Lipid-coated Magnesium Phosphate Nanoparticles for Intrapulmonary Protein Delivery in Mice

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

Lipid-coated Magnesium Phosphate Nanoparticles for Intrapulmonary Protein Delivery in Mice

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

The Art of Compounding

Works of art created students, faculty and staff as part of PharmArt in the Thomas J. Long School of Pharmacy and Health Sciences.https://scholarlycommons.pacific.edu/pharmart-2017/1022/thumbnail.jp

The Art of Compounding

Works of art created students, faculty and staff as part of PharmArt in the Thomas J. Long School of Pharmacy and Health Sciences.https://scholarlycommons.pacific.edu/pharmart-2017/1022/thumbnail.jp

Lipid-Coated, pH-Sensitive Magnesium Phosphate Particles for Intracellular Protein Delivery

PURPOSE: To develop cationic lipid-coated magnesium phosphate nanoparticles (LPP) for intracellular catalase (CAT) delivery. METHODS: Magnesium phosphate nanoparticles (MgP NP) were prepared by micro-emulsion precipitation and mixed with catalase-loaded cationic liposomes (DOTAP/cholesterol) to yield LPP formulation of catalase (LPP-CAT). The size and ζ-potential of LPP-CAT were measured by dynamic light scattering. The pH-sensitivity of LPP-CAT was determined by monitoring their degradation of hydrogen peroxide (H RESULTS: LPP-CAT were successfully prepared and carried an average diameter ofζ -potential of about +40 mV. At pH 5.5, LPP-CAT degraded H CONCLUSION: LPP represents a promising nano-system for intracellular protein delivery

Optimization of catalase-loaded liposomal magnesium phosphate nanoparticles for intracellular protein delivery

Protein therapeutics have great potential in treating human disease. Compared to small molecule drugs, proteins as therapeutic agents carry the potential advantages of exerting more complicated activities, higher specificity and higher biocompatibility. However, clinical use of intracellular proteins is hampered by their physicochemical properties such as large size, excess surface charge and relatively unstable structure. Such properties tend to induce their rapid denaturation and/or degradation, extensive uptake by the reticuloendothelial system (RES), and/or elimination by renal filtration. Furthermore, cell membranes also impose a formidable barrier for such proteins to cross in order to reach their intracellular target site. Apart from that, Proteins that enter cells through endocytosis will be eventually degraded in lysosomes if they do not escape the endosomal pathway before reaching lysosomes. Therefore, the development of protein delivery systems, including liposomal and/or polymeric nanoparticles would substantially facilitate the use of proteins in clinic

Liposomal Magnesium Phosphate Nanoparticles for Intracellular Delivery of Catalase

Protein therapeutics are being widely used for supplementing or modulating biological functions, targeting various cell types, vaccination and diagnostics. Compared to small molecule drugs, proteins are potentially advantageous in exerting more complicated functions, higher specificity and higher bio-compatibility. Nevertheless, proteins are prone to enzymatic degradation and elimination via renal and hepatic pathways and are too large to diffuse into cells. The development of protein delivery systems such as liposomal nanoparticles would substantially facilitate the use of proteins in clinic

Liposomal Magnesium Phosphate Nanoparticles for Intracellular Delivery of Catalase

Protein therapeutics are being widely used for supplementing or modulating biological functions, targeting various cell types, vaccination and diagnostics. Compared to small molecule drugs, proteins are potentially advantageous in exerting more complicated functions, higher specificity and higher bio-compatibility. Nevertheless, proteins are prone to enzymatic degradation and elimination via renal and hepatic pathways and are too large to diffuse into cells. The development of protein delivery systems such as liposomal nanoparticles would substantially facilitate the use of proteins in clinic

Intrapulmonary inoculation of multicellular spheroids to construct an orthotopic lung cancer xenograft model that mimics four clinical stages of non-small cell lung cancer

Introduction: Lung cancer leads in mortality among all types of cancer in US and Non-small cell lung cancer (NSCLC) is the major type of lung cancer. Mice models of lung cancer based on subcutaneous or orthotopic inoculation of cancer cell suspension do not adequately mimic the progression of lung cancer in clinic. Methods: A549-iRFP cells (human NSCLC adenocarcinoma) were cultured to form multicellular spheroids (MCS), which were then inoculated intrapulmonarily into male athymic nude mice. The xenograft cancer development was monitored by in vivo fluorescent imaging and validated by open-chest anatomy, ex vivo fluorescent imaging, and histological studies. Results: The newly developed orthotopic xenograft model of lung cancer simulated all four clinical stages of NSCLC progression over one month: Stage 1) localized tumor at the inoculation site, Stage 2) multiple tumor nodules or larger tumor nodule on the same side of the lung, Stage 3) cancer growth on heart surface, and Stage 4) metastatic cancer on both sides of the lung. The model yielded high rates of postoperative survival (100%) and parenchymal tumor establishment (88.9%). The roughness of the inoculated MCS associated negatively with the time needed to develop metastatic cancer (p = .0299). Discussion: This new orthotopic xenograft model of NSCLC would facilitate the development of medications to treat lung cancer

T cell engaging bispecific antibodies targeting CD33 IgV and IgC domains for the treatment of acute myeloid leukemia

Background Acute myeloid leukemia (AML) remains one of the most challenging hematological malignancies. Despite progress in therapeutics, majority of patients succumb to this neoplasm. CD33 is a proven therapeutic target, given its expression on most AML cells. Almost all anti-CD33 antibodies target the membrane distal immunoglobulin V (IgV) domain of the CD33 extracellular domain.Methods In this manuscript, we present data on three bispecific antibodies (BsAbs) against the CD33 IgV and membrane proximal immunoglobulin C (IgC) domains. We use in vitro binding and cytotoxicity assays to show the effect of these BsAbs on AML cell lines. We also use immunodeficient mice-bearing leukemias from cell lines and patient-derived xenografts to show the effect of these BsAbs in vivo.Results In vitro, the IgV-targeting BsAb had higher binding to AML cell lines using flow cytometry and delivered more potent cytotoxicity in T-cell-dependent cytotoxicity assays; importantly, the IgC domain-targeting outperformed the IgV domain-targeting BsAb in medullary and extramedullary leukemia animal models.Conclusions These data support further clinical development of this BsAb for first-in-human phase I clinical trial