Probal Banerjee

Core-shell hybrid nanogels for integration of optical temperature-sensing, targeted tumor cell imaging, and combined chemo-photothermal treatment.

We report a class of core-shell structured hybrid nanogels to demonstrate the conception of integrating the functional building blocks into a single nanoparticle system for simultaneously optical temperature-sensing, cancer cell targeting, fluorescence imaging, and combined chemo-photothermal treatment. The hybrid nanogels were constructed by coating the Ag-Au bimetallic NP core with a thermo-responsive nonlinear poly(ethylene glycol) (PEG)-based hydrogel as shell, and semi-interpenetrating the targeting ligands of hyaluronic acid chains into the surface networks of gel shell. The Ag-Au NP core can emit strong visible fluorescence for imaging of mouse melanoma B16F10 cells. The reversible thermo-responsive volume phase transition of the nonlinear PEG-based gel shell cannot only modify the physicochemical environment of the Ag-Au NP core to manipulate the fluorescence intensity for sensing the environmental temperature change, but also provide a high loading capacity for a model anticancer drug temozolomide and offer a thermo-triggered drug release. The drug release can be induced by both the heat generated by external NIR irradiation and the temperature increase of local environmental media. The ability of the hybrid nanogels to combine the local specific chemotherapy with external NIR photothermal treatment significantly improves the therapeutic efficacy due to a synergistic effect.

Chitosan-based responsive hybrid nanogels for integration of optical pH-sensing, tumor cell imaging and controlled drug delivery

We report a new class of chitosan-based hybrid nanogels by in-situ immobilization of CdSe quantum dots (QDs) in the chitosan-poly(methacrylic acid) (chitosan-PMAA) networks. The covalently crosslinked hybrid nanogels with chitosan chains semi-interpenetrating in the crosslinked PMAA networks exhibit excellent colloidal and structural stability as well as reversible physical property change in response to a pH variation cross the physiological condition. In contrast, the hybrid nanogels formed by non-covalent physical association exhibit a significant change in the structure and composition upon exposure to physiological pH. This distinction in the structural stability of hybrid nanogels produces very different outcomes for their biomedical applications. The covalently crosslinked hybrid nanogels are low-cytotoxic and could illuminate the B16F10 cells, sense the environmental pH change, and regulate the release of anticancer drug in the typical abnormal pH range of 5-7.4 found in pathological zone, thus successfully combine multiple functionality into a single nano-object. However, the physically associated hybrid nanogels exhibit a non-reversible pH-sensitive PL property and a significant cytotoxicity after 24 h treatment. It is critical to construct a highly stable biopolymer-QD hybrid nanogel, via a rational design for safe bionanomaterials, to simultaneously combine the biosensing, bioimaging, and effective therapy functions.

In-situ immobilization of quantum dots in polysaccharide-based nanogels for integration of optical pH-sensing, tumor cell imaging, and drug delivery

We report a class of polysaccharide-based hybrid nanogels that can integrate the functional building blocks for optical pH-sensing, cancer cell imaging, and controlled drug release into a single nanoparticle system, which can offer broad opportunities for combined diagnosis and therapy. The hybrid nanogels were prepared by in-situ immobilization of CdSe quantum dots (QDs) in the interior of the pH and temperature dual responsive hydroxypropylcellulose-poly(acrylic acid) (HPC-PAA) semi-interpenetrating polymer networks. The-OH groups of the HPC chains are designed to sequester the precursor Cd(2+) ions into the nanogels as well as stabilize the in-situ formed CdSe QDs. The pH-sensitive PAA network chains are designed to induce a pH-responsive volume phase transition of the hybrid nanogels. The developed HPC-PAA-CdSe hybrid nanogels combine a strong trap emission at 741nm for sensing physicochemical environment in a pH dependent manner and a visible excitonic emission at 592nm for mouse melanoma B16F10 cell imaging. The hybrid nanogels also provide excellent stability as a drug carrier, which cannot only provide a high drug loading capacity for a model anticancer drug temozolomide, but also offer a pH-triggered sustained-release of the drug molecules in the gel network.

