL emission: strong under 980 nm excitation (unchanged after coating/loading)

Component: Release Profile; Description: Accelerated OVA release at low pH (lysosomal pH 5.0) vs. physiological pH 7.4

Parameter: Cell Types; Details: • Primary DCs: Bone marrow-derived dendritic cells (BMDCs) from C57BL/6 mice (8-day culture with GM-CSF)<br>• Tumor cell lines: B16 melanoma, B16-OVA (OVA-expressing derivative)<br>• Primary macrophages: Peritoneal macrophages from immunized mice

Parameter: Animal Model; Details: C57BL/6 mice (8-week-old females); protocols approved by Soochow University Laboratory Animal Center

Parameter: DC Vaccination; Details: DCs incubated with OVA, UPP, or UPP@OVA (12 h, 10 μg/mL OVA); 2 × 10⁶ DCs injected intradermally at tail base; two immunizations at weekly intervals

Parameter: Controls; Details: • Untreated DCs (PBS)<br>• Free OVA-pulsed DCs<br>• UPP-treated DCs (no antigen)<br>• Free OVA alone (without DCs)<br>• UPP@OVA alone (without DCs)

Parameter: Experimental Groups; Details: n = 6 mice per group for vaccination studies

Parameter: Replicates; Details: All samples measured in triplicate; data presented as mean ± SEM

Analysis Category: Nanoparticle Characterization; Methods: • TEM, HR-TEM (FEI Tecnai F20)<br>• XRD (PANalytical)<br>• UCL spectra (FluoroMax 4 with 980 nm laser)<br>• Zeta potential/hydrodynamic size (Malvern Nano-ZS90)<br>• ICP-AES (UCNP concentration)<br>• BCA protein assay (OVA loading/release)

Analysis Category: Cellular Uptake & Localization; Methods: • Confocal microscopy (Olympus with 980 nm external laser) — UCL (red), FITC-OVA (green), DAPI (blue)<br>• LysoTracker Red staining (lysosomes)<br>• Flow cytometry (FACSCalibur) — FITC-OVA internalization kinetics

Analysis Category: DC Maturation; Methods: Flow cytometry: anti-CD11c-FITC, anti-CD86-PE, anti-CD80-APC (eBioscience); % CD11c⁺CD80⁺CD86⁺

Analysis Category: Cytokine Detection; Methods: ELISA: IL-1β, IL-12p70 (DC supernatants); TNF-α (serum)

Analysis Category: In Vivo UCL Imaging; Methods: Modified Maestro imaging system with 980 nm laser (1 W/cm², exposure 30 s); 850 nm short-pass emission filter; thermal monitoring (IR camera)

Analysis Category: DC Tracking Sensitivity; Methods: Subcutaneous injection of labeled DCs (50 to 50,000 cells) in hair-removed back

Analysis Category: DC Migration; Methods: Footpad injection; UCL imaging at 0, 12, 24, 36, 48 h; ex vivo lymph node imaging; immunofluorescence (Thy1.2 for T zone, B220 for B zone)

Analysis Category: T Cell Proliferation; Methods: CFSE staining; OVA peptide (257-264) restimulation (3 days); anti-CD3e staining; flow cytometry

Analysis Category: IFN-γ Production; Methods: ELISPOT (BD Biosciences); anti-IFN-γ antibody; AEC chromogenic substrate; spot-forming cells (SFC)/10⁶ cells

Analysis Category: CTL Cytotoxicity; Methods: Co-culture (4 h); splenocytes (effectors) vs. B16/B16-OVA targets (E:T = 100:1); LDH release assay (Promega); % specific lysis calculation

Analysis Category: Macrophage Phagocytosis; Methods: Fluorescent microbeads (12 h); flow cytometry

Analysis Category: Statistical Analysis; Methods: Student's t-test; P < 0.05 considered significant

Parameter: Zeta potential; UCNP: +12.5 mV; UPP: +40.1 mV; UPP@OVA: +29.4 mV

Parameter: Hydrodynamic size; UCNP: ~170 nm; UPP: ~180 nm; UPP@OVA: ~190 nm

Parameter: OVA loading capacity; UCNP: N/A; UPP: N/A; UPP@OVA: ~10% (w/w)

Parameter: Stability; UCNP: -; UPP: Stable in saline, medium, serum; UPP@OVA: Stable

Parameter: UCL emission; UCNP: Strong; UPP: Strong; UPP@OVA: Strong (unchanged)

Parameter: pH-responsive release; UCNP: N/A; UPP: N/A; UPP@OVA: Faster at pH 5.0 (lysosomal) vs. pH 7.4

Parameter: DC uptake (MFI, 4 h); Free OVA: Baseline; UPP@OVA: ~5× higher; Fold Enhancement: ~5-fold

Parameter: DC maturation (CD80⁺CD86⁺CD11c⁺); Free OVA: 41.9 ± 3.08%; UPP@OVA: 50.47 ± 3.22%; Fold Enhancement: Significant increase

