Nanoscale2016ResearchNon-viral Gene Delivery

Human immune cell targeting of protein nanoparticles – caveospheres

Joshua J. Glass, Daniel Yuen, James Rae, Angus P. R. Johnston, Robert G. Parton, Stephen J. Kent, Robert De Rose29 September 2026DOI 10.1039/c6nr00506c

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Summary

Investigations of nanoparticle targeting to immune cells are conventionally restricted to monocultured cell line models that lack the complexity of primary human blood cell populations, including non-specific association with, and competition from, diverse cell types. A more physiologically relevant system using mixed primary human blood cells is needed to evaluate nanoparticle targeting, particularly for therapeutically relevant non-phagocytic. ### Nanoparticle Characterisation | Parameter | Untargeted | αCD4 | αCD20 | |---------------|---------------|----------|-----------| | EM diameter (nm) | 42.7 ± 8.3 | 41.3 ± 8.3 | 45.3 ± 6.8 | | DLS hydrodynamic size.

Keywords

Nanoparticles T cells Immune cells Drug delivery Dendritic cells Biodistribution Endocytosis

Purpose: Investigations of nanoparticle targeting to immune cells are conventionally restricted to monocultured cell line models that lack the complexity of primary human blood cell populations, including non-specific association with, and competition from, diverse cell types. A more physiologically relevant system using mixed primary human blood cells is needed to evaluate nanoparticle targeting, particularly for therapeutically relevant non-phagocytic cells such as CD4⁺ T cells—a critical target for HIV therapeutics.

Hypothesis: Caveosphere nanoparticles (caveolin-1 protein-lipid vesicles) functionalised with monoclonal antibodies via a Protein A Z-domain will enable specific targeting to CD4⁺ T cells and CD20⁺ B cells within mixed populations of primary human peripheral blood mononuclear cells (PBMCs). Targeting caveospheres to CCR5 will enable internalisation into non-phagocytic CD4⁺ T cells—a critical cell type for HIV treatment—through receptor-mediated endocytosis. This targeting will be more sensitive and biologically relevant than cell line models.

Aims: 1. Generate and characterise caveosphere nanoparticles displaying a Protein A Z-domain for rapid antibody functionalisation 2. Demonstrate specific targeting of antibody-functionalised caveospheres to CD4⁺ T cells (anti-CD4) and CD20⁺ B cells (anti-CD20) in monocultured cell lines 3. Evaluate targeting specificity and efficiency in mixed populations of primary human PBMCs using flow cytometry and confocal microscopy 4. Achieve nanoparticle internalisation into non-phagocytic CD4⁺ T cells by targeting CCR5 (a rapidly internalising chemokine receptor) 5. Compare targeting efficiency in primary cell mixtures versus monocultured cell lines to assess biological relevance

Delivery system:

Component: Nanoparticle Type; Description: Caveospheres — protein-lipid vesicles (45-50 nm) assembled from recombinant human caveolin-1 expressed in E. coli

Component: Core Structure; Description: Hollow, biconcave spherical architecture; ~43 nm diameter by EM; ~60-70 nm hydrodynamic diameter (DLS)

Component: Surface Engineering; Description: Caveolin-1 fused to minimal Z-domain of Staphylococcal Protein A (binds IgG Fc); N and C termini oriented on outer surface

Component: Antibody Functionalisation; Description: Passive incubation of caveospheres with mouse anti-human IgG antibodies (1:2 mass ratio); ~10% of Z-domains occupied (~16 antibodies/particle)

Component: Targeting Antibodies; Description: • Anti-CD4 (OKT4, IgG2b) — CD4⁺ T cells • Anti-CD20 (2H7, IgG2b) — B cells • Anti-CCR5/CD195 (3A9, IgG2a) — CCR5⁺ memory T cells

Component: Fluorescent Labelling; Description: Alexa Fluor 647 succinimidyl ester (AF647) for tracking

Component: Cell Types; Description: • Cell lines: CEM.NKR-CCR5 (CD4⁺CCR5⁺ T cell line), 721.221 (CD20⁺ B cell line) • Primary cells: Human PBMCs (CD4⁺ T cells, CD19⁺ B cells, NK cells, monocytes)

Component: Biological Context; Description: 10% FBS or 0.5% BSA (protein corona present); mixed primary cell populations

Approach:

Parameter: Caveosphere Expression; Details: E. coli (Rosetta pLysS DE3) expressing caveolin-1-Z-domain fusion; IPTG induction (1 mM); cell disruptor lysis; amylose resin affinity chromatography

Parameter: Nanoparticle Characterisation; Details: • Negative stain EM (size, morphology) • DLS (hydrodynamic size) • Flow cytometry (Ab-capture microbeads for functionalisation confirmation)

Parameter: Cell Line Targeting; Details: CEM.NKR-CCR5 (CD4⁺) and 721.221 (CD20⁺); 1 μg NPs/10⁶ cells; 1 h on ice; flow cytometry and imaging flow cytometry

