Alterations in tumor infiltrating lymphocytes and myeloid cell populations following treatment with PSMAi-C’ dots and ICB in the Hi-Myc model (Day 10 post treatment). (a) Schematic of prostate cancer development and treatment before immunophenotyping of Hi-Myc GEM models. (b – o) Bar charts illustrating distinct T cell (b – i) and myeloid (j – o) populations harvested from Hi-Myc tumor-bearing mice (n = 3/group) treated with a multi-dose regimen (three doses every three days) of saline, ICB, PSMAi-C’dots or Dual Tx. Tumors were harvested 10 days after the final dose and dissociated to single cells, which were analyzed via multi-color flow cytometry. Data in (b – o) are presented as mean ± s.e.m. and a 1-way ANOVA with Tukey's multiple comparisons test was performed. *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001, ns, not significant.

Non-targeted C’ dots induce tumor-specific inflammatory responses in the Hi-Myc prostate cancer model. Ex vivo gene expression profiling of (a) tumor and (b) spleen harvested from Hi-Myc mice 4 days after the final dose of vehicle or non-targeted particles. Specific classes of genes (i.e., iron/ferroptosis-related, DAMPs, antigen presentation, and immune-related) were profiled. Transcripts were normalized to Gapdh and represented as fold changes, with significant changes indicated by values above 2 (dashed lines). Gene expression changes represented by bar plots at (c) 24 h and (d) 96 h post-particle administration in Hi-Myc mice, shown as log2 fold change transcript alterations relative to saline; significance indicated by values > 1 (solid lines).

PSMAi-C' dots trigger proinflammatory responses in prostate cancer and immune cells. (a) Gene expression levels, as fold changes over controls, using qRT-PCR and (b) normalized cytokine and chemokine expression levels by Proteome Profiler in Myc-CaP cells and supernatants, respectively, following incubation with 15 uM of PSMAi-C’ dots over 72 h. (c) IFN-a/b reporter B16 cell luminescence (OD620) after incubation with supernatants from particle-treated and untreated Myc-CaP cells. Percentage (%) of surface-expressed (d) IFNGR1, (e) MHC-I, (f) PD-L1, and (g) CD73 on vehicle- and particle-treated Myc-CaP cells by flow cytometry at 72-hour post-exposure. (h) Flow cytometry-based analyses of the %M1 and %M2 markers in vehicle- and particle-treated bone marrow-derived macrophages (BMDMs) over 72 h. (i) IFN-a/b reporter B16 cell luminescence (OD620) after incubation of BMDMs with supernatants from (h). Percentage of CD8+ T cell-specific (j) IFN-g and (k) TNF-a secretion from splenocytes by flow cytometry following a 72-hour incubation with or without PSMAi-C’ dots. (L) T cell cytotoxicity (expressed as luciferase (luc+) activity) following co-culture of mouse T cells with luc + Myc-CaP cells, with and without PSMAi-C’ dots for 48 h. All samples were run in triplicate. Numerical data are presented as mean ± s.e.m. 1-way ANOVA with Kruskal-Wallis test was performed in (a). Unpaired t-tests were performed in (d–g) and (i–l). 2-way ANOVA with Šídák's multiple comparisons test was performed in (c) and (h). *p < 0.05, **p < 0.01, ****p < 0.001.

Serum cytokine/chemokine analyses demonstrate PSMAi-C’ dots maintain a safe inflammatory profile relative to controls in the presence and absence of ICB. Proteome profiles of detectable cytokines and chemokines in serum from mice treated with saline, PSMAi-C' dots, ICB, and Dual Tx (a) 4 days and (b) 10 days following the final dose. All data are presented as mean ± s.e.m. (c) Corresponding body weights of mice over time (n = 5 per cohort). 1-way ANOVA with Tukey's multiple comparisons test was performed (***p < 0.005).

Pharmacokinetic and biodistribution studies in Myc-CaP tumor-bearing mice following i.v.-injection of 89Zr-DFO-PSMAi-C’ dots. (a) PD-10 elution profile of 89Zr-DFO-PSMAi-C’ dots. Pure radiolabeled product elutes between 2.5-5 mL. (b) Stability of 89Zr-DFO-PSMAi-C’ dots in human (n = 3 samples) and mouse serum (n = 3 samples) relative to phosphate buffered saline (PBS, n = 3 samples). (c) In vivo targeted PET imaging of 89Zr-DFO-PSMAi-C’ dots in one of four representative Myc-CaP tumor-bearing mouse over a 72-hour period post-injection. (d) Tumor- and organ-specific time-activity curves and activity-to-background ratios were obtained from all mice (n = 4) in the study (c). (e) Biodistribution (represented as injected dose per gram tissue, %ID/g) of 89Zr-DFO-PSMAi-C’ dots in mice from (c) 72 hr post-injection of particle tracer. Data in (b) and (d – f) are presented as mean ± s.e.m.

