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In JoVE (1)
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Articles by Shrirang Karve in JoVE
JoVE Bioengineering
Diblock polymeric नैनोकणों के Nanoprecipitation तकनीक के माध्यम से निरूपण
1Laboratory of Nano- and Translational Medicine, Department of Radiation Oncology, Lineberger Comprehensive Cancer Center, University of North Carolina School of Medicine, 2Carolina Center for Nanotechnology Excellence, University of North Carolina
यह लेख एक nanoprecipitation बहुलक आधारित diblock सह - पॉलिमर का उपयोग नैनोकणों synthesize विधि का वर्णन करता है. हम diblock सह पॉलिमर, nanoprecipitation तकनीक, और संभावित अनुप्रयोगों के संश्लेषण पर चर्चा करेंगे.
Other articles by Shrirang Karve on PubMed
Heterogeneous Domains and Membrane Permeability in Phosphatidylcholine-phosphatidic Acid Rigid Vesicles As a Function of PH and Lipid Chain Mismatch
Heterogeneous lipid membranes tuned by pH were evaluated at 37 degrees C in the form of PEGylated vesicles composed of lipid pairs with dipalmitoyl ( n = 16) and distearoyl ( n = 18) chain lengths. One lipid type was chosen to have the titratable moiety phosphatidic acid on its headgroup, and the other lipid type was chosen to have a phosphatidylcholine headgroup. The effect of pH on the formation of lipid heterogeneities and on membrane permeability was studied on vesicles composed of lipid pairs with matching and nonmatching chain lengths. The formation of lipid heterogeneities increases with decreasing pH in membranes composed of lipid pairs with either matching or nonmatching chain lengths. Increased permeability with decreasing pH was exhibited only by membranes composed of lipid pairs with nonmatching chain lengths. Permeability rates correlate strongly with the predicted extent of interfacial boundaries of heterogeneities, suggesting defective packing among nonmatching acyl chains of lipids. In heterogeneous mixtures with one lipid type in the fluid state ( n = 12), the dependence of membrane permeability on pH is weaker. In the presence of serum proteins, PEGylated gel-phase vesicles containing lipid pairs with nonmatching chain lengths exhibit faster release rates with decreasing pH compared to measured release rates in phosphate buffer, suggesting a second mechanism of formation of separated phases. PEGylated vesicles composed of lipid pairs with nonmatching chain lengths labeled with internalizing anti-HER2/neu antibodies that target overexpressed antigens on the surface of SKOV3-NMP2 ovarian cancer cells exhibit specific cancer cell targeting, followed by extensive internalization (more than 84% of bound vesicles) and fast release of contents intracellularly. These PEGylated vesicles composed of rigid membranes for long blood circulation times that exhibit pH-dependent release of contents intracellularly could become potent drug delivery carriers for the targeted therapy of solid tumors.
PH-dependent Formation of Lipid Heterogeneities Controls Surface Topography and Binding Reactivity in Functionalized Bilayers
During direct cell-to-cell communication, lipids on the extracellular side of plasma membranes reorganize, and membrane-associated communication-related molecules colocalize. At colocalization sites, sometimes identified as rafts, the local cell surface topography and reactivity are altered. The processes regulating these changes are largely unknown. On model lipid membranes, study of simplified processes that control surface topography and reactivity may potentially contribute to the understanding and control of related cell functions and associated diseases. Integration of these processes on nanometer-sized lipid vesicles used as drug delivery carriers would precisely control their interactions with diseased cells minimizing toxicities. Here we design such basic pH-dependent processes on model functionalized lipid bilayers, and we demonstrate reversible sharp changes in binding reactivity within a narrow pH window. Cholesterol enables tuning of the membrane reorganization to occur at pH values not necessarily close to the reported pK(a)'s of the constituent titratable lipids, and bilayer reorganization over repeated cycles of induced pH changes exhibits hysteresis.
The Use of PH-triggered Leaky Heterogeneities on Rigid Lipid Bilayers to Improve Intracellular Trafficking and Therapeutic Potential of Targeted Liposomal Immunochemotherapy
During endocytosis, pH-triggered release of encapsulated therapeutics from delivery carriers may accelerate their intracellular trafficking increasing therapeutic efficacy. To improve the therapeutic potential of targeted immunochemotherapy using anti-HER2/neu liposomal doxorubicin, we exploit the formation of leaky heterogeneities on rigid lipid bilayers to extensively release doxorubicin during endocytosis. We have previously demonstrated that pH-dependent formation of phase-separated lipid heterogeneities on the plane of a bilayer membrane increases the permeability of bilayers when they are composed of lipid pairs with rigid non-matching acyl chain lengths. This was suggested to be due to defective packing among lipids residing at the interfaces of lipid domains. Here we design nanometer-size antiHER2/neu-labeled PEGylated vesicles composed of lipid pairs with longer non-matching acyl chain lengths (n=18 and 21). These vesicles exhibit superior killing efficacy of cancer cells compared to established liposome formulations, and their killing efficacy is similar to the effect of combined free doxorubicin and free antiHER2/neu antibody. Other transport-related properties such as liposome blood circulation times, and specific binding and internalization by cancer cells are unaffected. These results demonstrate the potential of vesicles with pH-triggered leaky heterogeneities to increase the therapeutic potential of targeted immunochemotherapy.
