Date of Award
Doctor of Philosophy (Ph.D.)
Pharmaceutical and Chemical Sciences
First Committee Member
Bhaskara R. Jasti
Second Committee Member
Third Committee Member
Vyacheslav V. Samoshin
Fourth Committee Member
Proteins are a diverse category of biomolecules with great therapeutic potential. Intracellular delivery of proteins can augment the deficient activities of dysfunctional or poorly expressed innate proteins and therefore represents a promising strategy to treat the associated diseases. One major barrier to intracellular protein delivery is the translocation of the protein across the cellular membrane. Endocytosis provides an important pathway for protein nanocarriers to enter cells across the plasma membrane. However, the cargo protein must then promptly escape from the endosomes to avoid degradation in the lysosome and to exert its cellular function.
Previously, we reported a cationic lipid-coated magnesium phosphate nanoparticle (LPP) system for intracellular protein delivery. The intracellular delivery of catalase, an antioxidant enzyme, by LPP protected MCF-7 cells from a lethal level of exogenous H2O2 and lowered the reactive oxygen species (ROS) levels in EA.hy926 cells. These findings prompted us to further develop LPP to evaluate its protein delivery in animals.
Two categories of LPP formulations, catalase-encapsulated (CE) LPP and catalase-complexed (CC) LPP, were successfully prepared by a modular approach. Catalase-encapsulated liposomes (CE LP) were prepared by hydrating a thin-film of lipids with catalase solution followed by extrusion. However, extrusion of CE LP resulted in substantial loss of catalase activity. Catalase-complexed liposomes (CC LP) were prepared by first extruding cationic liposomes with a LIPEX extruder and then mixing with catalase solution. The resultant CC LP was much smaller than CE LP and preserved all the catalase activity. Magnesium phosphate nanoparticles (MgP NP) were prepared by the microemulsion precipitation technique. CE LP or CC LP were mixed with MgP NP to yield LPP formulations (CE LPP or CC LPP, respectively). The formulations were then rendered isotonic with glucose (5% w/v). Transmission electron microscopy (TEM) confirmed the proposed structure of LPP comprising a shell of lipid bilayers with a core of MgP NP. Furthermore, TEM showed drastic morphological changes of LPP formulations at acidic pH, consistent with an osmotic explosion.
The LPP formulations were administered by intravenous or intranasal routes to CD-1 mice. LPP formulations of fluorescently labeled catalase distributed substantially into the lung following intranasal administration, whereas intravenous administration of the same formulations caused catalase distribution mainly into the liver. In addition, intranasal administration of both the LPP formulations yielded higher pulmonary catalase activity and lowered the ROS levels in the healthy lung compared to free catalase solution. Based on these results, LPP’s antioxidant effects were further evaluated in mice with lipopolysaccharide-induced acute lung injury (ALI).
Lack of LPP distribution into the lung following intranasal administration indicated that intranasal dosing did not deliver catalase substantially into inflamed lungs. In corroboration, the inflammatory biomarker tumor necrosis factor-alpha (TNF-α) remained unchanged after intranasal dosing of LPP formulations. Intratracheal dosing of LPP formulations delivered the fluorescently labeled catalase deep into the lung and significantly reduced TNF-α production in the inflamed lungs compared to free catalase solution. CC LPP, which was smaller and which better preserved catalase activity than CE LPP, showed greater intrapulmonary catalase activity compared to CE LPP in both healthy and inflamed lungs. Taken together, LPP represents a promising nanocarrier for intracellular protein delivery.
Vadlamudi, Mallika. (2019). Lipid-coated Magnesium Phosphate Nanoparticles for Intrapulmonary Protein Delivery in Mice. University of the Pacific, Dissertation. https://scholarlycommons.pacific.edu/uop_etds/3631