Complete blood counts of male Hi-Myc mice injected with saline vehicle, PSMAi-C’ dots, PSMAi-C’ dots + ICB and ICB (αPD-1+ αCTLA-4)only (n = 3/group).

Immune cell population changes in Myc-CaP tumors following PSMAi-C' dot administration with or without TLR7/8 blockade. Bar plots show distinct T cell (a – g) and myeloid cell (i – n) populations in harvested tumors from all treatment cohorts (n = 3 per cohort) receiving a multi-dose (n = 3) regimen of saline vehicle, PSMAi-C’ dots, TLR7/8 antagonist (Enpatoran (M5049)) alone or in combination with PSMAi-C’ dots. Panels quantify ROS (o – q) and lipid peroxidation (r – t), the latter measured using C11 BODIPY in tumor-associated cells, including macrophages and T cells. Tumors were harvested ∼10 days after the final dose, dissociated into single-cell suspensions, and analyzed by multicolor flow cytometry. Data are presented as mean ± s.e.m. Statistical significance was determined using one-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001, ns, not significant.

Potency of PSMA-specific binding peptides and their corresponding sequences.

Antibodies for flow cytometry (T Cell Panel).

Complete automated differential blood counts of male Hi-Myc mice injected with saline vehicle, PSMAi-C’ dots, PSMAi-C’ dots + ICB and ICB (αPD-1+ αCTLA-4)only (n = 3/group).

Particle-driven triple therapy in Myc-CaP model induces additional proinflammatory and anti-tumor reprogramming of CD45+ cells over dual therapy. KEGG pathway enrichment shown by (a) bubble plot and (b) heatmap highlighting differentially expressed pathways and genes in CD45+ cells from Myc-CaP flank tumors 4 days after triple versus dual therapy.

Assessment of ferroptosis in immunodeficient mice. Myc-CaP flank tumor-bearing NSG mice were assigned to one of three treatment arms (n = 3 animals per arm): saline vehicle, PSMAi-C’ dots, and PSMAi-C’ dots plus liproxstatin-1 (LP-1). Tumor growth (a) and corresponding body weights of mice (b) monitored up to 10 days after the final treatment.

Tunable structural properties of ultrasmall PSMAi-PEG2-Cy5-C’ dots promote enhanced binding/uptake and potency in prostate cancer cell lines. (a) C’ dot-encapsulating deep red fluorescent dye Cy5 (1), surface functionalized with DFO chelator (2) and the PSMA targeting peptide, PSMAi (3). (b) Chemical structure of PSMAi-PEG2 peptide. (c) Cell binding of PSMAi-PEG-C’ dots and second generation PSMAi-PEG2-C’ dots in LNCaP cells. (d) FCS correlation curve and fit of PSMAi-PEG2-Cy5-C’ dots. (e) GPC elugram of PSMAi-PEG2-Cy5-C’ dots with Gaussian fit. (f) Competitive binding curves for various PSMAi-targeted C’ dot constructs versus PSMAi peptide in LNCaP cells. (g) Flow cytometry of concentration-dependent PSMAi-PEG2-Cy5-C’ dots particle uptake (4 h) in human and murine prostate cancer cell lines, expressed as mean fluorescence intensity (MFI). (h) Specific uptake of PSMAi-PEG2-Cy5-C’dots in LNCaP, Myc-CaP, and PC-3 cells without and with addition of anti-PSMA antibody by flow cytometry, expressed as MFI. (i) Differential PSMA expression in LNCaP, Myc-CaP, PC-3, and ID8 cells by western blot. (j) Confocal microscopy of PSMAi-PEG2-Cy5-C' dot uptake in LNCaP cells (Red: particle, Green: 70 kDa FITC-dextran, Blue: DAPI). All numerical data are presented as mean ± s.e.m. (n = 3); scale bar: 5 um. Unpaired t-tests were performed in (c) and (h). **p < 0.01, ***p < 0.005, ****p < 0.001, ns – not significant.