The PH-dependent Association with Cancer Cells of Tunable Functionalized Lipid Vesicles with Encapsulated Doxorubicin for High Cell-kill Selectivity
To enable selective cell-kill, we designed functionalized lipid vesicles with pH-triggered heterogeneous membranes and encapsulated doxorubicin that exhibit tunable surface topography. These vesicles "hide" (mask) the targeting ligands from their surface during circulation in the blood, and only progressively "expose" these ligands as they gradually penetrate deeper into the tumor interstitium, where after endocytosis they burst release their contents. The stimulus to activate the binding reactivity is the pH gradient between the blood stream (pH 7.4-7.0) and the increasingly acidic pH inside the tumor interstitium (pH 6.7-6.5). Doxorubicin release is activated at the endosomal pH 5.5-5.0. We show that tunable functionalized vesicles exhibit environmentally-dependent (pH-dependent) association with cancer cells resulting in high cell-kill selectivity. When lowering the extracellular pH from 7.4 to 6.5, tunable functionalized vesicles deliver doxorubicin to cancer cells that increases from 41% to 93% of maximum resulting in cancer cell killing that increases from 23 to 71% of maximum, respectively. This proof-of-concept shows the potential of tunable targeted liposomal chemotherapy to selectively kill cancer cells in an environmentally-dependent way.
Folate-targeted Nanoparticle Delivery of Chemo- and Radiotherapeutics for the Treatment of Ovarian Cancer Peritoneal Metastasis
Peritoneal metastasis is a major cause of morbidity and mortality in ovarian cancer. While intraperitoneal chemotherapy and radiotherapy have shown favorable clinical results, both are limited by their non-targeted nature. We aimed to develop a biologically targeted nanoparticle therapeutic for the treatment of ovarian cancer peritoneal metastasis. Folate-targeted nanoparticles encapsulating chemotherapy and/or radiotherapy were engineered. Paclitaxel (Ptxl) was used as the chemotherapeutic and yittrium-90 ((90)Y) was employed as the therapeutic radioisotope. Folate was utilized as the targeting ligand as most ovarian cancers overexpress the folate receptor. Nanoparticle characterization studies showed monodispersed particles with controlled Ptxl release. Folate targeting ligand mediated the uptake of NPs into tumor cells. In vitro efficacy studies demonstrated folate-targeted NPs containing chemoradiotherapy was the most effective therapeutic compared to folate-targeted NPs containing a single therapeutic or any non-targeted NP therapeutics. In vivo efficacy studies using an ovarian peritoneal metastasis model showed that folate-targeted NP therapeutics were significantly more effective than non-targeted NP therapeutics. Among the folate-targeted therapeutics, the NP containing chemoradiotherapy appeared to be the most effective. Our results suggest that folate-targeted nanoparticles containing chemoradiotherapy have the potential as a treatment for ovarian peritoneal metastasis.
Folate-targeted Polymeric Nanoparticle Formulation of Docetaxel is an Effective Molecularly Targeted Radiosensitizer with Efficacy Dependent on the Timing of Radiotherapy
Nanoparticle (NP) chemotherapeutics hold great potential as radiosensitizers. Their unique properties, such as preferential accumulation in tumors and their ability to target tumors through molecular targeting ligands, are ideally suited for radiosensitization. We aimed to develop a molecularly targeted nanoparticle formulation of docetaxel (Dtxl) and evaluate its property as a radiosensitizer. Using a biodegradable and biocompatible lipid-polymer NP platform and folate as a molecular targeting ligand, we engineered a folate-targeted nanoparticle (FT-NP) formulation of Dtxl. These NPs have sizes of 72 ± 4 nm and surface charges of -42 ± 8 mV. Using folate receptor overexpressing KB cells and folate receptor low HTB-43 cells, we showed folate-mediated intracellular uptake of NPs. In vitro radiosensitization studies initially showed FT-NP is less effective than Dtxl as a radiosensitizer. However, the radiosensitization efficacy is dependent on the timing of radiotherapy. In vitro radiosensitization conducted with irradiation given at the optimal time (24 h) showed FT-NP Dtxl is as effective as Dtxl. When FT-NP Dtxl is compared to Dtxl and nontargeted nanoparticle (NT-NP) Dtxl in vivo, FT-NP was found to be significantly more effective than Dtxl or NT-NP Dtxl as a radiosensitizer. We also confirmed that radiosensitization is dependent on timing of irradiation in vivo. In summary, FT-NP Dtxl is an effective radiosensitizer in folate-receptor overexpressing tumor cells. Time of irradiation is critical in achieving maximal efficacy with this nanoparticle platform. To the best of our knowledge, our report is the first to demonstrate the potential of molecularly targeted NPs as a promising new class of radiosensitizers.
