Moumita Rakshit1, Archana Gautam1, Li Zhen Toh2, Ying Shi Lee2, Hui Ying Lai1, Tina T Wong1,2, Kee Woei Ng1,3,4,5. 1. School of Materials Science and Engineering, Nanyang Technological University, Singapore 639798, Singapore. 2. Ocular Therapeutics and Drug Delivery, Singapore Eye Research Institute, Singapore 169856, Singapore. 3. Environmental Chemistry and Materials Centre, Nanyang Environment & Water Research Institute, Singapore 637141, Singapore. 4. Skin Research Institute of Singapore, Singapore 138648, Singapore. 5. Center for Nanotechnology and Nanotoxicology, Harvard T.H. Chan School of Public Health, Cambridge, MA 02115, USA.
Abstract
BACKGROUND: Hydroxyapatite (HA) [Ca5(PO4)3(OH)] is a naturally occurring calcium phosphate which makes up 60-70% of the dry weight of human bones. Nano-scale HA particles are increasingly being used as carriers for controlled and targeted delivery of bioactive agents like drugs, proteins, and nucleic acids due to their high porosity, negative charge, and biodegradability. PURPOSE: Although much effort has been devoted to understanding the delivery kinetics and effects of the payloads in such carriers, a thorough understanding of the influence of the carriers themselves is lacking. METHODS: HA particles (300 µg/mL) were administered to primary human dermal fibroblasts (HDFs). The uptake and intracellular localization of the particles were determined by flow cytometry, confocal imaging, and transmission electron microscopy (TEM). Immunological assays and PCR were performed to determine the levels of pro-inflammatory cytokines and collagens in cell lysates and media supernatant. RESULTS: The current study explores the effects of poly-dispersed HA particles on primary HDFs as a model system. The majority of the particles were determined to range between 150 and 200 nm in diameter. Upon exposure to HA suspensions, primary HDFs internalized the particles by endocytosis within 6 hours of exposure, showing maximum uptake at 72 hours following which the particles were exocytosed by 168 hours. This correlated to reduced secretion of various pro-inflammatory and pro-collagenic cytokines. Biochemical analysis further revealed a reduction in Type I collagen expression and secretion. CONCLUSION: HA particles have an immune-modulatory effect on dermal fibroblasts and reduce collagen production, which may impact the integrity of the extracellular matrix (ECM). This study demonstrates the need to consider the secondary effects of particulate carriers like HA, beyond basic cytotoxicity, in the specific tissue environment where the intended function is to be realized.
BACKGROUND: Hydroxyapatite (HA) [Ca5(PO4)3(OH)] is a naturally occurring calcium phosphate which makes up 60-70% of the dry weight of human bones. Nano-scale HA particles are increasingly being used as carriers for controlled and targeted delivery of bioactive agents like drugs, proteins, and nucleic acids due to their high porosity, negative charge, and biodegradability. PURPOSE: Although much effort has been devoted to understanding the delivery kinetics and effects of the payloads in such carriers, a thorough understanding of the influence of the carriers themselves is lacking. METHODS: HA particles (300 µg/mL) were administered to primary human dermal fibroblasts (HDFs). The uptake and intracellular localization of the particles were determined by flow cytometry, confocal imaging, and transmission electron microscopy (TEM). Immunological assays and PCR were performed to determine the levels of pro-inflammatory cytokines and collagens in cell lysates and media supernatant. RESULTS: The current study explores the effects of poly-dispersed HA particles on primary HDFs as a model system. The majority of the particles were determined to range between 150 and 200 nm in diameter. Upon exposure to HA suspensions, primary HDFs internalized the particles by endocytosis within 6 hours of exposure, showing maximum uptake at 72 hours following which the particles were exocytosed by 168 hours. This correlated to reduced secretion of various pro-inflammatory and pro-collagenic cytokines. Biochemical analysis further revealed a reduction in Type I collagen expression and secretion. CONCLUSION: HA particles have an immune-modulatory effect on dermal fibroblasts and reduce collagen production, which may impact the integrity of the extracellular matrix (ECM). This study demonstrates the need to consider the secondary effects of particulate carriers like HA, beyond basic cytotoxicity, in the specific tissue environment where the intended function is to be realized.