TRAMP-C2 cells exhibit concentration-dependent modulation of proinflammatory gene signatures following treatment with non-targeting C' dots. (a) Heatmap depicting different classes of genes (i.e., iron/ferroptosis-related, antioxidant, antigen presentation, DAMPS, and immune-related) and (b) correlative bars depict log fold transcript alterations in TRAMP-C2 cells exposed to increasing concentrations (0.1–15μM) of C’ dots for 72 h. Expression levels were normalized to GAPDH. All samples were run in triplicate.

Despite the considerable success of clinically approved immune-based therapies for treating advanced melanoma, a significant fraction of patients are not responsive owing to mechanisms engaged by the tumour to evade the immune system. Here we report the surprising finding that a clinically validated and tunable self-therapeutic ultrasmall silica nanoparticle prolongs survival in a highly resistant melanoma model in combination with interleukin-6 and PD-L1 inhibition through activation of the stimulator of interferon genes/interleukin-6/PD-L1 axis and reprogramming of the tumour microenvironment towards a pro-inflammatory phenotype. In a murine model, induction of significant cytotoxic and antitumour inflammatory responses leads to differential activation of immune cell populations in a CD8-dependent manner via type I/II interferon pathways after systemic particle injection. Importantly, these immunostimulatory responses accompany significant reductions in cell populations and receptors driving suppressive activities. Mechanistic insights highlight the potential clinical utility of this platform to maximize antitumour immunity and efficacy by subverting suppressive components in the tumour microenvironment.

Targeting both tumor cell surface receptors and soluble pro-angiogenic factors is a promising strategy to improve cancer treatment specificity and reduce therapy resistance. Human epidermal growth factor receptor 2 (HER2) and vascular endothelial growth factor (VEGF) are two clinically validated targets implicated in tumor growth, metastasis, and angiogenesis. To address limitations of conventional large-molecule therapeutics, we developed a modular bispecific protein scaffold based on Designed Ankyrin Repeat Proteins (DARPins) for precision nanobiotechnology applications. We engineered a bispecific HER2×VEGF DARPin containing a C-terminal cysteine for site-specific bioorthogonal conjugation. The construct was recombinantly expressed in Escherichia coli and purified to high monomeric purity. Subsequent azide functionalization enabled strain-promoted click conjugation with diverse payloads, including fluorescent dyes for imaging, radionuclide chelators for diagnostic and therapeutic isotope labeling, and a cleavable drug linker for cytotoxic payload delivery. All bioconjugates retained high structural integrity and dual-specific binding affinity to HER2 and VEGF, with dissociation constants in the low picomolar to nanomolar range as measured by surface plasmon resonance. Importantly, site-specific conjugation did not impair antigen recognition, highlighting the robustness of the scaffold. This study presents a scalable and versatile bispecific DARPin platform that enables modular, site-specific conjugation of imaging and therapeutic payloads without loss of binding function. The preserved dual-targeting capability and biochemical stability make it a promising candidate for nanobiotechnology-based diagnostics, radiotherapy, and targeted drug delivery in precision oncology.

Nanomedicines have created a paradigm shift in healthcare. Yet fundamental barriers still exist that prevent or delay the clinical translation of nanomedicines. Critical hurdles inhibiting clinical success include poor understanding of nanomedicines' physicochemical properties, limited exposure in the cell or tissue of interest, poor reproducibility of preclinical outcomes in clinical trials, and biocompatibility concerns. Barriers that delay translation include industrial scale-up or scale-down and good manufacturing practices, funding and navigating the regulatory environment. Here we propose the DELIVER framework comprising the core principles to be realized during preclinical development to promote clinical investigation of nanomedicines. The proposed framework comes with design, experimental, manufacturing, preclinical, clinical, regulatory and business considerations, which we recommend investigators to carefully review during early-stage nanomedicine design and development to mitigate risk and enable timely clinical success. By reducing development time and clinical trial failure, it is envisaged that this framework will help accelerate the clinical translation and maximize the impact of nanomedicines.

Cellular binding and internalization of cRGD-C' dots in glioma cells

Representative images of I-124-cRGD-C' dot uptake and distribution in primary human glioma; part of an ongoing Phase 1 trial

Table reviewing nanoparticle drug delivery systems in brain tumor models