Encapsulation of imaging agents and drugs in calcium phosphate nanoparticles (CPNPs) has potential as a nontoxic, bioresorbable vehicle for drug delivery to cells and tumors. The objectives of this study were to develop a calcium phosphate nanoparticle encapsulation system for organic dyes and therapeutic drugs so that advanced fluoresence methods could be used to assess the efficiency of drug delivery and possible mechanisms of nanoparticle bioabsorption. Highly concentrated CPNPs encapsulating a variety of organic fluorophores were successfully synthesized. Well-dispersed CPNPs encapsulating Cy3 amidite exhibited nearly a 5-fold increase in fluorescence quantum yield when compared to the free dye in PBS. FCS diffusion data and cell staining were used to show pH-dependent dissolution of the particles and cellular uptake, respectively. Furthermore, an experimental hydrophobic cell growth inhibitor, ceramide, was successfully delivered in vitro to human vascular smooth muscle cells via encapsulation in CPNPs. These studies demonstrate that CPNPs are effective carriers of dyes and drugs for bioimaging and, potentially, for therapeutic intervention.
The early diagnosis of cancer is the critical element in successful treatment and long term favorable patient prognoses. The high rate of mortality is mainly attributed to the tendency for late diagnoses as symptoms may not occur until the disease has metastasized, as well as the lack of effective systemic therapies. Late diagnosis is often associated with the lack of timely sensitive imaging modalities. The promise of nanotechnology is presently limited by the inability to simultaneously seek, treat and image cancerous lesions. This study describes the design and synthesis of fluorescent calcium phosphosilicate nanocomposite particles (CPNPs) that can be systemically targeted to breast and pancreatic cancer lesions. The CPNPs are a ~20nm diameter composite composed of an amorphous calcium phosphate matrix doped with silicate in which a near infra-red imaging agent indocyanine green (ICG) is embedded. In the present studies, we describe and validate CPNP bioconjugation of human holotransferrin, anti-CD71 antibody, and short gastrin peptides via an avidin-biotin-or a novel PEG-maleimide-coupling strategy. The conjugation of biotinylated human holotransferrin (diferric transferrin) and biotinylated anti-CD71 antibody (anti-transferrin receptor antibody) to avidin conjugated CPNPs (Avidin-CPNPs) permits targeting of transferrin receptors, which are highly expressed on breast cancer cells. Similarly, the conjugation of biotinylated pentagastrin to AvidinCPNPs and decagastrin (gastrin-10) to PEG-CPNPs via PEG-maleimide coupling permits targeting of gastrin receptors, which are over-expressed in pancreatic cancer lesions. These bioconjugated CPNPs have the potential to perform as a theranostic modality, simultaneously enhancing drug delivery, targeting and imaging of breast and pancreatic cancer tumors. Keywords bioconjugation; transferrin receptor; gastrin receptor; breast cancer; pancreatic cancer; calcium phosphate; whole animal imaging Calcium phosphate nanoparticles (CPNPs) have been engineered to be a non-toxic vehicle for the delivery of a diverse range of therapeutic and imaging agents in biological systems. [1][2][3][4] Previous studies have shown that encapsulation within CPNPs improved the lifetime and RESULTS AND DISCUSSION Physical Characterization of CPNPsCitrate functionalized CPNPs were utilized as a platform for functionalization, which allowed the characterization of bioconjugation via zeta potential analysis (Figure 1). Figure 1 shows the zeta potential distribution of Citrate-CPNPs prior to bioconjugation (blue line), and the zeta (violet). Prior to bioconjugation, the Citrate-CPNPs display a negative mean zeta potential value of −16 ± 1.3 mV, which is consistent with previous reports. 1 However, after bioconjugation, the Avidin-CPNPs displayed a relatively high positive mean zeta potential value of +29 ± 8.7 mV. The isoelectric point for avidin is pH 10. Thus, the shift from a negative zeta potential to a positive zeta potential distribution is strong evidence of avidin bioconjugation on...
Leukemia is one of the most common and aggressive adult cancers, as well as the most prevalent childhood cancer. Leukemia is a cancer of the hematological system and can be divided into a diversity of unique malignancies based on the onset of the disease as well as the specific cell lineages involved. Cancer stem cells, including recently identified leukemia stem cells (LSCs), are hypothesized to be responsible for cancer development, relapse, and resistance to treatment, and new therapeutics targeting these cellular populations are urgently needed. Nontoxic and nonaggregating calcium phosphosilicate nanoparticles (CPSNPs) encapsulating the near-infrared fluoroprobe indocyanine green (ICG) were recently developed for diagnostic imaging and drug delivery as well as for photodynamic therapy (PDT) of solid tumors. Prior studies revealed that specific targeting of CPSNPs allowed for enhanced accumulation within breast cancer tumors, via CD71 targeting, or pancreatic cancer tumors, via gastrin receptor targeting. In the present study, ICG-loaded CPSNPs were evaluated as photosensitizers for PDT of leukemia. Using a novel bioconjugation approach to specifically target CD117 or CD96, surface features enhanced on leukemia stem cells, in vitro ICG-CPSNP PDT of a murine leukemia cell line and human leukemia samples were dramatically improved. Furthermore, the in vivo efficacy of PDT was dramatically enhanced in a murine leukemia model by utilizing CD117-targeted ICG-CPSNPs, resulting in 29% disease-free survival. Altogether, this study demonstrates that leukemia-targeted ICG-loaded CPSNPs offer the promise to effectively treat relapsing and multidrug-resistant leukemia and to improve the life of leukemia patients.