Parameter: Primary PBMC Targeting; Details: PBMCs from 9 healthy donors; phenotyping with 6-antibody cocktail; 1 μg NPs/10⁶ cells; 1 h on ice; flow cytometry; confocal microscopy on sorted populations

Parameter: Internalisation Assay; Details: CEM.NKR-CCR5 or primary CD4⁺ T cells; 1 μg NPs; 90 min at 37°C or on ice; confocal microscopy z-stacks; surface vs. internal quantification

Parameter: Controls; Details: Untargeted caveospheres; irrelevant IgG-functionalised particles; temperature controls (ice, 37°C)

Parameter: Replicates; Details: n=3 independent experiments (cell lines); n=9 donors (PBMC targeting); >100 cells per condition for confocal quantification

Parameter: Statistical Tests; Details: Non-parametric one-way ANOVA (Friedman test) with Dunn's multiple comparisons test

Key methods:

Analysis Category: Nanoparticle Characterisation; Methods: Negative stain TEM (JEOL 1011, 80 kV); DLS (Nanopartica SZ-100); flow cytometry (Ab-capture microbeads)

Analysis Category: Cell Line Targeting; Methods: Flow cytometry (BD LSR Fortessa); imaging flow cytometry (Amnis ImageStream); 30,000 cells/sample

Analysis Category: Primary PBMC Targeting; Methods: Flow cytometry panel: CD3-AF488, CD8-BV570, CD14-PE-Cy7, CD19-BV650, CD45-V500, CD56-PE; 9 donors

Analysis Category: Confocal Microscopy; Methods: Zeiss LSM710; 20× and 63× oil objectives; ZEN 2012 Black; Imaris 7 (3D renders); z-stack quantification (>100 cells/condition)

Analysis Category: Cell Sorting; Methods: BD FACS ARIA III (100 μm nozzle) for confocal imaging of sorted populations

Analysis Category: Internalisation Quantification; Methods: Step-through z-stacks; count percentage of cells with surface-bound vs. internalised AF647-nanoparticles

Key results: ### Nanoparticle Characterisation

Parameter: EM diameter (nm); Untargeted: 42.7 ± 8.3; αCD4: 41.3 ± 8.3; αCD20: 45.3 ± 6.8

Parameter: DLS hydrodynamic size (nm); Untargeted: 70.1 ± 18.7; αCD4: 62.1 ± 15.9; αCD20: 61.2 ± 19.8

Parameter: Morphology; Untargeted: Spherical, hollow; αCD4: Unchanged; αCD20: Unchanged

Ab Functionalisation Confirmation:

Parameter: Ab-capture microbead binding (anti-light chain); Result: Positive for Ab-functionalised caveospheres

Parameter: Control microbead binding (BSA only); Result: Negative

Parameter: Functionalisation efficiency; Result: ~16 antibodies per particle (~10% of Z-domains occupied)

Cell Line Targeting:

Target: αCD4 → CEM.NKR-CCR5; Cell Line: T cell line; % Association (Flow): 90.9%; % Association (Imaging Flow): 71.0%

Target: αCD20 → 721.221; Cell Line: B cell line; % Association (Flow): 90.6%; % Association (Imaging Flow): 50.0%

Target: Untargeted → CEM.NKR; Cell Line: T cell line; % Association (Flow): <1%; % Association (Imaging Flow): -

Target: Untargeted → 721.221; Cell Line: B cell line; % Association (Flow): <2%; % Association (Imaging Flow): -

Target: αCD4 → 721.221 (off-target); Cell Line: B cell line; % Association (Flow): <5%; % Association (Imaging Flow): -

Target: αCD20 → CEM.NKR (off-target); Cell Line: T cell line; % Association (Flow): <5%; % Association (Imaging Flow): -

Primary PBMC Targeting (n=9 donors):

Target: αCD4; Targeted Cell Type: CD4⁺ T cells; % Cells with Bound NPs: 76.0 ± 11.3%; MFI Fold Increase vs. Untargeted: 43.9 ± 12.7×

Target: αCD20; Targeted Cell Type: CD19⁺ B cells; % Cells with Bound NPs: 79.3 ± 23.6%; MFI Fold Increase vs. Untargeted: 6.6 ± 1.0×

Target: Untargeted; Targeted Cell Type: CD4⁺ T cells; % Cells with Bound NPs: Lower; MFI Fold Increase vs. Untargeted: 1×

Target: Untargeted; Targeted Cell Type: B cells; % Cells with Bound NPs: 48.3 ± 13.0%; MFI Fold Increase vs. Untargeted: 1×

Target: αCD4 (off-target); Targeted Cell Type: B cells; % Cells with Bound NPs: 51.6 ± 10.6%; MFI Fold Increase vs. Untargeted: Lower than αCD20

