Characterization of biotin- and Cy5-loaded CDs. (A) MALDI mass spectrometry of CD (∼3k Da; blue), CD-Biotin (∼3.5k Da; black), and CD-Cy5 (∼4.2 kDa; red). (B) TEM images of unlabeled, biotin-loaded, and Cy5-loaded CDs. Scale bars are 20 nm. (C) SDS-PAGE chromatograph of CD, Cy5, CD mixed with Cy5, and two different preparations of CD-Cy5 (1 and 2) imaged under 488 nm (CD, pseudocolored cyan) and 647 nm (Cy5, pseudocolored magenta) excitation. CD-Cy5(1) and CD-Cy5(2) were purified using different conventional chromatography methods (1, size exclusion; 2, anion exchange). The far-right gel is a composite of merged images. The arrow indicates new molecular species that appear after EDC/NHS conjugation. The molecular weight (MW) ladder is in kDa.

pH and cargo influence CD fluorescence. (A) Titration curve of a 0.05 mg/mL CD suspension using HCl and NaOH. The two pKas found were pK1 = 3.4 and pK2 = 9.6. (B) CD excitation signal intensity was determined across different wavelengths. (C) CD emission spectra were recorded at 280, 333, and 460 nm excitation wavelengths at pH 6.4. (D-F) CD (D), CD-Cy5 (E), and CD-biotin (F) fluorescence spectra were obtained under 420 nm excitation light at pH values of 3.4 (black line) and 6.4 (red line). Absorbance (G) and emission (H) spectra of CD, CD-biotin, and CD-Cy5 show Cy5 absorption and emission peaks in the 600–700 nm range.

Conjugated CDs deliver cargo to bones. Caudal fins of zebrafish injected 4 days after amputation with (A) PBS, (B) CD, (C) CD-Cy5, (D) CD-Biotin, (E) CD and Streptavidin-Alexa 647, and (F) CD-Biotin and Streptavidin-Alexa 647 and imaging the following day. Deposition of CDs and cargo (Cy5 or Streptaviding-Alexa-647) was observed at 488 and 647 nm, respectively. Anterior is to the right and dorsal to the top. Arrows indicate areas of cargo deposition, and dashed lines are the planes of histological sectioning. Scale bar is 500 μm. (A’–F’) Cryosection images were observed under 360 nm (DAPI, nuclei), 488 nm (CD), and 647 nm (cargo) excitation light. Arrows indicate the areas of cargo deposition. BV is a blood vessel that shows blood autofluorescence. Scale bar is 50 μm.

Macrophages internalize CDs without activating inflammatory programs. Mouse RAW 264.7 macrophages internalize CDs. Macrophages were exposed for 6 h to (A) PBS, (B) CD, (C) CD-Cy5, and (D) CD-Biotin, costained with DAPI, and imaged at 360, 488, and 647 nm to respectively identify nuclei (blue), CDs (cyan), and Cy5 (cargo; magenta). While autofluorescence in the 488 nm channel precludes direct analysis of CD internalization, examination in the 647 nm channel revealed the internalization of CD-Cy5. Scale bar is 50 μm. (E) Nitric oxide (NO) production by mouse RAW 264.7 macrophages exposed to different CD concentrations. NO production was measured as released nitrate (μM). Lipopolysaccharide (LPS; immune response activator) was used as a positive control, and PBS as a negative control. Data represent the average ± SEM of three independent experiments and were analyzed for statistical significance using a one-way ANOVA followed by Tukey’s posthoc test (***p < 0.001).