Parameter: IL-12p70 secretion; Free OVA: Low; UPP@OVA: Significantly increased; Fold Enhancement: P < 0.05

Parameter: IL-1β secretion; Free OVA: Low; UPP@OVA: Significantly increased; Fold Enhancement: P < 0.05

Number of Labeled DCs Injected: 50,000 cells; UCL Detection: Strong signal

Number of Labeled DCs Injected: 5,000 cells; UCL Detection: Clear signal

Number of Labeled DCs Injected: 500 cells; UCL Detection: Detectable

Number of Labeled DCs Injected: 50 cells; UCL Detection: Clearly visible (detection limit)

Time Post-Injection: 0 h; UCL Signal in Popliteal Lymph Node: None (injection site only)

Time Post-Injection: 12-24 h; UCL Signal in Popliteal Lymph Node: Minimal/developing

Time Post-Injection: 36 h; UCL Signal in Popliteal Lymph Node: Appeared

Time Post-Injection: 48 h; UCL Signal in Popliteal Lymph Node: Quite strong

Time Post-Injection: Lymph node localization; UCL Signal in Popliteal Lymph Node: Exclusively in T cell zone (Thy1.2⁺); not in B cell zone (B220⁺)

Vaccine Group: PBS (control); T Cell Proliferation: Minimal; vs. OVA-DC: -

Vaccine Group: Untreated DCs; T Cell Proliferation: Low; vs. OVA-DC: -

Vaccine Group: OVA-pulsed DCs; T Cell Proliferation: Moderate; vs. OVA-DC: Baseline

Vaccine Group: UPP@OVA-pulsed DCs; T Cell Proliferation: Greater proliferation; vs. OVA-DC: Significantly enhanced

Vaccine Group: Free OVA alone; T Cell Proliferation: No significant enhancement; vs. OVA-DC: -

Vaccine Group: UPP@OVA alone; T Cell Proliferation: No significant enhancement; vs. OVA-DC: -

Parameter: IFN-γ production (ELISPOT); OVA-DC Vaccine: Moderate; UPP@OVA-DC Vaccine: Remarkably enhanced; Significance: Significant

Parameter: Serum TNF-α; OVA-DC Vaccine: Low; UPP@OVA-DC Vaccine: Significant increase; Significance: P < 0.05

Parameter: CTL-mediated lysis (B16-OVA); OVA-DC Vaccine: Moderate; UPP@OVA-DC Vaccine: Obviously enhanced; Significance: Specific to OVA-expressing cells

Parameter: CTL-mediated lysis (B16, no OVA); OVA-DC Vaccine: Minimal; UPP@OVA-DC Vaccine: Not significant; Significance: Antigen-specific

Parameter: Macrophage phagocytosis; OVA-DC Vaccine: Baseline; UPP@OVA-DC Vaccine: Enhanced; Significance: Increased bead engulfment

1. No therapeutic efficacy demonstrated: The study demonstrates immune responses (T cell proliferation, IFN-γ, CTL activity) but does not include a tumor challenge or therapeutic study to show actual tumor growth inhibition or survival benefit.

2. Model antigen only: OVA is a well-characterized model antigen but not clinically relevant; no demonstration with tumor-associated antigens (e.g., TRP-2, gp100) or patient-derived antigens.

3. No long-term safety/biodistribution data: While the authors cite previous work on UCNP biodistribution, this paper does not present systematic toxicity, clearance, or long-term retention data for UPP@OVA.

4. UCNP composition: Contains Gd (gadolinium), which has potential safety concerns; no demonstration of biodegradation or excretion pathways.

5. Laser exposure heating: The authors note heating was "insignificant" and "induced no harm," but thermal effects during repeated imaging sessions were not extensively characterized.

6. Primary DC variability: BMDCs derived from mice have inherent donor-to-donor variability; clinical translation would require robust protocols for human DC generation.

7. No human DC data: All experiments performed in murine cells; human DC compatibility, uptake, and immune activation not validated.

8. Ex vivo tracking only in lymph nodes: While DC homing to lymph nodes was demonstrated, migration to other tissues or tumor sites was not tracked.

9. No comparison to other DC tracking methods: The study does not benchmark UCL imaging against other modalities (MRI, PET, fluorescence) in the same experimental setup.

10. Vaccination schedule not optimized: Two doses at weekly intervals were used; dose-response, timing, and optimal prime-boost intervals were not systematically evaluated.

11. Macrophage phagocytosis as innate immunity readout: While increased macrophage phagocytosis was observed, direct evidence of enhanced innate anti-tumor immunity (e.g., NK cell activity) was not provided.

12. Mechanism of enhanced DC maturation not fully elucidated: UPP@OVA enhanced maturation beyond antigen delivery alone, suggesting potential adjuvant effects of the nanoparticles or polymers; mechanism (TLR signaling, etc.) was not investigated.

Report prepared based on the Just Accepted manuscript in ACS Nano. For full experimental details, supplementary figures, and final published version, please refer to the article at http://pubs.acs.org.