Target: αCD20 (off-target); Targeted Cell Type: CD4⁺ T cells; % Cells with Bound NPs: Lower; MFI Fold Increase vs. Untargeted: Lower

Target: Any caveosphere; Targeted Cell Type: NK cells; % Cells with Bound NPs: 28.8 ± 25.1% (low MFI); MFI Fold Increase vs. Untargeted: Low

Target: Any caveosphere; Targeted Cell Type: Monocytes; % Cells with Bound NPs: High association; MFI Fold Increase vs. Untargeted: Minimal targeting effect

Internalisation of CCR5-Targeted Caveospheres:

Condition: αCCR5 → CEM.NKR (37°C); Cell Type: T cell line; % Cell Association: 98.6 ± 0.7%; % Cells with Internalised NPs: 43.3 ± 4.6%

Condition: αCCR5 → CEM.NKR (ice); Cell Type: T cell line; % Cell Association: 97.4 ± 0.2%; % Cells with Internalised NPs: 0.98 ± 0.98%

Condition: Untargeted → CEM.NKR (37°C); Cell Type: T cell line; % Cell Association: 5.4 ± 1.2%; % Cells with Internalised NPs: 0%

Condition: Untargeted → CEM.NKR (ice); Cell Type: T cell line; % Cell Association: 2.5 ± 0.4%; % Cells with Internalised NPs: 0%

Condition: αCCR5 → primary CD4⁺ T cells (37°C); Cell Type: Primary T cells; % Cell Association: High; % Cells with Internalised NPs: Up to 34%

Condition: αCCR5 → primary CD4⁺ T cells (ice); Cell Type: Primary T cells; % Cell Association: Surface only; % Cells with Internalised NPs: 0%

Interpretation: The authors conclude that "caveospheres can actively target primary human immune cells within mixed blood cell populations and can be internalised by non-phagocytic primary immune cells." They emphasise that "this targeting model is more relevant and sensitive than cell line models, demonstrating enhanced non-specific binding." The authors state: "Investigating nanoparticle targeting in the complex environment of mixed populations of primary human blood cells has rarely been investigated," and their work demonstrates "efficient and flexible system of immune cell-targeted caveosphere nanoparticles holds promise for the development of advanced immunotherapeutics and vaccines." Importantly, "targeting caveospheres to CCR5 enabled caveosphere internalisation by non-phagocytic CD4⁺ T cells—an important therapeutic target for HIV treatment."

10. Limitations (Explicitly Stated or Evident):

1. Protein A Z-domain limitations in vivo: The authors note that "the high concentration of IgG (with strong affinity to Protein A) in human blood might compete off targeting IgG and render this system less suitable in vivo." They are "actively investigating chemical conjugation of IgG, which are better suited to future in vivo studies."

2. Bacterial origin and immunogenicity: The authors acknowledge that "by nature of their bacterial origin, caveospheres contain immunostimulatory molecules. Delivering anti-HIV-1 therapeutics will require nonimmunostimulatory caveospheres and efforts are underway to overcome this potential problem."

3. In vitro only: All experiments were performed in vitro with primary PBMCs; no in vivo targeting, biodistribution, or therapeutic efficacy studies were conducted.

4. No cargo delivery demonstrated: While the authors propose caveospheres for therapeutic delivery (e.g., HIV siRNAs or latency-reacting agents), actual cargo loading and functional delivery were not demonstrated.

5. Loading limitations: The authors note that "to load larger molecules such as proteins or RNA, caveosphere production may require cell-free protein expression systems" because only small molecules can pass through the bacterial outer membrane during expression.

6. Limited internalisation efficiency: Only 34% of primary CD4⁺ T cells internalised CCR5-targeted caveospheres, leaving room for improvement.

7. Targeting intensity difference: While αCD4 caveospheres showed 43.9-fold MFI increase, αCD20 only showed 6.6-fold, suggesting variable targeting efficiency depending on the receptor and antibody.

8. No evaluation of receptor downregulation: Targeting CCR5, which is known to internalise rapidly, could potentially downregulate the receptor and affect cell function—this was not assessed.

9. Donor variability: Data from 9 donors showed substantial variability (e.g., B cell binding SD of 23.6%), which may limit generalizability.

10. No active targeting to dendritic cells: The authors note their caveosphere platform "warrants investigation of targeting to more scarce dendritic cell populations" for vaccine applications, which was not performed.

11. Competition from serum proteins: While targeting was achieved in serum-containing media, the effect of a full protein corona on targeting efficiency was not systematically studied.

12. No therapeutic payload: The study focused solely on targeting and internalisation; functional outcomes (e.g., gene silencing, drug delivery efficacy) were not demonstrated.

Report prepared based on the published Nanoscale article. For full experimental details, supplementary data, and complete references, please refer to the original publication.

RSC pharmaceutics