Differential solubilization of lipids along with membrane proteins by different classes of detergents

Membrane proteins are typically extracted by detergent concentrations of 0.5-2.0%, using detergent/protein ratios of 1:1 to 3:1. We have compared the ability of 14 different detergents from seven different structural and ionic classes, at a concentration of 2.0% and a detergent/protein ratio of 2:1, to extract an integral membrane protein (the serotonin 5-HT1A receptor) in active form and have observed profound differences in both lipids and proteins. All extracts were freed from detergents and dialyzed to form vesicles containing 95-100% of the extracted lipids, prior to [3H]8-hydroxy-2-(N,N-di-n-propylamino)tetralin ([3H]8-OH-DPAT) binding. The most efficient detergents in extracting active 5-HT1A receptor protein were the zwitterionic 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) and 3-[(cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate (CHAPSO), followed by the neutral n-dodecyl-beta-D-maltoside. Zwitterionic detergents also produced the highest solubilized lipid/protein ratio (3.0 and 2.5, respectively) and in general the relative amounts of extracted lipids and proteins followed inverse profiles. Thus, hydrophobic detergents such as Tritons (with critical micelle concentrations similar to CHAPS) and Thesit (structurally similar to Lubrol) extracted the most protein, but relatively little lipid (ratios of less than 0.2) and very little active 5-HT receptor. Dramatic differences were also observed in the ratios of individual lipids extracted by the same concentrations of different detergents and resolved by high-performance thin-layer chromatography. For example, galactosylceramide (GalCer) content ranged from 2.7% (CHAPSO) to 13.4% (sodium cholate) of the total lipid extract and cholesterol ranged from 0% (digitonin) to 17.9% (Triton X-100). The detergent-extractability profile for phosphatidylethanolamine (PE) (range 15-40% of total lipid) paralleled that of phosphatidylinositol (PI) (range 4-10%), but was inverse to that for GalCer and cholesterol. Detergent-extractability profiles for phosphatidylcholine (PC) and phosphatidylserine (PS) also followed inverse profiles, with zwitterionic detergents giving high PS/PC and high PE/PC ratios (approximately 2:1), whereas the Tritons and digitonin gave ratios of 1:2. We believe that differential solubilization of lipids, as well as proteins, by detergents is important for the biological activity of the extracted proteins, and lipid extractability should be taken into account when purifying membrane proteins.

Externalization of Phosphatidylserine May Not Be an Early Signal of Apoptosis in Neuronal Cells, but Only the Phosphatidylserine‐Displaying Apoptotic Cells Are Phagocytosed by Microglia

Abstract: Earlier reports on nonneural cells have shown that the normally inner plasma membrane lipid, phosphatidylserine (PS), flip‐flops out during the early stages of apoptosis, whereas DNA laddering and plasma membrane permeabilization occur during the late stages. In this study, the applicability of these parameters to CNS‐derived neuronal cells was tested using hippocampal HN2‐5, cells that undergo apoptosis under anoxia. Because such insults on unsynchronized cells, e.g., undifferentiated HN2‐5 cells, result in both early and late apoptotic cells, we mechanically separated these cells into three fractions containing (a) cells that had completely detached during anoxia, (b) cells that remained weakly attached to the tissue culture dish and, once detached by trituration in serum‐containing medium, did not reattach, and (c) cells that reattached in 2–3 h. Fractions a and b contained cells that showed pronounced DNA laddering, whereas cells in fraction c did not show any DNA laddering. Double staining with fluorescein isothiocyanate‐annexin V (which binds to PS) and propidium iodide (which stains the DNA in cells with a permeable cell membrane) revealed that all cells in fraction a had a permeable cell membrane (propidium iodide‐positive) and PS molecules in the outer leaflet of the plasma membrane (fluorescein isothiocyanate‐annexin V‐positive). By contrast, fractions b and c contained cells with no externalized PS molecules. Cells in fractions a–c also showed, respectively, 50‐, 21‐, and 5.5‐fold higher caspase‐3 (CPP32) activity than that in healthy control cells. All these results show that fraction a contained late apoptotic cells, which also had the highest CPP32 activity; cells in fraction b were at an intermediate stage, when DNA laddering had already occurred; and fraction c contained very early apoptotic cells, in which no DNA laddering had yet occurred. Therefore, in the neuronal HN2‐5 cells, externalization of PS occurs only during the final stages of apoptosis when the cells have completely lost their adhesion properties. Further experiments showed that ameboid microglial cells isolated from neonatal mouse brain phagocytosed only the cells in fraction a. These results show that in CNS‐derived HN2‐5 cells, (a) PS externalization is a late apoptotic event and is concomitant with a complete loss of surface adhesion of the apoptotic cells and (b) PS externalization is crucial for microglial recognition and phagocytosis of the apoptotic HN2‐5 cells. Thus, PS externalization could be causally linked to the final detachment of apoptotic neuronal cells, which in turn prepares them for rapid phagocytosis by microglia.