The natural killer (NK) type of aggressive large granular lymphocytic (LGL) leukemia is a fatal illness that pursues a rapid clinical course. There are no effective therapies for this illness, and pathogenetic mechanisms remain undefined. Here we report that the survivin was highly expressed in both aggressive and chronic leukemic NK cells but not in normal NK cells. In vitro treatment of human and rat NK-LGL leukemia cells with cell-permeable, short-chain C₆-ceramide (C₆) in nanoliposomal formulation led to caspase-dependent apoptosis and diminished survivin protein expression, in a time- and dose-dependent manner. Importantly, systemic intravenous delivery of nanoliposomal ceramide induced complete remission in the syngeneic Fischer F344 rat model of aggressive NK-LGL leukemia. Therapeutic efficacy was associated with decreased expression of survivin in vivo. These data suggest that in vivo targeting of survivin through delivery of nanoliposomal C₆-ceramide may be a promising therapeutic approach for a fatal leukemia.
Autophagy, a catabolic survival pathway, is gaining attention as a potential target in cancer. In human liver and colon cancer cells, treatment with an autophagy inducer, nanoliposomal C6-ceramide, in combination with the autophagy maturation inhibitor, vinblastine, synergistically enhanced apoptotic cell death. Combination treatment resulted in a marked increase in autophagic vacuole accumulation and decreased autophagy maturation, without diminution of the autophagy flux protein P62. In a colon cancer xenograft model, a single intravenous injection of the drug combination significantly decreased tumor growth in comparison to the individual treatments. Most importantly, the combination treatment did not result in increased toxicity as assessed by body weight loss. The mechanism of combination treatment-induced cell death both in vitro and in vivo appeared to be apoptosis. Supportive of autophagy flux blockade as the underlying synergy mechanism, treatment with other autophagy maturation inhibitors, but not autophagy initiation inhibitors, were similarly synergistic with vinblastine. Additionally, knockout of the autophagy protein Beclin-1 suppressed combination treatment-induced apoptosis in vitro. In conclusion, in vitro and in vivo data support a synergistic antitumor activity of the nanoliposomal C6-ceramide and vinblastine combination, potentially mediated by an autophagic mechanism.
Ceramide is a sphingolipid metabolite that induces cancer cell death. When C6-ceramide is encapsulated in a nanoliposome bilayer formulation, cell death is selectively induced in tumor models. However, the mechanism underlying this selectivity is unknown. As most tumors exhibit a preferential switch to glycolysis, as described in the “Warburg effect”, we hypothesize that ceramide nanoliposomes selectively target this glycolytic pathway in cancer. We utilize chronic lymphocytic leukemia (CLL) as a cancer model, which has an increased dependency on glycolysis. In CLL cells, we demonstrate that C6-ceramide nanoliposomes, but not control nanoliposomes, induce caspase 3/7-independent necrotic cell death. Nanoliposomal ceramide inhibits both the RNA and protein expression of GAPDH, an enzyme in the glycolytic pathway, which is overexpressed in CLL. To confirm that ceramide targets GAPDH, we demonstrate that downregulation of GAPDH potentiates the decrease in ATP after ceramide treatment and exogenous pyruvate treatment as well as GAPDH overexpression partially rescues ceramide-induced necrosis. Finally, an in vivo murine model of CLL shows that nanoliposomal C6-ceramide treatment elicits tumor regression, concomitant with GAPDH downregulation. We conclude that selective inhibition of the glycolytic pathway in CLL cells with nanoliposomal C6-ceramide could potentially be an effective therapy for leukemia by targeting the Warburg effect.
The new oncologic paradigm of precision medicine is focused on identifying metabolic, proteomic, transcriptomic and genomic variabilities in tumors that can be exploited to tailor treatments and improve patient outcomes. Metabolic changes are a hallmark of cancer, and inhibition of metabolic pathways is now a major strategy in medicinal chemistry for targeting cancers. However, non-invasive biomarkers to categorize metabolic subtypes are in short supply. The purpose of this study was to characterize the intracellular and extracellular metabolic profiles of four prostate cancer cell lines with varying degrees of aggressiveness. We observed metabolic differences between the aggressive prostate cancer cell line PC3 and the even more aggressive, metastatic subline PC3M assessed by hyperpolarized in vivo pyruvate studies, nuclear magnetic resonance spectroscopy, and carbon-13 feeding studies. On further examination of the differences between these two cell lines, we found increased glutamine utilization in the metastatic PC3M subline that led directly to sensitivity to glutaminase inhibitor CB-839. Our study supports the theory that metastatic progression increases glutamine utilization and the inhibition of glutaminolysis could have clinical implications.
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