Capra cartilage-derived peptide delivery via carbon nano-dots for cartilage regeneration

Abstract

Targeted delivery of site-specific therapeutic agents is an effective strategy for osteoarthritis treatment. The lack of blood vessels in cartilage makes it difficult to deliver therapeutic agents like peptides to the defect area. Therefore, nucleus-targeting zwitterionic carbon nano-dots (CDs) have immense potential as a delivery vehicle for effective peptide delivery to the cytoplasm as well as nucleus. In the present study, nucleus-targeting zwitterionic CDs have been synthesized as delivery vehicle for peptides while also working as nano-agents towards optical monitoring of cartilage healing. The functional groups of zwitterion CDs were introduced by a single-step microwave assisted oxidation procedure followed by COL II peptide conjugation derived from Capra auricular cartilage through NHS/EDC coupling. The peptide-conjugated CDs (PCDs) allows cytoplasmic uptake within a short period of time (∼30 m) followed by translocation to nucleus after ∼24 h. Moreover, multicolor fluorescence of PCDs improves (blue, green, and read channel) its sensitivity as an optical code providing a compelling solution towards enhanced non-invasive tracking system with multifunctional properties. The PCDs-based delivery system developed in this study has exhibited superior ability to induce ex-vivo chondrogenic differentiation of ADMSCs as compared to bare CDs. For assessment of cartilage regeneration potential, pluronic F-127 based PCDs hydrogel was injected to rabbit auricular cartilage defects and potential healing was observed after 60 days. Therefore, the results confirm that PCDs could be an ideal alternate for multimodal therapeutic agents.

Keywords: carbon nano dots; cartilage regeneration; collagen II; peptide synthesis; zwitterion.

FIGURE 1

Workflow of MAP-based peptide isolation and PCD synthesis: Capra cartilage tissue was mixed with HNO₃ and microwave irradiated to generate zwitterion CDs. COL II peptides conjugate on the surface of CDs by NHS/EDC coupling followed by continuous stirring of the micelles to form peptide conjugated CD (PCD).

FIGURE 2

Characterization of synthesized CDs and isolated COLII peptides. (A) Hydrodynamic diameter, and (B) Zeta potential of synthesized CD assessed at different concentration of HNO3 treatment. (C) MAP based isolated COLII peptides identified by MALDI-TOF analysis.

FIGURE 3

Physico-chemical characterization of CD and PCD. (A) FTIR Spectroscopy, (B) UV absorption spectra, (C,D) Photoluminescence emission spectra, (E) Particles size distribution of PCD by AFM and (F) TEM images of the PCD.

FIGURE 4

Assessment of cytotoxicity and cyto-compatibility of PCD. (A) MTT assay results for CD and PCD at 1, 3, and 5 d, horizontal line indicating the p-value between two groups, (B–D) live-dead assay of PCD at 1, 3, and 5 d of culture.

FIGURE 5

PCD uptake study performed in ADMSCs. The fluorescence of PCD appeared in cytoplasm at 3 h, migrated to nucleus at 24 h, and complete relocation to nucleus observed at 48 h.

FIGURE 6

Chondrogenic differentiation of cells and cluster formation in PCD-F127 hydrogel. (A) Cell mono layers shrinking towards cluster formation, (B) Few clusters formed after 3 d culture and (C) Several clusters formed after 5 d culture, (D) Assessment of chondrogenic differentiation potential of PCD after 21 days’ spheroid culture, and (E) Estimated quantity of accumulated sGAG in the cells after 21 d.

FIGURE 7

Histological analysis of in-vivo cartilage regeneration induced by PCD: (A,B) Defected auricular cartilage with one control and the other treated with PCD-hydrogel, (C,E) Control area showing no noticeable presence of chondrocytes at 15 and 60 d, respectively, in the defect area in the center of the images (D,F) Presence of chondrocytes and cartilage regeneration at the PCD-treated area at 15 and 60 d, respectively (All images were taken at ×10 magnification).

FIGURE 8

Histochemical analysis of cartilage regeneration in the defect area. (A,D) No detectable cartilage regeneration in the control group at both 15 d and 60 d post-implantation, (B,C) Reduced defect size with new cartilage formation at the treated zone within 15 d, and (E,F) Neo-cartilage formation with presence of formed lacunae at the treated zone 60 d post-implantation (a, b, d, and e are ×10 magnified images; c and f are ×20).