Water-dispersible multifunctional hybrid nanogels for combined curcumin and photothermal therapy

We design a class of water-dispersible hybrid nanogels for intracellular delivery of hydrophobic curcumin. The core-shell structured hybrid nanogels were synthesized by coating the Ag/Au bimetallic nanoparticles (NPs) with a hydrophobic polystyrene (PS) gel layer as inner shell, and a subsequent thin hydrophilic nonlinear poly(ethylene glycol) (PEG)-based gel layer as outer shell. The uniqueness of these hybrid nanogels lies in the integration of the functional building blocks for combined curcumin and photothermal therapy to significantly improve the therapeutic efficacy. The Ag/Au core NPs cannot only emit strong fluorescence for imaging and monitoring at the cellular level, but also exhibit strong absorption in the near-infrared (NIR) region for photothermal conversion. While the inner PS gel layer is introduced to provide strong hydrophobic interactions with curcumin for high drug loading yields, the external nontoxic and thermo-responsive PEG analog gel layer is designed to trigger the release of the pre-loaded curcumin either by variation of surrounding temperature or exogenous irradiation with NIR light. Such designed multifunctional hybrid nanogels are well suited for in vivo studies and clinical trials, thereby likely to bring this promising natural medicine of curcumin to the forefront of therapeutic agents for cancers and other diseases.

Specific glucose-to-SPR signal transduction at physiological pH by molecularly imprinted responsive hybrid microgels

We design a class of imprinted hybrid microgels that can optically monitor glucose levels with high sensitivity and selectivity in complex media at physiological pH, acting like a glucose-indicator. Such imprinted hybrid microgels were made of Ag nanoparticles (NPs) in situ immobilized in molecularly imprinted glucose-responsive polymeric microgel templates containing phenylboronic acids in such a way that the Ag NPs were confined in the immediate vicinity to each other, thus enabling their efficient plasmon coupling. The glucose-responsive gel-actuated tunable plasmon coupling effects among the Ag NPs immobilized inside the microgels were investigated in both phosphate buffer solution and artificial tear fluid. The visually evident color shift from yellow to red of the hybrid microgel dispersion in response to a glucose concentration change from 0 to 20.0 mm allows one to see the glucose levels without instrumental aid. The surface plasmon resonance (SPR) response of the imprinted hybrid microgels at appropriate loading amount of Ag NPs is free of the significant interferences from the major non-glucose constituents, enabling the optical glucose sensing in artificial tear fluids with the achieved root-mean-squared error of predication (RMSEC) as low as 13.7 μM (~0.2 mg/dL) over a clinically relevant glucose concentration range of 0.1-20 mm (1.8-360 mg/dL). The highly versatile imprinted hybrid microgels could potentially be used for continuous glucose monitoring in clinical diagnostic and bioprocess applications.

Multi-functional core-shell hybrid nanogels for pH-dependent magnetic manipulation, fluorescent pH-sensing, and drug delivery.

Remotely optical sensing and drug delivery using an environmentally-guided magnetically-driven hybrid nanogel particle could allow for medical diagnostics and treatment. Such multifunctional hybrid nanogels (<200 nm) were prepared through the first synthesis of magnetic Ni NPs, followed by a moderate growth of fluorescent metallic Ag on the surface of Ni NPs, and then a coverage of a pH-responsive copolymer gel shell of poly(ethylene glycol-co-methacrylic acid) [p(EG-MAA)] onto the Ni-Ag bimetallic NP cores (18 ± 5 nm). The introduction of the pH-responsive p(EG-MAA) gel shell onto the magnetic and fluorescent Ni-Ag NPs makes the polymer-bound Ni-Ag NPs responsive to pH over the physiologically important range 5.0-7.4. The hybrid nanogels can adapt to surrounding pH and regulate the sensitivity in response to external magnetic field (such as a small magnet of 0.1 T), resulting in the accumulation of the hybrid nanogels within the duration from hours to a few seconds as the pH value decreases from 7.4 to 5.0. The pH-dependent magnetic response characteristic of the hybrid nanogels were further integrated with the pH change to fluorescent signal transduction and pH-regulated anticancer drug (a model drug 5-fluorouracil) delivery functions. The hybrid nanogels can overcome cellular barriers to enter the intracellular region and light up the mouse melanoma B16F10 cells. The multiple responsive hybrid nanogel that can be manipulated in tandem endogenous and exogenous activation should enhance our ability to address the complexity of biological systems.

Microarray Analysis of Oxidative Stress Regulated Genes in Mesencephalic Dopaminergic Neuronal Cells: Relevance to Oxidative Damage in Parkinson’s Disease

Oxidative stress and apoptotic cell death have been implicated in the dopaminergic cell loss that characterizes Parkinsons disease. While factors contributing to apoptotic cell death are not well characterized, oxidative stress is known to activate an array of cell signaling molecules that participate in apoptotic cell death mechanisms. We investigated oxidative stress-induced cytotoxicity of hydrogen peroxide (H2O2) in three cell lines, the dopaminergic mesencephalon-derived N27 cell line, the GABAergic striatum-derived M213-20 cell line, and the hippocampal HN2-5 cell line. N27 cells were more sensitive to H2O2-induced cell death than M213-20 and HN2-5 cells. H2O2 induced significantly greater increases in caspase-3 activity in N27 cells than in M213-20 cells. H2O2-induced apoptotic cell death in N27 cells was mediated by caspase-3-dependent proteolytic activation of PKCdelta. Gene expression microarrays were employed to examine the specific transcriptional changes in N27 cells exposed to 100 microM H2O2 for 4 h. Changes in genes encoding pro- or anti-apoptotic proteins included up-regulation of BIK, PAWR, STAT5B, NPAS2, Jun B, MEK4, CCT7, PPP3CC, and PSDM3, while key down-regulated genes included BNIP3, NPTXR, RAGA, STK6, YWHAH, and MAP2K1. Overall, the changes indicate a modulation of transcriptional activity, chaperone activity, kinase activity, and apoptotic activity that appears highly specific, coordinated and relevant to cell survival. Utilizing this in vitro model to identify novel oxidative stress-regulated genes may be useful in unraveling the molecular mechanisms underlying dopaminergic degeneration in Parkinsons disease.

Magnetic iron oxide–fluorescent carbon dots integrated nanoparticles for dual-modal imaging, near-infrared light-responsive drug carrier and photothermal therapy

Multifunctional hybrid nanoparticles (NPs, ∼100 nm) that combine magnetic Fe3O4 nanocrystals and fluorescent carbon dots (CDs) in porous carbon (C) were successfully synthesized using a one-pot solvothermal method by simply increasing the H2O2 concentration. The resultant Fe3O4@C-CDs hybrid NPs not only demonstrate excellent magnetic responsive properties (Ms = 32.5 emu g-1) and magnetic resonance imaging ability (r = 674.4 mM-1 s-1) from the Fe3O4 nanocrystal core, but also exhibit intriguing photoluminescent (quantum yield ∼6.8%) properties including upconversion fluorescence and excellent photostability from the CDs produced in the porous carbon. The hybrid NPs can enter the intracellular region and illuminate mouse melanoma B16F10 cells under different excitation wavelengths. Meanwhile, the mesoporous carbon shell and hydrophilic surface functional groups endow the hybrid NPs with high loading capacity (835 mg g-1) for the anti-cancer drug doxorubicin and excellent stability in aqueous solutions. More importantly, the hybrid NPs can absorb and convert near-infrared (NIR) light to heat due to the existence of CDs, and thus, can realise NIR-controlled drug release and combined photothermo/chemotherapy for high therapeutic efficacy. Such nanostructured Fe3O4@C-CDs hybrid NPs demonstrate great promise towards advanced nanoplatforms for simultaneous imaging diagnostics and high efficacy therapy.